ISPP2009 Final Program

FINAL PROGRAMand ABSTRACTS

www.ispp2009.com

August 9 to 14, 2009

Hotel Delta Centre-VilleMontréal, QC, Canada

08:30

Registration

Welcome &Opening KeynoteA.E. Walsby

Plenary 1R. BlankenshipA. MulkidjanianV. Gorlenko

Plenary 2B. BergmanA. Grossman

Poster Mounting07:30 to 10:00

MONDAYAugust 10

SUNDAYAugust 9

WEDNESDAYAugust 12

TUESDAYAugust 11

FRIDAYAugust 14

THURSDAYAugust 13

OralCommunications

1 & 2

OralCommunications

3 & 4

PosterSession

LunchRégence C& Foyer

Coffee Break

Plenary 3E. FLores

M. HagemannK. Forchhammer

Plenary 4S. GoldenP.C. Wolk

OralCommunications

5 & 6

OralCommunications

7 & 8

PosterSession

LunchRégence C orTour de Ville /

Check your Ticket inyour badge holder

Coffee Break

Plenary 6C. BauerD. KirilovskyB. Masepohl

PosterSession

OralCommunications

11 & 12

OralCommunications

13 & 14

Plenary 7I. SuzukiJ.T. Beatty

LunchRégence C orTour de Ville /

Check your Ticket inyour badge holder

Coffee Break

Plenary 8C. HarwoodP. TamagniniK. Sivonen

OralCommunications

15 & 16

OralCommunications

17 & 18

Closing KeynoteF.R. Tabita

LunchRégence C& Foyer

Coffee Break

Plenary 5F. DaldalL. ShermanF. Partensky

OralCommunications

9 & 10

FreeAfternoon

ISPP 2009Party

Sucreriede la Montagne

Buses leave at17:30 from HotelDelta Centre-ville

Coffee Break

OpeningReception

09:00

07:30

08:00

09:30

10:00

10:30

11:00

11:30

12:00

12:30

13:00

13:30

14:00

14:30

15:00

15:30

16:00

16:30

17:00

17:30

18:00

18:30

19:00

19:30

20:00

20:30

21:00

21:30

22:00

August 9 to 14, 2009Hotel Delta Centre-VilleMontréal, QC, Canada

WEEK-AT-A-GLANCE

Table of Contents

General Information

Floor Plans 2

Welcome / Previous ISPP Symposia 3

Committees 4

Symposium Information 5

About Montréal & Useful Information 6

Social Program 7

Scientific Program 9

How This Program Is Organized 10

Instructions to Oral and Poster Presenters 10

Invited Faculty 11

Detailed Program

Sunday, August 9 13

Monday, August 10 15

Tuesday, August 11 19

Wednesday, August 12 23

Thursday, August 13 25

Friday, August 14 29

Exhibitors 147

Presenter Index 149

Abstracts

Keynotes and Plenaries 33

Oral Communications 45

Posters 85

Symposium Secretariat

c/o IS Event Solutions1334 Notre-Dame Street West, 2nd Floor

Montréal, Québec, Canada H3C 1K7Tel.: (514) 392-7703Fax: (514) 227-5083

E-mail: [email protected]

August 9 to 14, 2009 • Montréal, QC, Canada 1

13th International Symposium on Phototrophic Prokaryotes Final Program and Abstracts

Printed in Canada, August 2009Recycled paper

Floor Plans

Site Plan Floor C

2 August 9 to 14, 2009 • Montréal, QC, Canada

Final Program and Abstracts 13th International Symposium on Phototrophic Prokaryotes

CARTIER

A

B

VITRÉ ROYERBONSECOURS

FOYER

RÉGENCE

B AC

TÉLÉPHONES

TELEPHONES

VESTIAIRE

CLOAKROOM

Welcome to MontréalWelcome to Montréal to ISPP 2009, the 13th International Symposium on Phototrophic Prokaryotes. This is only thesecond time that the ISPP series finds itself in North America, and we hope that you find Montréal, a major culturaland scientific hub of North America, a pleasant and exciting blend of Old World charm and New World vitality.

This is one of my favourite conference series to attend because not only does it inform my specific researchpassions, it broadly educates me in ways that improve my teaching and increase my breadth of understanding in avariety of outside areas. Indeed, the ISPP series brings together a broad spectrum of interests, techniques, anddisciplines. The planned plenary and parallel symposia sessions will permit oral communications by bothestablished researchers and newcomers to this field, as well as promoting active audience participation and livelydiscussions. Poster sessions will complement the oral program.

In addition to the planned social events, we hope that the venue, and the surrounding city filled with small cafésand bistros, will be conducive to productive and insightful informal discussions. One of the highlights of pastconferences in this series is the high level of active participation by attendees. We count on you to make ISPP 2009a great success.

Enjoy!

Patrick HallenbeckISPP 2009 Conference ChairUniversité de MontréalMontréal, Quebec, Canada

Previous ISPP

1st 1973 Freiburg, Germany

2nd 1976 Dundee, United Kingdom

3rd 1979 Oxford, United Kingdom

4th 1982 Bombannes, France

5th 1985 Grindelwald, Switzerland

6th 1988 Noordwijkerhout, The Netherlands

7th 1991 Amherst, USA

8th 1994 Urbino, Italy

9th 1997 Vienna, Austria

10th 2000 Barcelona, Spain

11th 2003 Tokyo, Japan

12th 2006 Pau, France



Local Organizing Committee

Patrick Hallenbeck, Université de Montréal; Chair

Tom Beatty, University of British Columbia

David Bird, Université de Québec à Montréal

Doug Campbell, Mount Allison University

George Espie, University of Toronto

George Owttrim, University of Alberta

Charles Trick, University of Western Ontario

Vladimir Yurkov, University of Manitoba

International Scientific Committee

Chair: Jörg Overmann, Germany

Group I. Reaction Centres, Antennae, Bioenergetics

Robert Blankenship, USA

Richard J. Cogdell, United Kingdom

David B. Knaff, USA

Mette Miller, Denmark

Conrad Mullineaux, United Kingdom

Wim Vermaas, USA

Group II. Metabolism, Biosynthesis, Physiology

Christiane Dahl, Germany

Antonia Herrero, Spain

Neil Hunter, United Kingdom

Masahiko Ikeuchi, Japan

Oded Beja, Israel

Paula M. Tamagnini, Portugal

Group III. Cell Structure and Biology, Development

Dave G. Adams, United Kingdom

Judith Armitage, United Kingdom

Cheryl Kerfeld, USA

Katsumi Matsuura, Japan

Frederic Partensky, France

Satoshi Tabata, Japan

Group IV. Taxonomy, Ecology and Evolution

Patrizia Albertano, Italy

Ferran Garcia Pichel, USA

Akira Hiraishi, Japan

Jörg Overmann, Germany

Annick Wilmotte, Belgium

Vladimir Yurkov, Canada

Committees



Symposium Information

Book of AbstractsThe abstracts have been included in this publication,immediately following the detailed program section. Abstractsare also available online.

Business CentreThe business centre can take care of photocopies, sendingfaxes etc. It is located one level below the lobby in theadministrative offices of the hotel.

Eco-friendly EventWe incorporated the following considerations into theplanning of this conference:• Online abstract review requiring no printing• Onine registration system requiring no printing• E-mail marketing requiring no printing• Pitchers instead of bottled drinks• Milk and sugar containers instead of individual packaging• Recyclable and environmentally-friendly delegate bags

The hotel is a 4 green key’s property which means that it hasshown national industry leadership and commitment toprotecting the environment through wide ranging policies andpractices. The hotel has mature programs in place that involvemanagement, employees, guests, and the public, and whichhave shown substantial and measurable results.

Emergency/First AidFor any emergency or first aid services inside the Hotel DeltaCentre-Ville, please dial “0” for the operator from anyavailable house phone.

InformationShould you require any assistance during the Conference,please call 514-991-3851. The Conference Services Desk willbe located in the foyer on Sunday and Monday. As of Tuesday,the information desk is located in room Royer (across fromPlenary room).

Internet AccessWireless Internet is available for delegates carrying a laptopand can be accessed in the lobby area. Laptops must be wi-fienabled. Some Internet stations are available inside roomRoyer. This room will be open during conference hours.

LanguageAlthough French is the official language of Quebec, visitorswill have no problem communicating in English throughoutMontréal. English is the official language of the congress.

Lost and FoundAny lost and found items will be held at the information deskin room Royer for the duration of the event. For any unclaimeditems after the conference, please contact IS Event Solutions.

LuggageDuring the ISPP conference, whether you are staying at theHotel Delta Centre-Ville or at a different hotel, you can leaveyour suitcases with the concierge.

Meals and RefreshmentsCoffee breaks will be served daily in the morning andafternoon in the foyer area. For times of service, please referto the detailed program section.

Lunch will be served in in different locations:

Monday and Friday – Buffet lunch in Régence C and foyer

Tuesday and Thursday – Régence C and Tour de Ville (checkyour lunch ticket!)

Poster AreaPosters are installed in room Cartier AB. While delegates canvisit posters at their convenience, we have scheduled specificposter sessions on Monday, Tuesday and Thursday.

Public NoticeThe Hotel Delta Centre-Ville specifically and all Quebec publicspaces including terraces in general are smoke-freeenvironments. Smoking is permitted only outside of buildings.

RegistrationRegistration is located in the foyer area of Régence. As ofTuesday, the information desk will be available in the roomRoyer.

Registration Hours

Sunday, August 9 15:00-20:00 foyer

Monday, August 10 07:30-17:00 foyer

Tuesday, August 11 07:30-17:00 Royer

Wednesday, August 12 08:00-12:00 Royer

Thursday, August 13 08:00-17:00 Royer

Friday, August 14 08:00-15:00 Royer

Name Badges

For identification and security purposes, delegates mustwear their name badges when onsite at the Hotel DeltaCentre-Ville.



Rogers CupAugust 8-16 • Jarry Park StadiumThe best tennis players in the world meet in one of the mostprestigious tournaments on the men's professional tour.

International Vestibules of MontréalAugust 12-16 • Downtown10th edition of Relève en Blues contest

AquaOngoing • Montréal Science CentreThe global vision that reflects the interdependence betweennature and human beings will unfold as the story of water isshared. AQUA is a voyage into the world of water; a non-stopadventure through three distinct spaces where the spectator iscompletely immersed in the magic of water without a singleword being spoken. A signature experience by Guy Laliberté’sONE DROP foundation.

Les Tam-tams du Mont-RoyalOngoing • Mont-RoyalEvery Sunday, this free, unofficial festival takes place in the vastgreen spaces of Mount Royal Park on Avenue du Parc, exactlyhalf-way between Avenue des Pins and rue Mont-Royal.

Piknic ÉlectronikOngoing • Park Jean-DrapeauEvery Sunday, discover the best of the electronic music scene inan environment created to stimulate the senses.

Pirates, Privateers and FreebootersOngoing • Pointe-à-CallièreDiscover the amazing stories of these terrors of the high seas asyou delve into the history of piracy.

Aires LibresOngoing • DowntownSainte-Catherine Street turns into a pedestrian zone for artistsand visitors between Berri and Papineau Streets.

Credit CardsIn order to report lost or stolen credit cards, these numbers willprovide assistance.

American Express 1-800-268-9805Mastercard 1-800-263-2263Visa 1-800-847-2911

Getting Around MontrealClean, safe and comfortable, the underground subway system,called the METRO, is an ideal and economical way to reachmany of Montreal’s attractions. You can also connect to the citybus system with the same fare. A 1-day pass costs $9 CAD anda 3-day pass costs $17 CAD.

Dress CodeThe dress code during the conference is casual.

Important NumbersInformation Desk 514-991-3851Delta Centre-Ville 514-879-1370Taxi Service 514-725-9885Tourism Montréal 514-873-2015Montréal Trudeau Airport 514-394-7377

ShoppingMontreal’s reputation as an international fashion and shoppingcentre is well established. With major department stores andcountless boutiques lining the city’s downtown streets and theunique underground city, Montreal offers creations by the best-known names on the local, national and international fashionscene. Find upscale boutiques, art galleries and jewellers a fewblocks north, near the Museum of Fine Arts on SherbrookeStreet.

A 29-kilometre network of underground passageways providesaccess to 1,700 boutiques, department stores, restaurants,movie houses, theatres and exhibition halls as well as 7 majorhotels, 1,615 housing units and thousands of offices.



About Montreal – ActivitiesYou will love Montréal in August. This European-style, multicultural city invites you to enjoy the warm balmy days of summer when Canadianslove to be outside enjoying parks and the lively cityscapes. We invite you to enjoy a contagious joie de vivre with a vibrant social life incafés and on outdoor terraces! The Hotel Delta Centre-Ville is located downtown, close to many attractions, restaurants and shopping.

About Montreal – Useful Information

Sunday, August 9, 2009

Welcome to Montréal

Opening Reception

18:30-20:00

Hotel Delta Centre-Ville, Foyer of Régence,777 University Street

(Included in full conference registration fee for delegatesand registered accompanying persons)

Dress Code: Casual

An exciting evening awaits as ISPP 2009 officially opens with akeynote address followed by a reception with light horsd’oeuvres and beverages. Come and network with yourcolleagues to set the tone for the meeting.

Wednesday, August 12, 2009

Conference Party at

Sucrerie de la Montagne, Rigaud, Québec

19:00 to 23:00

Buses leave at 17:30 from Hotel Delta Centre-ville– approx. 45 minute bus ride

Buses return around 22:00

(Reservation fee of $25 CDN applies.)

Dress Code: Comfortable wear!

REMEMBER TO BRING YOUR TICKET!

What’s a Sucrerie? It is a traditional Sugar Shack.

In Quebec, sugaring-off is a hearty, typically French Canadiancelebration. A traditional event that dates back to pioneerdays, it celebrates the end of a hard winter. This activity is heldin a sugar bush. The hosts have recreated the rural setting ofthe last century, with buildings made of authentic barn timbersand fieldstone ovens. This establishment was singled out bythe New-York Times Food Editors for its typical decor and thequality of its sugartime feast. Quebecois folklore at its best! AllQuebec food specialties are yours to eat as much as you like.A traditional bakery is in operation and a horse drawn calecheride is part of the fun. Musicians will set the mood.

The traditional Sugar Shack meal includes:

• Green Salad• Farm-Style Crusty Bread• Canadian Salted Back Bacon• Québécois Maple-Smoked Ham• Wood-Fired Baked Beans• Farm-Style Omelet• Traditional Meatball Stew• Country-Style Sausages• Old-style Mashed Potatoes• Meat Pie from Quebec's Beauce Region• Homemade Fruit Ketchup & Pickles• Dessert: Pancakes with Maple Syrup, Sugar pie, teaand coffee

Vegetarian options are available.



Social Program and Excursions



TOUR 1:

OLD MONTRÉAL WALK AND VISIT TOTHE MUSEUM OF ARCHEOLOGYDuration: 3 hours

Departing from Hotel Delta Centre-Ville, you will first walkthrough the historic quarter into the life of the first settlers ofMontréal. You will stroll through a maze of narrow cobblestonelanes and old buildings, providing a perfect opportunity todiscover the history and charm of Old Montréal. En route youwill see Place d'Armes, Jacques Cartier Square and MontréalCity Hall. One of the highlight of this tour is the museum ofArcheology, where you will discover the history of Montréal,starting with the multimedia show, and watch thousands ofyears of history spin by in 18 fascinating minutes. You will thenexplore the remains in the Museum at your own pace, through avibrant and colourful exhibition. You will walk back to the hotel.

TOUR 2:

CITY TOUR:MONTRÉAL C'EST MAGNIFIQUE!Duration: 3 hours

This exciting tour will introduce you to various parts of the citythat make Montréal what it is today. You will have the chance tosee how culturally diverse Montréal is, as well as visit landmarksand new developments that are so very important in makingMontréal such a unique and wonderful city.

See Montréal's elegant upper class communities, ouruniversities and part of our Underground City. Also, the PlateauMont-Royal, one of the top four "hippest" neighbourhoods inNorth America, known for its distinctive Montréal architecture,with its spiral staircase and finely wrought cornices. Along theway, the tour includes a stop at the summit of Mt. Royal Park fora panoramic view of the city and a guided visit of Notre-DameBasilica, an outstanding example of neo-gothic architecture.

TOUR 3:

BOTANICAL GARDEN and BIODÔMEDuration: 4 hours

Travel along the St. Lawrence River to the second largestBotanical Garden in the world displaying 20,000 species offlowers and plants in 30 outdoor gardens and 10 greenhouses.Of special interest: a remarkable collection of bonsai andorchids. You can choose to visit the Exhibition Greenhouses, theJapanese Pavilion, the Chinese Garden, the Arboretum or theInsectarium.

The next visit will bring you to the the Biodôme, a uniqueconcept of a "living" museum. Wander through four full scalenatural ecosystems of the Americas: the Tropical Forest, the St.Lawrence Marine, the Laurentian Forest and the Polar World,complete with landscapes, waterfalls, rivers, flora and birds,animals and fish.

Social Program and Excursions

Departure from hotel at 1:30 pm. Buses leave from St. Jacques Street.



SCIENTIFICPROGRAM



ALL ORAL PRESENTATIONS(INVITED SPEAKERS AND ABSTRACT PRESENTERS)

All sessions have been coded to facilitate indexing andreference to presentations in the book of abstracts. The codesconsist of two letters (representing the session type) followedby a number from 1 to 18 (2 Keynote, 9 Plenary and 18 OralCommunication sessions in total), followed by a numberdesignating the sequence of presentations.

LEGEND OF ORAL SESSIONSKeynote Speakers= KNPlenary Speakers = PLOC = Oral Communications

OC- 1. 1

Oral Communications Session First PresentationNumber in Session

LEGEND OF POSTER SESSIONS

P = Poster Session

Poster sessions are coded as follows:

P. 18

Poster Poster Number

Posters have been organized by Topic, followed by AlphabeticOrder.

How This Programme Is Organized

Invited Speakers and Oral AbstractPresenters who Uploaded TheirPresentation Pre-SymposiumEven if you have successfully uploaded your presentation, wewant to make sure that your presentation functions smoothlyonsite. Therefore, we have assigned a review time period foryou where you should present yourself at the technician’s tablein the room Le Royer. The technician will open yourpresentation for you to review the functionality of all slides.There won’t be time for you to make major changes. As back-up, we request that you bring your presentation on a USB keyor CD-rom.

Please proceed to the session room 15 minutes before the startof your session in case that the program needs to be adjusteddue to last-minute cancellations.

Invited Speakers and Oral AbstractPresenters who Did NOT Upload TheirPresentation Pre-ConferenceSpeakers MUST check in and submit their PowerPointpresentation at least three hours prior to their presentation, wehave assigned two computers for you where you could brieflyupload and preview your slides. Please see the registration stafffor instructions.

You will be required to provide your presentation on USB key orCD-Rom.

PLEASE NOTE THAT ALL PRESENTATIONS WILL BE DELETEDFROM THE SERVER AT THE END OF THE DAY.

INDIVIDUAL LAPTOP COMPUTERS ARE NOT PERMITTED FORPRESENTATIONS!

Poster PresentersPoster presenters should mount their posters in the designatedposter area (room Cartier) between 07:30-10:00 on Monday,August 10 and remove their posters no later than 18:00 onThursday, August 13. Conference staff is available to assist.Velcro will already be attached to your board when you arrive.

Any posters that are not taken down by 18:00 on Thursday willbe taken down by conference staff and stored at theregistration desk. Posters unclaimed by 18:00 on Friday, August14 will be discarded.

Instructions for Oral and Poster Presenters



Carl BauerProfessor of BiologyIndiana UniversityBloomington, IN, USA

J. Thomas BeattyProfessor, Microbiology & ImmunologyUniversity of British ColumbiaVancouver, BC, Canada

Brigitta BergmanProfessor, Department of BotanyStockholm UniversityStockholm, Sweden

Robert BlankenshipProfessor of Biology and ChemistryWashington UniversitySt. Louis, MO, USA

Fevzi DaldalProfessor of BiologyUniversity of PennsylvaniaPhiladelphia, PA, USA

Enrique FloresProfessor of ResearchInstituto de Bioquímica Vegetal y FotosíntesisCSIC-Universidad de SevillaSevilla, Spain

Karl ForchhammerProfessor and HeadDepartment of Microbiology / Organismic InteractionsUniversity TübingenTübingen, Germany

Susan GoldenProfessor Division of Biological SciencesDepartment of BiologyUniversity of California-San DiegoLa Jolla, CA, USA

Vladimir GorlenkoProfessor and Head of LaboratoryEcology and Geochemical Activity of MicroorganismsWinogradsky Institute of MicrobiologyRussian Academy of SciencesMoscow, Russia

Arthur GrossmanProfessor, Carnegie Institution of Washington and Departmentof Biological SciencesStanford UniversityStanford, CA, USA

Martin HagemannBioscience Institute, Plant Physiology DepartmentUniversität RostockRostock, Germany

Caroline HarwoodProfessor of MicrobiologyUniversity of WashingtonSeattle, WA, USA

Diana KirilovskyInstitut de Biologie et Technologies de SaclayCEA and CNRSGif sur Yvette, France

Bernd MasepohlChair, MicrobiologyRuhr-Universität BochumBochum, Germany

Armen Y. MulkidjanianUniversity of OsnabrueckOsnabrueck, Germany

Frederic PartenskyMarine Photosynthetic Prokaryotes TeamStation Biologique, CNRS and Université Paris06 (UMR 7144)Roscoff, France

Louis ShermanProfessor, Biological SciencesPurdue UniversityWest Lafayette, IN, USA

Kaarina SivonenProfessor, Applied Chemistry and MicrobiologyUniversity of HelsinkiHelsinki, Finland

Iwane SuzukiAssociate ProfessorUniversity of TsukubaTsukuba, Ibaraki, Japan

F. Robert TabitaProfessor; Ohio Eminent Scholar of MicrobiologyOhio State UniversityColumbus, OH, USA

Paula M. TamagniniDepartamento de Botânica, Fac. CiênciasIBMC – Instituto de Biologia Molecular e CelularUniversidade do PortoPorto, Portugal

Anthony E. WalsbyEmeritus Professor of MicrobiologySchool of Biological SciencesUniversity of BristolBristol, United Kingdom

Peter C. WolkProfessorMichigan State UniversityE. Lansing, MI, USA

Invited Faculty

Notes



Sunday, August 9, 2009

17:30–18:30 OPENING KEYNOTE RÉGENCE AB

WELCOME TO ISPP 2009 AND INTRODUCTION OF KEYNOTE SPEAKER.Patrick Hallenbeck, Symposium Chair and Université de Montréal, Montréal, QC.

WELCOME FROM THE CHAIR OF THE INTERNATIONAL SCIENTIFIC COMMITTEE.Jörg Overmann, Planegg-Martinsried, Germany.

KN-1.1 A SCIENTIST OF THE FLOATING WORLD.Anthony E. Walsby, University of Bristol, United Kingdom.

Biography

I trained as a botanist at the University of Birmingham and moved to Westfield College London for research with G.E. Fogg on cyanobacteria, which at that time masqueraded as blue-green algae.

My first paper, with Peter Fay in 1966, described the serendipitous isolation of heterocysts. Bill Stewart saw theopportunity to test N2-fixation by isolated heterocysts; my contribution was to measure 15N labelling by massspectrometry. This technique led to my analysing the gas in gas vesicles of cyanobacteria and a life-time of researchon these fascinating structures. In the early 70s I spent two years on sabbatical leave in Berkeley, where I met andmarried Fausta Segrè. Returning to the UK, I spent another seven years with Fogg in marine science at theUniversity College of North Wales and then, despite never having worked on a real plant, I moved to the Chair ofBotany at the University of Bristol.

Throughout my career chasing gas vesicles I sought collaborations with experts in other fields: on crystallographywith Allen Blaurock in London; microscopy with Daniel Branton at Berkeley; protein sequencing with John Walker atCambridge; and mathematical physics with John Simpson at Menai Bridge. My machines for measuring gas vesiclessuggest inspiration by Heath Robinson (or Rube Goldberg). The results were the mechanics of gas vesicles,measurements of cell turgor pressure and estimates of gas diffusion rates into heterocysts. At Bristol I enjoyed along collaboration with Paul Hayes and some excellent students. We took the physiology and molecular biology ofcyanobacteria from the laboratory to analyse populations in lakes, seas and salterns. We had many nicecollaborations with local ecologists, including those with Ferdinand Schanz on Lake Zürich, Lucas Stal on the BalticSea, and Colin Reynolds on the English Lakes. Sometimes I returned with unexpected trophies: Dactylococcopsisfrom Solar Lake, the square archeon Haloquadratum from the Sinai, and hundreds of Planktothrix strains from LakeZürich.

18:30–20:00 WELCOME RECEPTION FOYER – RÉGENCE

See page 7 for details.





Notes



Monday, August 10, 2009

08:30–10:00 PLENARY 1: PHYLOGENY, TAXONOMY AND DIVERSITY RÉGENCE AB

Co-Chairs: Johannes Imhoff, Kiel, Germany and Min Chen, Sydney, Australia

08:30 PL-1.1 EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHIC BACTERIA DEDUCTED FROM WHOLE GENOMECOMPARISONS.Robert Blankenship, Washington University, St. Louis, MO, USA.

09:00 PL-1.2 ORIGIN AND EVOLUTION OF PHOTOSYNTHETIC SYSTEMS.Armen Y. Mulkidjanian, University of Osnabrueck, Osnabrueck, Germany.

09:30 PL-1.3 ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA: DIVERSITY, NEW TAXONS.Vladimir Gorlenko, Russian Academy of Sciences, Moscow, Russia.

10:00–10:30 HEALTH BREAK FOYER

10:30–12:30 ORAL COMMUNICATIONS 1: PHYLOGENY TAXONOMY AND DIVERSITY RÉGENCE C

Chair: Robert Blankenship, St. Louis, MO, USA

OC-1.1 SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETIC MARKERS FOR PURPLE SULFUR BACTERIA.Marcus Tank, Vera Thiel, Johannes F. Imhoff.IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University), Kiel, Germany.

OC-1.2 BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTING GENES IN MARINE PHYTOPLANKTON.Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2.1School of Ocean and Earth Sciences, National Oceanography Centre, Southampton, United Kingdom; 2School ofBiological Sciences, University of Sydney, Sydney, New South Wales, Australia.

OC-1.3 PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THE CYANOBACTERIAL GENERA ANABAENOPSIS ANDCYANOSPIRA.Stefano Ventura, Cristina Mascalchi, Claudio Sili.Institute of Ecosystem Study, National Research Council, Sesto Fiorentino, Italy.

OC-1.4 CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATS OF A TROPICAL LAGOON, TIKEHAU ATOLL,TUAMOTU ARCHIPELAGO.Katarzyna A. Palinska1, Raeid M. M. Abed2†, Katja Wendt1, Maria Łotocka3, Loic Charpy4 & Stejpko Golubic5.1Institute of Chemistry and Biology of the Marine Environment, Geomicrobiology Dep. and CvO University ofOldenburg, Oldenburg, Germany; 2College of Science-Biology Department, Sultan Qaboos University, Al-Khod,Muscat, Sultanate of Oman; 3Institute of Oceanology, Polish Academy of Sciences, Powsta�ców Warszawy, Sopot,Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167 (CYROCO), Traverse de la Batterie des Lions, Marseille,France; 5Biological Science Center, Boston University, Boston, MA, USA.

OC-1.5 METAGENOMIC AND PHYLOGENETIC ANALYSES OF CYANOBACTERIAL MATS IN EXTREME HIGH ARCTICENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICAL FUNCTION.Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3, Warwick F. Vincent1.1Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté de Médecine, Université Laval; 3Québec-Océan,Dép. de Biologie, and Institut de biologie intégrative et des systèmes (IBIS); Université Laval; Québec, QC, Canada.

OC-1.6 GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENON SPP. AND ANABAENA SPP. IN NORTHEASTGERMAN LAKES.Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1.1Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes,Neuglobsow; 2Federal Environmental Agency, Berlin; 3Brandenburg Technical University, Department of FreshwaterConservation, Bad Saarow; Germany.




10:30–12:30 ORAL COMMUNICATIONS 2: ECOLOGY I RÉGENCE AB

Chair: Mary Allen, Wellesley, MA, USA

OC-2.1 MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS IN PHOTOTROPHIC CONSORTIA.Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller.Section Microbiology, Department Biology I, University of Munich, Planegg-Martinsried, Germany.

OC-2.2 LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBIC ANOXYGENIC PHOTOTROPHS IN A SEMIARIDREALM.J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov.Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.

OC-2.3 ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MAT- NITROGEN FIXATION.Ina Severin, Lucas J. Stal.Department of Marine Microbiology, NIOO-Center for Marine and Estuarine Ecology, Yerseke, The Netherlands.

OC-2.4 BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCED PHOTOSYNTHETIC MICROBES.Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer.SRI International, Menlo Park, CA, USA.

OC-2.5 DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACE WATERS OF MARINE AND FRESHWATERENVIRONMENTS WITH AN EMPHASIS ON CYANOBACTERIA.Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, Torsten Staller2, Rüdiger Schulz2, Jens Appel1.1School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Botanisches Institut, Universität Kiel, Kiel,Germany.

OC-2.6 HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRING MICROBIAL MATS DOMINATED BY THEFILAMENTOUS ANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUS AGGREGANS.H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1.1Department of Biology, Tokyo Metropolitan University, Tokyo; 2Institute for Biological Resources and Functions,National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki; Japan.

12:30–14:00 LUNCH RÉGENCE C AND FOYER




14:00–15:00 ORAL COMMUNICATIONS 3: ECOLOGY II RÉGENCE AB

Chair: Vladimir Gorlenko, Moscow, Russia

OC-3.1 CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROM THE HALOCLINE OF THE SALINEMEROMICTIC LAKE, LAKE SUIGETSU, JAPAN.Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya.Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan.

OC-3.2 THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIA PROCHLOROCOCCUS.Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA.

OC-3.3 THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTAL CYANOBACTERIA COMMUNITY AND ITSCONTRIBUTION TO TOXICITY IN A TROPICAL BRAZILIAN RESERVOIR.Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos.Department of Botany, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), BeloHorizonte, MG, Brazil.

14:00–15:00 ORAL COMMUNICATIONS 4: PHYSIOLOGY I RÉGENCE C

Chair: David Adams, Leeds, United Kingdom

OC-4.1 ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUM TEPIDUM: GENES, METABOLITES AND LINKAGESTO LIGHT HARVESTING.Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan, Rachael Morgan-Kiss.College of Marine and Earth Studies and DBI, Newark, DE, USA.

OC-4.2 CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS.Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E. Blankenship1.1Departments of Biology and Chemistry; 2Department of Energy, Environment and Chemical Engineering;Washington University, St. Louis, MO, USA.

OC-4.3 STRUCTURAL AND FUNCTIONAL STUDIES OF A PHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGHCOMPARATIVE METATRANSCRIPTOME ANALYSIS.Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt, Lynn Tomsho, Stephan C. Schuster,David M. Ward, Donald A. Bryant.The Pennsylvania State University, University Park, PA, USA.

15:00–17:00 POSTER SESSION CARTIER AB

17:00–18:00 PLENARY 2: ECOLOGY RÉGENCE AB

Co-Chairs: Jörg Overmann, Planegg-Martinsried, Germany and Vladimir Yurkov, Winnipeg, MB, Canada

17:00 PL-2.1 MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMIC AND PROTEOMIC ANALYSESBrigitta Bergman, Department of Botany, Stockholm University, Stockholm, Sweden.

17:30 PL-2.2 PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATION AND POTENTIAL INTERACTIONSArthur Grossman, Department of Plant Biology, The Carnegie Institution, Stanford, CA, USA.



Notes



Tuesday, August 11, 2009

08:30–10:00 PLENARY 3: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES RÉGENCE AB

Chairs: Jack Meeks, Davis, CA, USA and James Golden, La Jolla, CA, USA

08:30 PL-3.1 MULTICELLULARITY IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA.Enrique Flores, Professor of Research, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla,Sevilla, Spain.

09:00 PL-3.2 EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY GLYCOLATE METABOLISM IN CYANOBACTERIA.Martin Hagemann, Plant Physiology, University Rostock, Inst. Biosciences, Rostock, Germany.

09:30 PL-3.3 THE NETWORK OF INTERACTIONS OF PII SIGNALING IN CYANOBACTERIA.Karl Forchhammer, Department of Microbiology, University Tübingen, Tübingen, Germany.


10:30–12:30 ORAL COMMUNICATIONS 5: PHYSIOLOGY II RÉGENCE AB

Chairs: David Knaff, Lubbock, TX, USA and Ann Magnuson, Uppsala, Sweden

OC-5.1 HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOC PUNCTIFORME DESTINED TO BECOMEHORMOGONIA.H. Christman, E.L. Campbell, J.C. Meeks.Section of Microbiology, University of California, Davis, CA, USA.

OC-5.2 PLANT CELL WALL EPITOPES ARE EXPRESSED BY CYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS.Owen Jackson, J. Paul Knox, David G. Adams.University of Leeds, West Yorkshire, United Kingdom.

OC-5.3 REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE IN HETEROCYST-FORMING CYANOBACTERIA.Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, Enrique Flores2, David G. Adams3.1School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; 2Institutode Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty of Biological Sciences,Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.

OC-5.4 SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS IN ANABAENA SP. STRAIN PCC 7120.Jinjie Liu, C. Peter Wolk.MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA.

OC-5.5 THE INVOLVEMENT OF TWO SIGMA FACTORS IN CYANOBACTERIAL AKINETE FORMATION.Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers.Department of Biology, California State University, Northridge, CA, USA.

OC-5.6 SEQUENCES REGULATING NITROGENASE GENE EXPRESSION IN THE CYANOBACTERIUM ANABAENAVARIABILIS.Teresa Thiel, Justin L. Ungerer.Department of Biology, University of Missouri St. Louis, St. Louis, MO, USA.




10:30–12:30 ORAL COMMUNICATIONS 6: BIOENERGETICS I RÉGENCE C

Chairs: Michael Summers, Northridge, CA, USA

OC-6.1 THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES AT THE MONOMER-MONOMER INTERFACEOF PHOTOSYSTEM II IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley.Department of Biochemistry, University of Otago, Dunedin, New Zealand.

OC-6.2 PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO IN SYNECHOCYSTIS.Danny Yao, Dan Brune, Wim Vermaas.School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA.

OC-6.3 IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FOR THE OXYGEN DEPENDANCE OF THEPHOTOSYNTHETIC ACTIVITY OF ROSEOBACTER DENITRIFICANS.Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and Andreas Labahn1.1Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany; 2Department of Cell Biology, HarvardMedical School, Boston MA, USA.

OC-6.4 VARIABLE FLUORESCENCE IN HELIOBACTERIUM MODESTICALDUM CELLS: OBSERVATION ANDEXPLANATION.Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, Stefano Santabarbara1,2, Robert Blankenship3.1Arizona State University, Dept. of Chemistry and Biochemistry, Tempe, AZ, USA; 2Institut de BiologiePhysico-Chimique, Paris, France; 3Washington University, Departments of Biology and Chemistry, St Louis, MO, USA.

OC-6.5 NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM II PROTECTS THE CYANOBACTERIUMMICROCOLEUS SP. AGAINST EXCESS LIGHT STRESS.Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2.Departments of 1Biological Chemistry, 2Plant and Environmental Sciences and 3The Interuniversity Marine Institute,Eilat, The Hebrew University of Jerusalem, Israel.

OC-6.6 THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THE CYANOBACTERIUM SYNECHOCYSTISSP. PCC 6803.Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-Mari Aro.Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland.

12:30–14:00 LUNCH RÉGENCE C AND TOUR DE VILLEPlease check for a ticket inside your badge holder.

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14:00–15:00 ORAL COMMUNICATIONS 7: PHYSIOLOGY III RÉGENCE AB

Chairs: Enrique Flores, Sevilla, Spain

OC-7.1 PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA (PRRA), TRANSCRIPTIONAL REGULATORS OFTHE CBB OPERONS (CO2 FIXATION) IN RHODOBACTER SPHAEROIDES.Andrew W. Dangel, F. Robert Tabita .

Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University,Columbus, OH, USA.

OC-7.2 PpsR AS A NEW TYPE OF HEMIN SENSOR.Liang Yin, Vladimira Dragnea, Carl Bauer.Indiana University, Bloomington, IN, USA.

OC-7.3 ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN: AN EXPERIMENTAL SCENARIO FOR ASTRICT ANAEROBIC PHOTOTROPH.Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg, Françoise Reiss-Husson, Chantal Astier,Soufian Ouchane.CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; Université Paris-Sud, Orsay; Université Pierreet Marie Curie, Paris; France.

14:00–15:00 ORAL COMMUNICATIONS 8: BIOENERGETICS II, PROTEINS, GENOMICS II RÉGENCE C

Chair: Susan Golden, La Jolla, CA, USA and Willem Vermaas, Tempe, AZ, USA

OC-8.1 CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BY FERREDOXIN:THIOREDOXIN REDUCTASE,FERREDOXIN AND THIOREDOXIN M.David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, Masakazu Hirasawa, Marcellus Ubbink.Texas Tech University, Lubbock, TX, USA.

OC-8.2 CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA.Shinichi Takaichi1, Mari Mochimaru2.1Department of Biology, Nippon Medical School, Kawasaki; 2Department of Natural Sciences, Komazawa University,Setagaya, Tokyo; Japan.

OC-8.3 EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROM PURPLE BACTERIA AND CYANOBACTERIA.Gerhard Sandmann, Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Frankfurt, Germany.


16:30–17:30 PLENARY 4: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES RÉGENCE AB

Chair: Fevzi Daldal, Philadelphia, PA, USA

16:30 PL-4.1 A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME.Susan Golden, Department of Biology, University of California-San Diego, La Jolla, CA, USA.

17:00 PL-4.2 EFFECTS OF INSERTION SEQUENCES ON THE GENOME OF ANABAENA SP. STRAIN PCC 7120.Peter C. Wolk, Michigan State University, E. Lansing, MI, USA.



Notes




08:30–10:00 PLENARY 5: BIOENERGETICS, PROTEINS AND GENOMICS RÉGENCE AB

Chairs: F. Robert Tabita, Columbus, OH, USA

10:00 PL-5.1 CYTOCHROMES STRUCTURE FUNCTION AND BIOGENESIS.Fevzi Daldal, University of Pennsylvania, Philadelphia, PA, USA.

10:30 PL-5.2 BETTER LIVING THROUGH CYANOTHECE-UNICELLULAR DIAZOTROPHIC CYANOBACTERIA WITH HIGHLYVERSATILE METABOLIC SYSTEMS.Louis Sherman, Professor, Biological Sciences, Purdue University, West Lafayette, IN, USA.

11:00 PL-5.3 PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENT EVOLUTION SCHEMES FROM A COMMONANCESTRAL GENOME.Frederic Partensky, Marine Photosynthetic Prokaryotes Team, Station Biologique, CNRS and Université Paris,Roscoff, France.


10:30–12:30 ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES RÉGENCE AB

Chairs: Bernd Masepohl, Bochum, Germany

OC-9.1 THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM – A STRUCTURAL MODEL BASED ONSOLID-STATE NMR DATA COMBINED WITH MOLECULAR SIMULATION STUDIES OF CD AND ABSORPTIONSPECTRA.Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1, Peter Højrup2, Anders Giessing2, JuhaLinnanto3, Niels-Ulrik Frigaard4, Mette Miller2 and Niels Chr. Nielsen1.1Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department ofChemistry, University of Aarhus, Århus, Denmark; 2Department of Biochemistry and Molecular Biology, University ofSouthern Denmark, Odense, Denmark; 3Department of Chemistry, University of Jyväskylä, Finland; 4Department ofBiology, University of Copenhagen, Copenhagen, Denmark.

OC-9.2 THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLS IN CHLOROSOMES OFCHLOROBACULUM TEPIDUM.Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, Michael Reus, Aline Gomez Gomez Maqueo Chew,Alfred R. Holzwarth, Egbert J. Boekema, Huub J. M. de Groot.Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.

OC-9.3 MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANES IN CYANOBACTERIA.Sawsan Hamad, Wim Vermaas.School of Life Sciences and the Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA.

OC-9.4 PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OF FUNCTION IN THE CARBOXYSOME.Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C. Bagby3, Sabine Heinhorst4, Fei Cai4, GordonCannon4, Sallie W. Chisholm3.1US Department of Energy, Joint Genome Institute, Walnut Creek, CA; 2Department of Plant and Microbial Biology,University of California, Berkeley, CA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge,MA; 4Department of Biochemistry, University of Southern Mississippi, Hattiesburg, MS; USA.




10:30–12:30 ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES RÉGENCE AB

Chairs: Bernd Masepohl, Bochum, Germany

OC-9.5 PROTEOMIC ANALYSIS OF THE DEVELOPING INTRACYTOPLASMIC MEMBRANE DURING CHROMATICADAPTATION IN RHODOBACTER SPHAEROIDES.Kamil Woronowicz, Robert A. Niederman.Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA.

OC-9.6 BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODING THE MEMBRANE-ASSOCIATEDTHIOREDOXIN PROTEIN PLAYS AN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OF PS I.Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1.1Department of Biochemistry and Molecular Biology; 2Department of Chemistry; The Pennsylvania State University,University Park, PA, USA.

10:30–12:30 ORAL COMMUNICATIONS 10: PHYSIOLOGY IV RÉGENCE C

Chair: J. Thomas Beatty, Vancouver, BC, Canada

OC-10.1 A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THE APPARENT PHOTOSYNTHETIC AFFINITY FOR CO2

BY MODULATING LOW-CO2-INDUCED GENE EXPRESSION.Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2, Yukako Hihara2, Ariel Gaathon3, MarioLebendiker4, Aaron Kaplan1.1Dept. of Plant and Environmental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; 2 Dept. ofBiochemistry and Molecular Biology, Saitama University, Saitama, Japan; 3Bletterman Laboratory, InterdepartmentalEquipment Unit, Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre,Hebrew University of Jerusalem, Jerusalem, Israel.

OC-10.2 THE EFFECTS OF TEMPERATURE AND OXYGEN ON NITROGENASE ACTIVITY IN THE THERMOPHILICCYANOBACTERIUM FISCHERELLA SP.Lucas J. Stal, Department of Marine Microbiology, Netherlands Institute of Ecology, Yerseke, The Netherlands.

OC-10.3 PHOSPHATE SCAVENGING IN AN UNPREDICTABLE ENVIRONMENT, HOW DO CYANOBACTERIA MEET THECHALLENGE?Frances D. Pitt, David J. Scanlan.Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom.

OC-10.4 REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY IN CYANOBACTERIA. IDENTIFICATION OF THEREGIONS INVOLVED IN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS.Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J. Florencio.Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla-CSIC, Sevilla, Spain.

OC-10.5 BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS.Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A. Bryant1.1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2

Department of Biological Sciences, University of New Orleans, New Orleans, LA; USA.

OC-10.6 ACCUMULATION OF TREHALOSE IN RESPONSE TO DESICCATION AND CONTROL OF TREHALASE IN THETERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE.Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima.Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan.

FREE AFTERNOON AND EXCURSIONS

17:30 ISPP 2009 PARTY (SEE PAGE 7)



Thursday, August 13, 2009

08:30–10:00 PLENARY 6: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION RÉGENCE AB

Chairs: Donald A. Bryant, University Park, PA, USA and Kaori Inoue-Sakamoto, Ishikawa, Japan

08:30 PL-6.1 OXYGEN AND LIGHT CONTROL OF TETRAPYRROLE BIOSYNTHESIS IN RHODOBACTER SPECIES.Carl Bauer, Indiana University, Bloomington, IN, USA.

09:00 PL-6.2 A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHT INTENSITY SENSOR IN CYANOBACTERIAPHOTOPROTECTION.Diana Kirilovsky, Institut de Biologie et Technologies de Saclay, CEA and CNRS, Gif sur Yvette, France.

09:30 PL-6.3 NITROGEN AND MOLYBDENUM CONTROL OF NITROGEN FIXATION.Bernd Masepohl, Chair, Microbiology, Ruhr-Universität Bochum, Bochum, Germany.


10:30–12:30 ORAL COMMUNICATIONS 11: RÉGENCE ABENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I

Chairs: Iwane Suzuki, Tsukuba, Ibaraki, Japan

OC-11.1 FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAIN IN RHODOSPIRILLUM CENTENUM PPR.J.A. Kyndt, T.E. Meyer, M.A. Cusanovich.Biochemistry Dept., University of Arizona, Tucson, AZ, USA.

OC-11.2 SHORT-TERM LIGHT ADAPTATION STRATEGIES OF PHYCOBILISOME-CONTAINING PHOTOSYNTHETICS,CYANOBACTERIA AND RED ALGAE.Igor Stadnichuk, PA.N.Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.

OC-11.3 REGULATORY RNAS IN CYANOBACTERIA AND BEYOND.W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1, J. Vogel2, A. Wilde3.1Freiburg Initiative in Systems Biology and Faculty of Biology, University Freiburg; 2Max-Planck-Institute for InfectionBiology Berlin; 3Justus-Liebig University Giessen, Institute of Microbiology and Molecular Biology; Germany.

OC-11.4 FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUM PROCHLOROCOCCUS:WHAT CAN WE LEARN?C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, Joerg Vogel3, Wolfgang Hess1.1University of Freiburg, Freiburg, Germany; 2University of Algarve, Faro, Portugal; 3Max Planck Institute for InfectionBiology, Berlin, Germany.

OC-11.5 REGULATION BETWEEN PHOTOAUTOTROPHIC AND PHOTOMIXOTROPHIC GROWTH AND ITSDEPENDENCE ON THE CO2 LEVEL IN SYNECHOCYSTIS PCC 6803.Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy Lieman-Hurwitz1, Aaron Kaplan1.1Plant and Environmental Sciences, The Hebrew University of Jerusalem; Israel; 2Department of Biochemistry andMolecular Biology, Saitama University, Japan.

OC-11.6 CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY IN OSCILLATORIA.Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson, Dave Adams.University of Leeds, Leeds, West Yorkshire, United Kingdom.




10:30–12:30 ORAL COMMUNICATIONS 12: RÉGENCE CENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I

Chairs: Karl Forchhammer, Tübingen, Germany and Soufian Ouchane, Gif-sur-Yvette, France

OC-12.1 GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGEN CONDITIONS AND THE ROLE OF THE HISTIDINEKINASE, HIK31, IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.Tina C. Summerfield1, Louis A. Sherman2.1Botany Department, University of Otago, PO Box 56, Dunedin, New Zealand; 2Purdue University, Department ofBiological Sciences, West Lafayette, IN, USA.

OC-12.2 INCREASING HYDROGEN PRODUCTION BY PERTURBING FERMENTATIVE METABOLISM IN THE MARINE,UNICELLULAR CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002.Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G. Charles Dismukes.Princeton University, Princeton, NJ, USA.

OC-12.3 PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATION TRANSFER TO PHOTOSYSTEM I IN THEFILAMENTOUS CYANOBACTERIUM NOSTOC PUNCTIFORME.Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring, Ann Magnuson.Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden.

OC-12.4 GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC 7942.Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, Yasunori Nakamura.Akita Prefectural University, Akita, Japan.

OC-12.5 IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTIS AERUGINOSA UNDER IRON LIMITATION.Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1.1School of Biotechnology and Biomolecular Sciences; 2School of Civil and Environmental Engineering; University ofNew South Wales, Sydney, Australia.

OC-12.6 THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITE WITH ECOPHYSIOLOGICAL ROLES.S.B. Watson1, F. Juttner2.1Environment Canada, Canada Centre for Inland Waters, Burlington, ON, Canada; 2Limnological Station,University of Zurich, Switzerland.

12:30–14:00 LUNCH RÉGENCE C AND TOUR DE VILLEPlease check for a ticket inside your badge holder.

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14:00–15:00 ORAL COMMUNICATIONS 13: PHYSIOLOGY VI RÉGENCE C

Chair: Peter Lindblad, Uppsala, Sweden

OC-13.1 COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND PURPLE SULFUR BACTERIA.N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3.1Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Plant Biology Department, Universityof Geneva, Geneva, Switzerland; 3Department of Biochemistry and Molecular Biology, Pennsylvania State University,State College, Pennsylvania, USA.

OC-13.2 THE GENOME OF THE TOXIC BLOOM-FORMING CYANOBACTERIUM ANABAENA SP. 90.David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2, Kaarina Sivonen1.1Department of Microbiology and Applied Chemistry, University of Helsinki, FIN-00014, Helsinki, Finland;2Beijing Institute of Genomics of the Chinese Academy of Sciences, Beijing Genomics Institute, Beijing, China.

OC-13.3 CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPMENT AND BEHAVIOR OFHORMOGONIA OF NOSTOC PUNCTIFORME.Jack Meeks, Elsie Campbell, Rui Chen.Department of Microbiology, University of California, Davis, CA, USA.

14:00–15:00 ORAL COMMUNICATIONS 14: BIOENERGETICS IV RÉGENCE AB

Chair: Peter C. Wolk, East Lansing, MI, USA and Teresa Thiel, St. Louis, MO, USA

OC-14.1 COMPARISON OF NONCODING FEATURES OF CYANOBACTERIAL GENOMES.Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, José Costa5.1Virginia Commonwealth University, VA, USA; 2University of California at Davis, CA, USA; 3University of Pennsylvania,PA, USA; 4Uppsala University, Uppsala, Sweden; 5University of Porto, Porto, Portugal.

OC-14.2 HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) AS ELECTRON DONOR? SEARCHING FOR THEANSWER IN THE MEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM 263.Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, Ulrich Fischer1.1Zentrum für Umweltforschung und nachhaltige Technologien (UFT) and Fachbereich Biologie/Chemie, AbteilungMarine Mikrobiologie, Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie; 3FachbereichBiologie/Chemie, Laboratorium für Allgemeine Mikrobiologie, Universität Bremen; Bremen, Germany.

OC-14.3 PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS: INSIGHTS INTO THE SULFUR METABOLISM OFA PHOTOTROPHIC GREEN BACTERIUM.Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, Anders Mellerup2, Kirsten S. Habicht3, Raymond P. Cox1, JensS. Andersen1 & Niels-Ulrik Frigaard2.1Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK5230 Odense;2Department of Biology, University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center for Earth Evolution andInstitute of Biology, University of Southern Denmark, Odense; Denmark.




15:00–16:00 PLENARY 7: ENVIRONMENT AND BIOENERGETICS RÉGENCE AB

Chairs: Davide Zannoni, Bologna, Italy and Igor Stadnichuk, Moscow, Russia

15:00 PL-7.1 HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVES THE ENVIRONMENTAL STIMULI?Iwane Suzuki, University of Tsukuba, Tsukuba, Ibaraki, Japan.

15:30 PL-7.2 GENOMICS AND METAGENOMICS APPROACHES TO THE EVOLUTION AND REGULATION OF GENETRANSFER AGENTS (GTAS).J. Thomas Beatty, University of British Columbia, Vancouver, BC, Canada.


18:00 POSTER REMOVAL CARTIER AB

Friday, August 14, 2009

08:30–10:00 PLENARY 8: BIOREMEDIATION, SECONDARY METABOLITES, RÉGENCE ABAND APPLIED ASPECTS (BIOFUELS AND CLIMATE CHANGE)

Chairs: Jens Appel, Tempe, AZ, USA and Anatoly Tsygankov, Pushchino, Russia

08:30 PL-8.1 PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGEN PRODUCTION.Caroline Harwood, University of Washington, Seattle, WA, USA.

09:00 PL-8.2 THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICAL APPLICATIONS.Paula M. Tamagnini, Departamento de Botânica, Fac. Ciências , IBMC – Instituto de Biologia Molecular e Celular,Universidade do Porto, Porto, Portugal.

09:30 PL-8.3 CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES, ACTIVITIES AND BIOSYNTHESIS.Kaarina Sivonen, University of Helsinki, Helsinki, Finland.


10:30–12:30 ORAL COMMUNICATIONS 15: APPLIED ASPECTS I RÉGENCE AB

Chairs: Caroline Harwood, Seattle, WA, USA and Hidehiro Sakurai, Hiratsuka, Japan

OC-15.1 A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALE PHOTOBIOLOGICAL HYDROGENPRODUCTION UTILIZING CYANOBACTERIA.Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue.Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka, Kanagawa, Japan.

OC-15.2 SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTION AMONG DIVERSE HETEROCYSTOUSCYANOBACTERIA.Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples, Polly Berseth, Tommy Sessions.Savannah River National Laboratory, Aiken, SC, USA.

OC-15.3 THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLE BACTERIA WITH DARK FERMENTATIVEHYDROGEN PRODUCTION AND HYDROGEN ELECTRODE.A. Tsygankov, Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino,Moscow Region, Russia.

OC-15.4 HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUM RUBRUM UNDER RESPIRATORY DARKCONDITIONS.Lisa Zeiger, Christiane Rudolf, Hartmut Grammel.Max-Planck-Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

OC-15.5 DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA IN MISSISQUOI BAY, QUEBEC, USING Q-PCR.Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, Frances Pick3, David Bird4, Charles W. Greer1.1National Research Council, Biotechnology Research Institute, QC; 2Environmental Health Center, Health Canada,ON; 3Biology Department, University of Ottawa, ON; 4Biological Sciences, University of Quebec at Montreal, QC,Canada.

OC-15.6 EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENES AND THE GENETIC POPULATIONSTRUCTURE OF TOXIC CYANOBACTERIA.Rainer Kurmayer, Guntram Christiansen.Austrian Academy of Sciences, Institute for Limnology, Mondsee, Austria.




10:30–12:30 ORAL COMMUNICATIONS 16: PHYSIOLOGY VII RÉGENCE C

Chairs: Louis Sherman, West Lafayette, IN, USA and Kaarina Sivonen, Helsinki, Finland

OC-16.1 THE RESPIRATORY TERMINAL OXIDASES OF THE CYANOBACTERIUM SYNECHOCOCCUS SP. STRAINPCC7942.Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder, Dominik Aschenbrenner.Institute of Physical Chemistry, Vienna, Austria.

OC-16.2 PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA: STRUCTURE AND FUNCTION OF ENZYMESINVOLVED IN POST-TRANSLATIONAL MODIFICATION.Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1, Gaozhong Shen2, Donald A. Bryant2, Wendy M.Schluchter1.1Department of Biological Sciences, University of New Orleans, New Orleans, LA; 2Department of Biochemistry andMolecular Biology, The Pennsylvania State University, University Park, PA; USA.

OC-16.3 SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVEL CYANOBACTERIOCHROME-TYPEPHOTORECEPTOR AnPixJ.Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose, Tomoo Shiba, Shigeru Itoh, Genji Kurisu,Masahiko Ikeuchi.University of Tokyo, Meguro, Tokyo, Japan.

OC-16.4 CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OF SYNECHOCOCCUS ELONGATUS REVEALSA NEW ANTIDOTE MOLECULE.Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz.Bar-Ilan University, Ramat-Gan, Israel.

OC-16.5 HOW CYANOBACTERIA BORE (AND WHY LATERAL HETEROCYSTS EXIST).F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao.School of Life Sciences, Arizona State University, Tempe, AZ, USA.

OC-16.6 FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEEN IRON HOMEOSTASIS AND OXIDATIVESTRESS.Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2, Nir Keren1.1The Alexander Silberman Institute of Life Sciences, Department of Plant and Environmental Sciences, HebrewUniversity, Givat Ram, Jerusalem, Israel; 2Department of Biological Sciences Purdue University, West Lafayette, IN,USA; 3Department of Botany, University of Otago, Dunedin, New Zealand.

12:30–14:00 LUNCH RÉGENCE C AND FOYER




14:00–15:00 ORAL COMMUNICATIONS 17: APPLIED ASPECTS II RÉGENCE C

Chair: Paula Tamagnini, Porto, Portugal

OC-17.1 RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS, HARBORING THE CRTIREPORTER GENE TO MONITOR ENVIRONMENTAL TOXIC METALS.Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid.Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.

OC-17.2 THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTER CAPSULATUS CELLS VIA ACETATE PERMEASE.Roberto Borghese, Davide Zannoni.Department of Biology, University of Bologna, Bologna, Italy.

OC-17.3 CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CR-PLATING INDUSTRY WITHEXOPOLYSACCHARIDE-PRODUCING CYANOBACTERIA.Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis.Deparment of Agricultural Biotechnology, University of Florence, Firenze, Italy.

14:00–15:00 ORAL COMMUNICATIONS 18: BIOENERGETICS V AND APPLIED ASPECTS III RÉGENCE AB

Chair: Peter Lindblad, Uppsala, Sweden

OC-18.1 TRANSCRIPTIONAL REGULATION AND MATURATION OF CYANOBACTERIAL HYDROGENASES.Peter Lindblad, Department of Photochemistry & Molecular Science, Uppsala University, Uppsala, Sweden.

OC-18.2 PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803.Carrie Eckert, Jianping Yu, and Pin-Ching Maness.National Renewable Energy Laboratory, Golden, CO, USA.

OC-18.3 ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATION OF THE BIDIRECTIONAL [NIFE]-HYDROGENASE FROM SYNECHOCYSTIS SP. PCC 6803.Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2, Rüdiger Schulz3 and Frauke Germer3.1School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Max-Volmer-Laboratorium, TechnischeUniversität, Berlin, Germany; 3Botanisches Institut, Universität Kiel, Kiel, Germany.

15:00–16:00 CLOSING KEYNOTE RÉGENCE AB

Chair: Patrick Hallenbeck, Montréal, QC, Canada

KN-2.1 INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN, AND SULFUR METABOLISM: THE CENTRALROLE OF THE CALVIN-BENSON-BASSHAM CYCLE.F. Robert Tabita, Ohio State University, Columbus, OH, USA.





Notes

KN-1.1

A SCIENTIST OF THE FLOATING WORLD.

Anthony E. Walsby, School of Biological Sciences, University of Bristol,United Kingdom.

My invitation to give the Keynote Address proposed a lecture withreflections and a history of research on phototrophs. Beforeaccepting, I ought to have reflected on Kazuo Ishiguro’s novel AnArtist of the Floating World, in which Mr Ono, an aging painter, looksback on his life noticing how his reputation has faltered and attitudestowards him and his work have changed.

The Floating World in which I wandered for over forty years is that ofplanktonic prokaryotes that derive buoyancy from gas vesicles. I waslaunched into this by the discovery of Bowen & Jensen (1965) that gasvacuoles of cyanobacteria were made up of cylindrical gas vesicles,packed like the cells of a honeycomb. Their report ended a hiatus ofover twenty years since the scholarly review on gas vacuoles by G.E.Fogg, then a 21 year-old graduate student of F. E. Fritsch. HansKlebahn had been the dominant figure but his fêted ‘hammer, corkand bottle’ experiment, demonstrating the loss of gas vauoles underpressure, belonged to Ahlborn (1894). Years later, the report ofHouwink (1956) of gas vesicles in halobacteria was also eclipsed, bythe later investigations on cyanobacteria. Discoveries made inisolation can become consigned to obscurity.

I was using a mass spectrometer for 15N2-fixation studies in 1965 and Ithought I would use the technique to identify the gas-vacuole gas.Two years later, using a modified “blood-gas manometer” of Barcroftand Haldane (1902), I showed that gas vesicles were freely permeableto gases. This led to investigations of the gas vesicles’ collapse underpressure, which affected many aspects of these structures. Forexample, cell turgor presure which presses on gas-vesicles, could bedetermined from the modified collapse pressure. The rigid, proteinwall of the gas vesicle, little more than one molecule thick, obeyed theprinciples of engineering used in making pipes and submarines,principles also relevant to the provision of buoyancy of cyanobacteriain lakes, where gas vesicles must survive the pressure increasing withdepth. In deeper lakes are cyanobacteria that evolved stronger gasvesicles, which are necessarily narrower and more expensive to make;in shallow salty pools, where cells have no turgor pressure, theweakest, widest and cheapest gas vesicles suffice.

In my researches on gas vesicles I depended on collaborations withspecialists in diverse disciplines – the mathematical physicist JohnSimpson, the freeze-etching pioneer Daniel Branton, thecrystallographer Allen Blaurock, and the molecular biologist JohnWalker; Paul Hayes, who started as my apprentice in things molecular,soon became my mentor, the co-superviser of some clever studentsand collaborator with visitors to our lab. Other valued contributerswere the biologists drawn into my obsession with gas vesicles andbuoyancy when I visited their institutes – Colin Reynolds in the EnglishLake District; Lucas Stal, who organised our Baltic cruises; andFerdinand Schanz who sampled Planktothrix for a decade from LakeZürich.

I suspect that the fruitless attempts of Fogg (1941) to induce gas-vacuole formation in Anabaena, led to his discovery that fixednitrogen inhibits heterocyst formation. My career began as hisgraduate student with papers on the first isolation not of gas vesicles,but of heterocysts (with Peter Fay), and then with Bill Stewart, Fay andFogg, the theory of nitrogen fixation by heterocysts, now one ofmicrobiology’s forgotten discoveries. As Mr Ono discovered, attitudeschanged.

KN-2.1

INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN,AND SULFUR METABOLISM: THE CENTRAL ROLE OF THE CALVIN-BENSON-BASSHAM CYCLE.

F. Robert Tabita, Ohio State University, Columbus, OH, USA

For many years our laboratory has studied the biochemistry andregulation of CO2 assimilation in phototrophic prokaryotes. In this talk,the central role that RubisCO and the Calvin-Benson-Bassham (CBB)pathway play in controlling many different aspects of metabolism willbe stressed. Some 15 years ago, we discovered that the molecularcontrol of key processes such as CO2 fixation, nitrogen fixation,hydrogen metabolism, and sulfur oxidation/reduction are linked inphotosynthetic bacteria. Indeed, when the primary means by whichthese organisms balance their intracellular redox state is blocked (viainactivating the CBB reductive pentose pathway such that thepreferred metabolic electron acceptor, CO2 , is no longer used)interesting adaptive mutant strains may be selected. One adaptive“trick” that we discovered in four separate organisms is that thenitrogenase complex may be derepressed so that this enzyme systemacts exclusively as a hydrogenase and reduces protons to balanceexcess redox equivalents that are produced via the oxidation oforganic carbon during photosynthetic growth. Such strains eructatemassive quantities of molecular hydrogen, even in the presence offixed nitrogen; some strains more than others. However, it is clear thatthe carbon assimilatory process influences this capability. It will also beshown that RubisCO and the control of the CBB cycle plays a centralrole in mediating other aspects of normal metabolism of theseorganisms.



PL-1.1

EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHICBACTERIA DEDUCTED FROM WHOLE GENOME COMPARISONS.

Robert Blankenship, Departments of Biology and Chemistry,Washington University, St. Louis, USA.

Photosynthesis is a central biological process that has a long andcomplex evolutionary history. The photosynthetic machineries found inthe existing groups of phototrophs have both common and divergentcharacters, suggesting an evolutionary process that combines de novogene appearance, gene duplication, gene and pathway recruitmentand loss, with both vertical and horizontal genetic transfer. The earliestphototrophs were almost certainly anoxygenic and were in existenceon Earth by at least 3.4 billion years ago and possibly somewhatearlier. Oxygenic phototrophs were undoubtedly in existence by 2.4billion years ago when free molecular oxygen, the waste product ofoxygenic photosynthesis, began to accumulate in the atmosphere.These organisms may have been present up to several hundredmillion years before that time. Several lines of evidence, includingmolecular evolution analysis, structural comparisons, and biochemicaland biophysical data, suggest that all modern photosynthetic reactioncenters are derived from a single ancient common ancestor and thatthe anoxygenic phototrophs preceded oxygenic ones. The transitionfrom anoxygenic to oxygenic photosynthesis was accompanied by anumber of evolutionary innovations, including multiple geneduplication and divergence events, modification of the pigmentbiosynthesis pathways from anaerobic to oxygen-requiring, inventionof the oxygen evolution center and a dramatic increase in the reactioncenter protein subunit complexity. The precise evolutionary pathwaythat led to the current diversity of different types of phototrophs wasnot linear and involved significant amounts of horizontal gene transfer.

PL-1.2

ORIGIN AND EVOLUTION OF THE PHOTOSYNTHETIC FUNCTION.

Armen Y. Mulkidjanian1,2, Michael Y. Galperin3, Eugene V. Koonin3.1School of Physics, University of Osnabrueck, Osnabrueck, Germany;2A.N. Belozersky Institute of Physico-Chemical Biology, Moscow StateUniversity, Moscow, Russia; 3NCBI, NLM, National Institutes of Health,Bethesda, MD, USA.

Introduction: Based on a comparative analysis of 15 cyanobacterialgenome sequences as well as complete genomes of the anoxygenicphototrophic bacteria Chlorobium tepidum, Rhodopseudomonaspalustris, Chloroflexus aurantiacus and Heliobacillus mobilis, we havesuggested that the (bacterio)chlorophyll-based photosynthesis hadoriginated in the anoxygenic ancestors of cyanobacteria (‘pro-cyanobacteria’) and then spread to other phyla via lateral genetransfer [1].

Results: This scheme will be considered in a broader context of theevolution of complex cellular systems and, in particular, of the energy-converting machinery [2-4]. It will be argued that whether or not anincreased complexity of a cellular system would be preserved in thecourse of evolution is determined by a trade-off between the gainsthat are brought by the system and its maintenance costs.

Conclusions: The evolution of the primeval pro-cyanobacterialphotosynthetic machinery can be considered as going in twodirections, namely (1) towards the sophisticated photosyntheticapparata of the modern cyanobacteria that, besides being able tocapture the energy of light, can exploit water as an unlimited source ofelectrons and 2) towards the “spare” photosynthetic appliances ofother bacteria that have tighter energy budgets.

References:

1. Mulkidjanian, A.Y., E. V. Koonin, K. S. Makarova, S. L. Mekhedov, A.Sorokin, Y. I. Wolf, A. Dufresne, F. Partensky, H. Burd, D. Kaznadzey, R.Haselkorn, M. Y. Galperin (2006) The cyanobacterial genome coreand the origin of photosynthesis, Proc. Natl. Acad. Sci. U.S.A., 103:13126-13131.

2. Mulkidjanian, A.Y., M.Y. Galperin, K.S. Makarova, Y.I. Wolf, E.V.Koonin (2008) Evolutionary primacy of sodium bioenergetics, BiolDirect 3 13.

3. Mulkidjanian, A.Y., P. Dibrov, M.Y. Galperin (2008) The past andpresent of the sodium energetics: May the sodium-motive force bewith you, Biochim. Biophys. Acta, 582, 238-242.

4. Mulkidjanian, A.Y., M.Y. Galperin, E.V. Koonin (2009) Co-evolution ofmembranes and membrane bioenergetics, Trends Biochem. Sci., inpress, DOI: 10.1016/j.tibs.2009.01.005.



PL-1.3

ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA:DIVERSITY AND NEW TAXONS.

Vladimir Gorlenko, Irina Bryantseva, Ekaterina Boldareva.

Vinogradsky Institute of Microbiology RAS, Moscow, Russia.

The first isolates of alkaliphilic purple sulfur bacteria (PSB) obtainedfrom the Vadi Natrun soda lakes included moderately halophilicmembers of the genus Ectothiorhodospira (Ect. haloalkalophila, Ect.vacuolata, and Ect. shaposhnicovii) and extremely halophilicHalorhodospira species (Hlr. halophila, Hlr. abdelmalekii, and Hlr.halochloris). We have described two new alkaliphilic genera of thefamily Ectothiorhodospiraceae, Thiorhodospira andEctothiorhodosinus. The members of these new genera PSB differedfrom the known species in the topography of their photosyntheticmembrane structures (PS). Recent research revealed big purple spirilla,“Ect. magna”, sp. nov., closely related to Ect. shaposhnicovii. Simialrto Thiorhodospira sibirica, their PS contained irregular intracellularlamellae and precipitated sulfur in the periplasm, like to members ofthe family Chromatiaceae. Microorganisms with gas vacuoles,described as Ect. variabilis, are widespread in the soda lakes wherethe mineralization is higher than ion seawater; this species isphylogenetically different from Ect. vacuolata. Thus, our findingsconfirm the low taxonomic value of the presence of intracellular gasvacuoles. Bacteria morphologically and phylogenetically related toEct. shaposhnicovii were recently isolated from the Mono Lake(California); they are capable of a new type of photosynthesis witharsenite as an electron donor. The first alkaliphilic member ofChromatiaceae, Thioalkalicoccus limnaeus (containing BChl b), wasisolated from Transbaikalian soda lakes. We investigated new strains ofthis species of alkaliphilic phototrophic bacteria. The second memberof Chromatiaceae, Thiocapsa imhoffii (containing BChl a), was recentlydescribed. In the lakes with low salinity, alkalitolerant members of thegenera Allochromatium and Thiocapsa are also present; thesystematic position of these species has not yet been determined.Among nonsulfur purple bacteria (NPB), two Rhodobaca species arealkaliphilic. The new NPB Rubribacter polymorphus, was isolated froma soda lake in the Barguzin Valley. Some species of the genusRhodovulum were also shown to be alkaliphilic. Close phylogeneticsimilarity was revealed between NPB and anaerobic,bacteriochlorophyll a-containing bacteria (ABC) of the genusRoseinatronobacter; this finding indicates their evolutionaryrelatedness. Similarity exists between NPB and ABC in their reactioncenters and the electron transfer system. The new members of thegenus Roseococcus, Rsc. suduntuensis and Rsc. vulcaneus weredemonstrated to be alkaliphilic (natronophilic) but not halophilic. Thealkaliphilic genus Heliorestis contains three natronophilic species. Itmay be concluded that obligate alkaliphily and natrononophilydeveloped as the result of evolutionary selection caused by prolongedexistence of the microorganisms under the alkaline conditions of sodaenvironments. The taxonomic rank of obligate alkaliphiles is usuallythat of a genus. Such secondary adaptations as active motility bymeans of flagella, formation of gas vesicles and of cyst-likemicrocolonies are important from the ecological point of view. Theintracellular membranes are diverse and correlate with the cell sizeand bacteriochlorophyll type.

PL-2.1

MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMICAND PROTEOMIC ANALYSES.Birgitta Bergman, Department of Botany, Stockholm University,Stockholm, Sweden.

It has become increasingly clear that diazotrophic cyanobacteria play acrucial role in maintaining life in oceans, major players being membersof the genus Trichodesmium. We are interested in understanding thestructural and molecular background related to the maintenance ofdiazotrophy in Trichodesmium IMS101, a non-heterocystouscyanobacterium fixing nitrogen in light/day. We have approached thisby examining events at the structural, transcriptomic and proteomiclevel, with focus on cellular dynamics during the development of thenitrogen-fixing cells, the diazocytes. The data obtained demonstrateda diurnal separation of basic physiological processes, with nitrogen-fixation and photosynthesis confined to the light and cell division anddiazocyte development to the dark period. When non-diazotrophiccultures were subject to nitrogen depletion a subset of cellsdifferentiated into nitrogenase containing diazocytes, while addition ofcombined nitrogen abolished development. Comparative proteomicanalysis revealed that ninty-four proteins were differentially expressedin diazotrophic (with diazocytes), as opposed to in the non-diazotrophic cultures, such as the nitrogenase enzyme and proteinsrelated to supporting reducing equivalents and lowering oxygentensions. Some recent metagenomic analyses of size fractionatedmicrobes from a transect spanning the Indian Ocean will alsopresented.



PL-2.2

PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATIONAND POTENTIAL INTERACTIONS.

Arthur R. Grossman1, Anne-Soisig Steunou2, Rosario Gomez-Garcia1,Melissa Adams1, Michael Kühl3, John Heidelberg4, Devaki Bhaya1.1The Carnegie Institute for Science, Department of Plant Biology,Stanford, CA, USA; 2CNRS, Centre de Génétique Moléculaire, Gif-sur-Yvette, France; 3University of Copenhagen, Marine Biology Laboratory,Department of Biology, Helsingør, Denmark; 4University of SouthernCalifornia, Department of Biological Sciences, Avalon, CA, USA.

Introduction: Hot spring microbial mats are natural biofilmscomposed of oxygenic photosynthetic cyanobacteria as well asheterotrophic microbes. These organisms are distributed alonghorizontal thermal and vertical light and oxygen gradients. The matsthat we have been studying are from Octopus and Mushroom Springsof Yellowstone National Park. They are generally 1-2 cm thick, withdensely packed cyanobacteria of the genus Synechococcus in theupper mat layer. The Synechococcus of the hot spring mats canharvest light energy for photosynthetic CO2 fixation at temperaturesexceeding 70oC.

Methods: Our work involves sequencing genomes from specificSynechococcus ecotypes that reside at different temperatures in themat and also generating metagenome sequence information.Moreover, we have analyzed the abundances of specific transcriptsusing reverse transcriptase quantitative PCR and measured levels(immunologically) and activities of specific proteins over the diel cyclein situ.

Results and Conclusions: The genomes of two Synechococcusvariants from the hot springs of Yellowstone National Park weresequenced, and while they have similar gene content, they areremarkably different with respect to gene arrangement. Furthermore,the genomes of both of the cyanobacterial isolates harbor a fullcomplement of nearly identical nitrogen-fixation (nif) genes. The nifgenes are expressed in the mat toward evening, when the matbecomes anoxic, although the highest in situ N2 fixation rates aremeasured in early morning, when the mat is still anoxic but light startsto drive photosynthesis and the production of ATP. The matcommunity also switches from oxidative metabolism in the day, whencells are photosynthesizing, to fermentation metabolism in theevening. This metabolic switching is reflected by rapid changes ingene expression in response to anaerobic/aerobic conditions. Wehave also cultured Synechococcus variants from the hot spring matsand show that they can use various forms of nutrients, includingphosphonates as a sole source of phosphate. I will discuss thephysiology, metabolism and energetic constraints within the matenvironment over the diel cycle.

PL-3.1

MULTICELLULARITY IN THE HETEROCYST-FORMINGCYANOBACTERIUM ANABAENA.

Enrique Flores, Instituto de Bioquímica Vegetal y Fotosíntesis,CSIC-Universidad de Sevilla, Sevilla, Spain.

Filamentous, heterocyst-forming cyanobacteria have been describedto reproduce by random trichome breakage implying that their unit ofgrowth is the filament. Under combined nitrogen deprivation, growthof filaments depends on the activity of two different types of cells,nitrogen-fixing heterocysts and photosynthetic vegetative cells, whichexchange nutrients and regulatory compounds making the filamentstrue multicellular organisms. They carry a Gram-negative type of cellenvelope in which, as shown by electron microscopy, the outermembrane is continuous along the filament without entering the septabetween cells. The periplasmic space, which lies between thecytoplasmic and outer membranes, is therefore continuous along thefilament, and this is so not only between vegetative cells but alsobetween vegetative cells and heterocysts. In the model organismAnabaena sp. PCC 7120, a periplasmic form of the green fluorescentprotein produced in (pro)heterocysts can be detected at a distance inthe periplasm of vegetative cells showing that the periplasm is alsofunctionally continuous and suggesting that it could constitute acommunication conduit between the cells in the filament. Recentstudies have shown that the Anabaena outer membrane is apermeability barrier for some sugars and amino acids that areimportant in the diazotrophic physiology of these organisms. On theother hand, electron microscopy also shows the presence in theintercellular septa of thin structures perpendicular to the cytoplasmicmembranes of adjacent cells. In Anabaena sp. PCC 7120, a proteinlocated at the cell poles in the intercellular septa, which consists of apredicted N-terminal extracytoplasmic domain and a C-terminalpermease domain, has been identified and named SepJ, and twoproteins, FraC and FraD, required for focused localization of SepJhave also been identified. Mutants of the genes encoding theseproteins show a filament fragmentation phenotype indicating that theycontribute to keep cells together in the filament. A method for testingintercellular molecular exchange in filamentous cyanobacteria hasbeen developed that consists in loading of a fluorescent dye, calcein,in the cytoplasm of the cells and measuring calcein fluorescence inFluorescence Recovery After Photobleaching (FRAP) experiments.Calcein exchange takes place rapidly between the cells in Anabaenafilaments but is impaired in sepJ, fraC and fraD mutants suggestingthat these proteins are involved in intercellular molecular exchange.Our current view of the Anabaena filament is that of a string of cellswhich are encapsulated by a continuous outer membrane and sharethe periplasm, and that are connected by proteinaceous structuresthat could also be involved in intercellular communication. Identifyingwhich compounds move in the filament through the periplasm or thecell-to-cell joining structures is a major topic for future research.



PL-3.2

EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY2-PHOSPHOGLYCOLATE METABOLISM IN CYANOBACTERIA.

Martin Hagemann, University of Rostock, Institute of BiologicalSciences, Plant Physiology, Rostock, Germany.

Introduction: Cyanobacteria fix most of the carbon using the Calvin-Benson cycle with RubisCO as the carboxylating enzyme. However,Rubisco has a rather low affinity to CO2 and exhibits in the presence ofoxygen also an oxygenase activity resulting in the toxic byproduct 2-phosphoglycolate (2PG). This intermediate is detoxified by thephotorespiratory 2PG cycle in plants. Cyanobacteria evolved aninorganic carbon concentrating mechanism (CCM) allowing them tocope with low carbon concentrations in the presence of oxygen.Active uptake systems for inorganic C accumulate high internalbicarbonate amounts. Moreover, RubisCO is concentrated incarboxysomes, where bicarbonate is transferred to CO2 leading tohigh CO2 concentration in the vicinity of RubisCO making oxygenaseactivity in cyanobacteria unlikely. Accordingly, it was widely acceptedthat cyanobacteria do not perform photorespiratory 2PG metabolism.During the last years we analyzed the mode of 2PG metabolism andits possible diverse function with the cyanobacterial model strainSynechocystis sp. PCC 6803.

Methods:Defined mutants of Synechocystis defective in candidategenes for glycolate metabolism were generated by interposonmutagenesis (1, 3). Moreover, microarray analyses were used to searchfor genes coding for alternative routes in the glycolate metabolism (2).

Results:Single-mutants in candidate genes for 2PG metabolism ofSynechocystis showed only small changes, while the generation andcharacterization of double- and triple-mutants showed that an active2PG metabolism employs three different routes: A) Glycolate isdegraded by a plant-like C2 cycle. B) A route exists corresponding tothe bacterial glycerate pathway. C) Additionally, the completedecarboxylation of glycolate via oxalate and formate occurs. Acomplete block in all three postulated routes resulted in a high-CO2-requiring (HCR) phenotype as it is known from photorespiratorymutants in higher plants. Bioinformatic analyses were used to searchfor corresponding genes in the completed genomes of othercyanobacterial strains, which indicated that generally all cyanobacteriaare able to metabolize 2PG despite the existing strain-specificdifferences in the occurrence of single pathways. The photorespiratorymetabolism contributes also to high light acclimation in Synechocystis,since a combined mutation of 2PG metabolism and the so-calledMehler reaction resulted in higher light sensitivity.

Conclusions: We could show that cyanobacteria perform an activephotorespiratory metabolism despite the activity of the CCM. The2PG metabolism uses three different routes in Synechocystis. Acomplete block of 2PG metabolism resulted in a HCR phenotype,which shows that this metabolism is essential for organismsperforming oxygenic photosynthesis in the present day atmosphere.These findings gave rise to the hypothesis that an earlyphotorespiratory has evolved already in ancient cyanobacteria andwas conveyed endosymbiontically into plants. The main function ofthe glycolate metabolism is directed to the detoxification of criticalintermediates. However, it is also involved in the acclimation to highlight conditions.

1 – Eisenhut et al. (2006) Plant Physiol 142:333-3422 – Eisenhut et al. (2007) Plant Physiol 144:1946-19593 – Eisenhut et al. (2008) PNAS 105:17199-17204

PL-3.3

THE NETWORK OF PII SIGNALING PROTEIN INTERACTIONS INCYANOBACTERIA .

Karl Forchhammer, Institut für Mikrobiologie der Eberhard-Karls-Universität Tübingen, Tübingen, Germany.

Introduction: The family of PII signal transduction proteins isrepresented in all domains of life, and is especially abundant inorganisms capable of inorganic nitrogen assimilation. The elementarymode of PII action is sensing the state of central metabolism/nitrogenassimilation and in response, orchestrating cellular processes relatedto nitrogen assimilation by binding to key factors involved in nitrogencontrol. Sensing the state of central metabolism is achieved byinterdependent binding of effector molecules to PII, in particular, 2-oxoglutarate and adenyl-nucleotides (ATP and ADP), and eventuallyby additional covalent modification of PII by modifying enzymes.

In most cyanobacteria, one highly conserved gene, termed glnB, en-coding a PII homologue has been identified in the genomic se-quences. Some strains, such as Gloeobacter violaceus, Synechococcussp. WH5701 or Acharyochloris marina harbor one or two further po-tential glnB paralogues. Functions and properties of cyanobacterial PIIsignalling were mostly worked out with the proteins from strains Syne-chococcus elongatus and Synechocystis sp. PCC 6803. There, PII iscovalently modified by S49 phosphorylation, but this apparently doesnot apply to all cyanobacteria. Here, I will present interaction studiesof PII with different target molecules, PII-phosphatase PphA, N-acetyl-L-glutamate kinase (NAGK) and PipX.

PII - phosphatase interaction:

The structure of the phospho-PII phosphatase PphA from Thermosyne-chococcus elongatus was resoved recently (1). Based on this structure,mutants were generated to elucidate the basis of substrate recogni-tion of PphA. Further, an assay was established to stabilize PII-PphA in-teraction intermediates. Metal 1 (M1) of the trinuclear metal centre ofPphA, a PPM family phosphatase, is absolutely required for bindingPII-P. Furthermore, several residues at the periphery of the enzymecould be identified to be required for productive PII-P binding, butwithout affecting the activity of the catalytic centre towards artificialsubstrates. PphA binding of PII responds specifically to the presenceof PII effector molecules ATP/ADP and 2-oxoglutarate, indicating thatthe entry of the phosphorylated T-loop of PII into the catalytic cavity iscontrolled by the effector molecules.

PII-NAGK interaction:

NAGK, the controlling enzyme of arginine biosythesis, by binding PII,is catalytically activated and relieved from arginine inhibition. Thestructure of the PII-NAGK complex from Synechococcus elongatus wasresolved (2) and the catalytic activation of NAGK by PII was investi-gated in detail. 2-oxoglutarate antagonizes the PII mediated activationof NAGK. Compared to PII/NAGK from Arabidopsis, the principalmode of PII activation - in particular its response to 2-oxoglutarate - isconserved in these oxygenic phototrophs (3). By random mutagenesiscombined with bacterial-two hybrid screening, we could identify sev-eral PII mutants with novel phenotypes regarding NAGK and effectormetabolite binding, leading to novel insights in PII function. This in-volves in particular residues at the B-loop of PII, which appears to playa key role in the intra-molecular transmission of metabolite signals.

PII-PipX interaction:

A further PII target, PipX, appears to be a transcriptional co-activatorof NtcA. PII competes with NtcA for binding to PipX, thereby antago-nizing the activation of NtcA (Espinosa et al., 2007). Whereas bindingof PipX to NtcA requires 2-oxoglutarate, 2-OG in concert with ATP im-



pairs PipX binding to PII. When PII and NtcA compete for PipX in pres-ence of constant 2-oxoglutarate levels, PipX swaps form PII to NtcAwhen ATP is added. The NtcA-PipX complex binds to DNA, with PipXnot affecting the DNA binding properties of NtcA. When PipX andNAGK compete for PII binding in absence of 2-OG, PII preferentiallybinds to NAGK.

Other PII interactions:

Further to the above-mentioned targets, PII was shown to bind to aMscS-like protein from Synechocystis PCC 6803, PamA (5). In contrastto PipX or NAGK, PamA is not highly conserved in cyanobacteria, andit remains to be elucidated what the physiological function of this pro-tein is. Mutants in the PII signalling system show impaired ability toregulate nitrate utilization (6) implying PII interactions with compo-nents of the nitrate utilization pathway.

The overall organization of the PII signal transduction network will bediscussed.

1. Schlicker et al., 2008 J. Mol. Biol. 376: 570-581

2. Llacer et al, 2007 Proc. Natl. Acad. Sci. U.S.A. 104: 17644-17649

3. Beez et al., 2009 J Mol Biol. 389: 748-758

4. Espinosa et al., 2007 Microbiology 153: 711-718

5. Osanai et al., J. Biol. Chem. 280: 34684–34690

6. Kloft & Forchhammer, 2005 J. Bacteriol. 187: 6683-6690

PL-4.1

A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME.

Susan Golden, Department of Biology, Professor Division of BiologicalSciences University of California-San Diego, La Jolla,CA, USA.

Cyanobacteria, like diverse eukaryotes, possess circadian clocks thatallow cells to coordinate physiological processes with the predictabledaily patterns of environmental fluctuations on Earth. An autonomousoscillator comprised of three distinctive proteins, KaiA, KaiB, andKaiC, underpins the circadian clock of the cyanobacterial modelorganism Synechococcus elongatus PCC 7942. Our work appliesgenetic, genomic, biochemical, and structural approaches, integratedwith cell biology, to achieve a comprehensive understanding of howthe S. elongatus cell harnesses this oscillator to control cellularactivities. The emerging picture is one of a clock that is set daily bysampling the cellular redox environment as a proxy for light and whichuses protein-protein interactions and subcellular localization tocoordinate gene expression, cell division, and chromosome dynamics.A functional genomics project has produced inactivation alleles ofnearly all S. elongatus loci. Gene expression assays of the 700 mutantstested so far suggest that about 10% of loci affect circadian period orphasing.

PL-4.2

EFFECTS OF INSERTION SEQUENCES ON THE GENOME OFANABAENA SP. STRAIN PCC 7120.

C. Peter Wolk, Sigal Lechno-Yossef.

Michigan State University, East Lansing, MI, USA.

Introduction: Many cyanobacteria closely related to Anabaena sp.strain PCC 7120 make akinetes and gas vacuoles, but PCC 7120 lacksthose properties despite having homologs of the akinete-marker geneavaK (1) and gas vacuole-related orfs all2247 through asl2254. It bearsca. 145 orfs that are annotated as encoding transposases(http://bacteria.kazusa.or.jp/cyanobase/), which are diagnostic ofinsertion sequences (ISs). Some of the ISs surely transpose (2-4, andcompare 5 and 6), and can thereby mutagenize. Our laboratory earlieridentified three evidently IS891-interrupted regulatory genes inAnabaena sp. (7). Using the newly available sequence data for variouscyanobacteria, we have returned to an in-silico attempt to examinefeatures of the genome of PCC 7120 as it may have appeared prior toentry of those ISs.

Methods: To “remove” an IS, one must know how far it extendsoutwards from its transposase orf(s) and how many base pairs itreplicates upon insertion. Such information is provided by:transposition of ISs to known genetic backgrounds; alignments ofsequences containing related ISs to see where their commonalityceases, with the ISs often terminating in inverted repeats andfrequently bracketed by direct repeats; and the finding that in otherorganisms, sequences that are nearly identical to those that flank ISsare joined.

Results: Depending on whether neighboring transposase orfs are partof the same IS, PCC 7120 may have ca. 100-150 ISs. We have excisedmost, in silico, with convincing accuracy; and have identified furtherelements that, although lacking an annotated transposase, appear tobe mobile. Once ISs are removed, we find: nearby orfs extended byreplacement of their initiation or termination codons; flanking orfsfused in-frame; orfs previously unrecognized, including those ofunderlying ISs, found; orfs lost that consisted principally of ends of ISs;and extended intergenic regions that appear empty of orfs. Anabaenavariabilis orthologs of two of the kinase genes intercepted in PCC7120 may also have been intercepted by ISs.

Conclusions:We have not identified a phenotype that is surelyattributable to the presence of an IS. However, our “archeologicalgenomics“ permits a view of at least part of the genome of PCC 7120in a more ancestral form. We have identified presumptive orfs,operons, and regulatory genes whose restoration might enlarge PCC7120‘s phenotype, including its ability to protect itself against enteringDNA. Finally, we have added to knowledge of the ISs of PCC 7120(and other bacteria): have they inverted terminal repeats, do theygenerate direct repeats upon insertion, and how widespread is theirtaxonomic dispersion? The excavation continues.

References: 1. R Zhou and CP Wolk, J Bacteriol 184: 2529 (2002). 2. YCai and CP Wolk, J Bacteriol 172: 3138 (1990). 3. J Alam et al., JBacteriol 173: 5778 (1991). 4. Y Cai, J Bacteriol 173: 5771 (1991). 5. IBancroft and CP Wolk, J Bacteriol 171: 5949 (1989). 6. T Kaneko et al.,DNA Res 8: 205 (2001). 7. M Ohmori et al., DNA Res 8: 271 (2001).



PL-5.1

CYTOCHROME C MATURATION, DISULFIDE BOND FORMATIONAND PERIPLASMIC PROTEIN DEGRADATION IN RHODOBACTERCAPSULATUS.

Ozlem Onder, Serdar Turkarslan, Carsten Sanders, Fevzi Daldal.

University of Pennsylvania, Department of Biology,Philadelphia, PA, USA.

Current knowledge on cytochrome c maturation process in thefacultative phototrophic bacterium Rhodobacter capsulatus will bereviewed, and the crucial role of periplasmic thio-redox homeostasis(DsbA-DsbB versus CcdA-CcmG) involved in forming and breaking ofapocytochrome c disulfide bonds during this process will bedescribed. In addition, we will report the unusual observation thatDsbA-null mutants are proficient in photosynthesis but are defective inrespiration, especially in enriched growth medium at 35 ºC. Usingcombined proteomic and molecular genetic approaches wedemonstrated that the respiratory defect of R. capsulatus DsbA-nullmutants originate from the overproduction of the periplasmic proteaseDegP, which render them temperature sensitive for growth. The DsbA-null mutants revert frequently to overcome this growth defect bydecreasing, but not completely eliminating, their DegP activity. Wehave shown that overproduction of DegP abolishes the newly restoredrespiratory growth ability of the revertants in all growth media.Structural localizations of the reversion mutations in DegP revealed theregions and amino acids that are important for the protease-chaperone activity. Remarkably, although R. capsulatus DsbA-null orDegP-null mutants are viable, DegP-null DsbA-null double mutants arelethal at all growth temperatures. We found that this is unlike E. coli,indicating that in the absence of DsbA some DegP activity is requiredfor survival of R. capsulatus. Absence of a DegQ protease homologuein some bacteria together with major structural variations amongst theDegP homologues, including a critical disulfide bond-bearing region,correlate well with the differences seen between various species like R.capsulatus and E. coli, and illustrate the occurrence of two related butdistinct periplasmic protease families in bacterial species.

PL-5.2

BETTER LIVING THROUGH CYANOTHECE--UNICELLULARDIAZOTROPHIC CYANOBACTERIA WITH HIGHLY VERSATILEMETABOLIC SYSTEMS.

Louis A. Sherman1, Hongtao Min1, Jorg Toepel1, Michelle Liberton2,Hanayo Sato2, Jana Stöckel2, Himadri B. Pakrasi2.1Dept. of Biological Sciences, Purdue University, West Lafayette, IN;2Dept. of Biology, Washington University, St. Louis, MO; USA.

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophiccyanobacterium with a versatile metabolism and very pronounceddiurnal rhythms. Since nitrogen fixation is exquisitely sensitive tooxygen, Cyanothece utilizes temporal regulation to accommodatethese incompatible processes in a single cell. When grown under 12hlight-dark (LD) periods, it performs photosynthesis during the day andN2 fixation and respiration at night. During this process,carbohydrates and amino acids are compartmentalized in granules inthe light and dark, respectively. In essence, Cyanothece creates an O2-limited intracellular environment to perform oxygen-sensitiveprocesses such as N2-fixation and H2 production during the night. Thisstrain also grows exceedingly well heterotrophically (on glycerol). Theexcellent synchrony of a culture under LD diazotrophic conditionspermits analysis of cellular morphology, mRNA levels, proteomics andmetabolomics as a function of time.

The genome sequence of Cyanothece sp. ATCC 51142 (Welsh et al(2008) PNAS 105: 15094-99) and that of 5 other Cyanothece sp. arecomplete and that of a seventh is in progress. Cyanothece sp. ATCC51142 has 2 chromosomes—a 4.9 Mb circular chromosome and a 0.43Mb chromosome with a total of 5,300 genes. This strain has thecapability of producing high-energy compounds, such as ethanol andhydrogen and Cyanothece sp. PCC 7822 can produce thesecompounds as well as rather large quantities of parahydroxyalkanotes(PHAs). We are particularly interested in the regulation of thesemetabolic processes and the way in which these organisms respond toenvironmental cues such as light, the lack of combined nitrogen andchanging O2 levels. Analysis at both the transcriptomics (withmicroarrays, Toepel et al (2008) J Bact 190:3904 and Stockel et al(2008) PNAS 105:6156) and proteomics levels in Cyanothece sp.ATCC 51142 has demonstrated the relationship of the metabolicsynchrony with gene expression and has given us insights into diurnaland circadian regulation throughout a daily cycle.

The Cyanothece strains (51142 and 7822 have been the most studied)produce copious amounts of H2 under different types of physiologicalconditions. Both the bidirectional hydrogenase (encoded by the hoxgenes) and the nitrogenase (nif genes) are capable of producingmeasurable hydrogen under appropriate conditions. However,nitrogenase produces far more H2 than the hydrogenase, in partbecause the nitrogenase levels are extremely high under N2-fixingconditions. With Cyanothece 51142 cultures grown in NO3-freemedia, either photoautotrophically or mixotrophically with glycerol, wehave obtained rates of H2 produced of 150 mmoles/mg Chl/hr and300 mmoles/mg Chl/hr, respectively. We anticipate even highervalues once we are able to construct specific mutants and developoptimal physiological conditions. This work was sponsored by grantsfrom the DOE and from the EMSL at the Pacific Northwest NationalLabs.



PL-5.3

PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENTEVOLUTION SCHEMES FROM A COMMON ANCESTRALGENOME.

Frédéric Partensky1, Alexis Dufresne1, 2, Martin Ostrowski3, David J.Scanlan3 and Laurence Garczarek1 .1UMR 7144 CNRS & Université Paris 6, Station Biologique, Roscoff,France; 2UMR 6553 EcoBio, Université Rennes, Rennes, France;3Department of Biological Sciences, University of Warwick, Coventry,United Kingdom.

Prochlorococcus and Synechococcus are the two most abundantoxyphototrophs in the ocean. These two marine picocyanobacteria arephylogenetically closely related and together make a branch wellseparated from all other cyanobacteria, including freshwaterSynechococcus but also marine cyanobacteria of larger cell size,including Trichodesmium thiebautii and Crocosphaera watsonii. Thelarge number of genomes of Prochlorococcus (13) and marineSynechococcus (11) that have recently been sequenced, covers mostof the diversity known within these groups. This makes it possible todraw insights about the mechanisms that have presided genomeevolution in these ecologically important groups. It is now clear thatProchlorococcus is a fairly recent group, which is highly specialized tolife in warm, oligotrophic waters. It appeared quasi concomitantly withthe highly diversified Synechococcus subcluster 5.1, whose membersare seemingly the best represented in the ocean nowadays. Light hasbeen a key environmental factor in the evolution of both genera.Prochlorococcus has first colonized the bottom of the euphotic zonethen, more recently, the upper layer of oligotrophic zones. As a result,Prochlorococcus has differentiated itself into low- and high lightecotypes, a phenomenon that has allowed this genus to colonize thewhole sunlit layer. For Synechococcus, however, light quality hasseemingly been the key factor, since the diversification of its pigmentcontent has allowed this genus to colonize the whole range ofenvironments over the coastal to offshore gradient. But otherenvironmental factors, including salinity, temperature or nutrientavailability are also important in shaping the genomes.

In this talk, current scenarios for genome evolution in these two keyorganisms that originate from a common ancestor will be presented.In particular, the impact of environmental factors (mainly light) on thegenome content and structure of the different ecotypes within eachgenus will be discussed.

PL-6.1

OXYGEN AND LIGHT CONTROL OF TETRAPYRROLEBIOSYNTHESIS IN RHODOBACTER SPECIES.

Carl Bauer, Department of Molecular and Cellular Biochemistry,Indiana University, Bloomington, IN, USA.

The purple photosynthetic bacteria Rhodobacter capsulatus andRhodobacter sphaeroides synthesizes three tetrapyrrole endproducts,bacteriochlorophyll, heme and cobalamine (vitamin B12). All threetetrapyrroles are the product of a complex pathway that isresponsible for controlling synthesis of these endproducts in responseto light intensity and oxygen tension. Over the past decade ourlaboratory has identified numerous transcription factors that areresponsible for the control of tetrapyrrole gene expression. Recentbiochemical analysis of several of these transcription factors hasdemonstrated that many also utilize heme or cobalamine as cofactors.Our current understanding of the control of tetrapyrrole biosynthesisin these species will be presented as well as what is known about thebiochemical properties of key regulators.

PL-6.2

A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHTINTENSITY SENSOR IN CYANOBACTERIA PHOTOPROTECTION.

Adjélé Wilson, Clémence Boulay, Claire Punginelli, Diana Kirilovsky.

Commissariat à l’Energie Atomique (CEA), Institut de Biologie etTechnologies de Saclay (iBiTecS) and Centre National de la RechercheScientifique (CNRS), 91191 Gif sur Yvette, France.

Because too much light can be lethal for photosynthetic organisms,photoprotection against excess absorbed light energy is an essentialand universal attribute of oxygenic photosynthetic organisms. In plantsand algae, the membrane embedded chlorophyll antenna, LHCII,reversibly switchs from a very efficient energy collection state into aphotoprotective state. This state by converting the excess energy intoheat decreases the energy arriving at the reaction centers under highlight conditions. The existence of an equivalent process incyanobacteria, which use the extramembranal phycobilisomes toharvest energy instead of LHCII, was only recently discovered. Thestudy of the photoprotective thermal energy dissipation mechanism inphycobilisome- and Photosystem II-mutants of the cyanobacteriumSynechocystis PCC6803 by fluorescence measurements allowed todemonstrate that this mechanism involves a specific decrease of thefluorescence emission of the phycobilisomes and a decrease of theenergy transfer from the phycobilisomes to the reaction centers. Theprocess is induced by the absorbance of blue-green light by a solubleorange carotenoid protein, the Orange-Carotenoid-Protein (OCP). InSynechocystis , the OCP is encoded by the slr1963 gene which isconstitutively expressed. OCP genes appear to be highly conservedamong phycobilisome-containing cyanobacteria with few exceptions,indicating that the OCP related photoprotective mechanism iswidespread. The strains containing a whole OCP gene can performthe blue-light induced photoprotective mechanism. In contrast, strainscontaining only N-terminal and/or C-terminal OCP like genes or noOCP like genes at all lack this mechanism and they were moresensitive to high light illumination. These strains to longer surviveunder stress conditions degrade the phycobiliproteins very fast toavoid the appearance of a population of dangerous, functionallydisconnected phycobilisomes. The OCP containing strains increasethe expression of the OCP and as a consequence increase theconversion to heat of the excess energy absorbed by thephycobilisomes.



The OCP is a photoactive protein. The absorbance of blue-green lightby the OCP induces structural changes in the carotenoid and theprotein, converting its dark stable orange form into a relativelyunstable active red form. The presence of the red OCP form isessential for the induction of the photoprotective mechanism. In theabsence of the formation of the red form due to point mutations inthe OCP, no fluorescence quenching is induced by strong white orblue-green light. OCP is fully active when binds hydroxyechinenone orechinenone. When these carotenoids are absent, OCP bindszeaxanthin but light is unable to photoconvert the dark form into alight active form. In the strains containing zeaxanthin-OCP, blue-greenlight did not induce the photoprotective mechanism. These resultsstrongly suggest that the presence of the carotenoid carbonyl groupthat distinguishes echinenone and hydroxyechinenone fromzeaxanthin is essential for the OCP activity. Moreover, Trp288 and Tyr201 forming hydrogen bonds to the carbonyl are essential for thephotoconvertion and cannot be replaced by other amino-acid withoutloosing activity. The OCP is the first photoactive protein containing acarotenoid as the photoactive chromophore and its photocycle iscompletely different from those of other photoactive proteins.

PL-6.3

NITROGEN AND MOLYBDENUM CONTROL OF NITROGENFIXATION.

Bernd Masepohl, Alexandra Müller, Jessica Wiethaus.

Ruhr University Bochum, Bochum, Germany.

The phototrophic purple bacterium Rhodobacter capsulatus has thecapacity to grow with atmospheric dinitrogen (N2) as sole source ofnitrogen. Reduction of N2 to ammonia is catalyzed by twonitrogenases, molybdenum-nitrogenase (Mo-nitrogenase) and analternative iron-only nitrogenase (Fe-nitrogenase). Fe-nitrogenaseexhibits lower specific activity than Mo-nitrogenase, and thus, Mo-nitrogenase is the preferred enzyme as long as molybdenum isavailable. Upon Mo-depletion, R. capsulatus synthesizes the high-affinity Mo-uptake transporter ModABC. Since nitrogen fixation is ahighly energy-demanding process, synthesis and activity of bothnitrogenases is tighly controlled. Only in the absence of ammoniumthe sensor kinase NtrB autophosphorylates, and subsequently, phos-phorylates its cognate response regulator NtrC. In turn, NtrC activatestranscription of nifA and anfA coding for the transcriptional activatorsof all the other nitrogen fixation genes. Transcription of anfA isrepressed by the Mo-responsive regulators MopA and MopBpreventing expression of Fe-nitrogenase in the presence ofmolybdenum. Finally, activity of both nitrogenases is controlled by aswitch-off/switch-on mechanism involving reversible covalentmodification, thus enabling R. capsulatus to respond very fast tosubtle changes in ammonium availability.

Like many other bacteria, R. capsulatus contains two genes, glnB andglnK, coding for PII-like signal transduction proteins, which playcentral roles in regulation of nitrogen fixation and assimilation. GlnBand GlnK control activity of many target proteins by transientinteraction in response to changes in the cellular nitrogen status.Among these target proteins are the sensor kinase NtrB, thetranscription activator NifA, and the switch-off protein DraT. Affinity ofGlnB and GlnK to their target proteins is modulated by binding ofoxoglutarate and ATP, and uridylylation at a conserved tyrosineresidue. The uridylylation state of GlnB and GlnK reflects the cellularglutamine concentration, with both proteins being uridylylated whenglutamine concentrations are low. Upon ammonium addition both PIIproteins are sequestered to the membrane by binding to theammonium transporter AmtB.

The Mo-responsive regulators MopA and MopB are modular instructure consisting of an N-terminal DNA-binding and a C-terminalMo-binding domain. They bind to conserved palindromic promotersequences, so-called Mo-boxes. Affinity of the regulators to theirtarget promoters is clearly enhanced by molybdenum. MopA andMopB can substitute for each other in repressing transcription ofseveral target genes including the mopA-modABC operon and theanfA gene. In addition to its role as a repressor, MopA acts as anactivator of the mop gene coding for the molbindin-like Mop protein.The genes coding for the Mo-responsive regulators, MopA and MopB,belong to two divergently transcribed operons, mopA-modABC andmopB. While transcription of the mopA operon is repressed bymolybdenum, mopB is constitutively expressed, suggesting that theMopA-MopB ratio changes in response to the cellular Mo status.MopA and MopB form homodimers and heteromers. Formation ofheteromers is thought to deplete the pool of homodimers, and thusmight represent a fine-tuning mechanism controlling expression ofMo-regulated genes.

PL-7.1

HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVESENVIRONMENTAL STIMULI?

Iwane Suzuki.

University of Tsukuba, Tsukuba, Ibaraki, Japan.

Introduction: Two-component systems, which generally consist of asensory histidine kinase and a response regulator, are major signalingpathways in most of bacteria. In general, histidine kinases possess aconserved transmitter domain associating kinase activity at the C-terminus and a unique signal input domain (SID) at the N-terminus.The SIDs in the histidine kinases are thought to play important roles inperception of the specific stimuli and regulation of the kinase activityof the transmitter domain, however, the actual molecular mechanismsof signal perception are not clarified yet. I developed a system toexpress chimeric histidine kinases containing a transmitter domain ofSphS, a phosphate-deficient sensor, and SID from other histidinekinase in the cyanobacterium Synechocystis sp. PCC 6803. It might bea useful strategy to investigate the functions of the histidine kinases.

Methods: I constructed genes for chimeric sensors containingtransmitter domain of SphS, which include either the N-terminalregion of a Ni2+-sensor, NrsS, or SID of Hik33. The chimeric histidinekinases were driven by the native promoter of the sphS gene. Effectsof the chimera proteins on the activity of SphS transmitter domainwere examined by measuring expression levels of the phoA gene foran alkaline phosphatase as an internal reporter.

Results: The N-terminal region of SphS contains both a hydrophobicregion and a PAS domain. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of thenative promoter were analyzed for in vivo AP activity. Deletion of theN-terminal hydrophobic region resulted in loss of AP activity underboth Pi-deficient and Pi-sufficient conditions. Substitution of thehydrophobic region of SphS with that from NrsS resulted in the sameinduction characteristics as SphS. Deletion of the PAS domain resultedin the constitutive induction of AP activity regardless of Pi-availability.These results indicated that the presence of a transmembrane helix inthe N-terminal region of SphS is critical for activity and the PASdomain is involved in perception of Pi-availability.

This strategy was also applied to characterize the SID in Hik33, whichregulate gene expression under cold, high light, oxidative, high salt,and hyperosmotic stress conditions. The SID includes twotransmembrane helices, a periplasmic loop, a HAMP and a PAS



domain. When the SID was fused with the transmitter domain of SphS,it activated the phoA expression under the standard growth conditionand repressed under the stress conditions. The repression ofexpression of phoA was dependent on the PAS domain in SID. Thedetail results will be discussed in the presentation.

Conclusions: The chimeric sensor system may be a useful approach toinvestigate the function of SID of the histidine kinases.

PL-7.2

EVOLUTION AND REGULATION OF THE RHODOBACTERCAPSULATUS GENE TRANSFER AGENT.

J. Thomas Beatty1, Molly Leung1, Sarah Florizone1, Jeanette A.Johnson1, Ryan Mercer2, and Andrew S. Lang2.

Department of Microbiology & Immunology, the University of BritishColumbia1, BC; Department of Biology, Memorial University ofNewfoundland2, NF; Canada.

Introduction: The gene transfer agent (GTA) produced by the purplenon-sulfur bacterium Rhodobacter capsulatus is a model for severalvirus-like elements that seem to function solely for mediating geneexchange. Some GTA structural genes in R. capsulatus are related tobacteriophage genes, but the cellular regulatory mechanisms thatcontrol GTA production indicate that GTA is more than just a defectiveprophage. Genome sequencing projects have shown that mostprokaryotes contain prophage-like gene clusters, but possiblerelationships to the GTA gene cluster were unclear. The R. capsulatusGTA is produced maximally in stationary phase cultures, but it was notknown what aspect of stationary phase induces the expression of GTAstructural genes.

Our research was done to investigate the presence of GTA genes in avariety of prokaryotes, and to study factors that affect the stationaryphase production of GTA in R. capsulatus.

Methods: R. capsulatus GTA protein sequences were used as queriesin BLAST searches of genome sequences, and the genome context ofhigh-scoring hits was subsequently evaluated by eye. Sequenceswere aligned with Clustal X v1.81 and the alignment was used toconstruct neighbor-joining trees with PAUP* v4.0. Cultures of R.capsulatus were grown photoheterotrophically, and cells separatedfrom GTA by centrifugation and filtration through a 0.2 µm filter. Theproduction of GTA was evaluated in gene transduction experiments,or by probing western blots with antiserum raised against the GTAmajor capsid protein over-expressed in E. coli.

Results: Complete genome sequences were searched for GTA genehomologues to identify candidate GTA-producing species. Althoughhomologous GTA gene clusters are widespread, they are foundexclusively within the α-proteobacteria. A phylogenetic treecomparing 16S rRNA and GTA gene homologues shows that thesetwo types of gene have evolved in concert.

Work on R. capsulatus GTA regulatory gene mutants indicates threeindependent pathways that affect the production of extracellular GTAparticles. One pathway involves the response-regulator CtrA, which isessential for transcription of GTA structural genes. A second pathwaytransmits a quorum-sensing signal, produced by a homoserine lactonesynthase, which is needed for maximal induction of transcription ofGTA genes. A third pathway requires the sensor-kinase homologueCckA for release of GTA particles from cells.

An evaluation of the effects of carbon, nitrogen and phosphorusdeprivation on the synthesis and release of GTA particles indicatesthat different environmental signals affect the production of GTA,similarly to the effects of regulatory mutations.

Conclusions: It appears that GTA arose in the α-proteobacteria afterdivergence of this line from other prokaryotes. The similarity of 16SrRNA and GTA gene descent to present-day species indicates that thetransmission of GTA was largely vertical, with little or no horizontalgene transfer.

We propose a speculative model in which three environmental signalsaffect GTA production: 1) the availability of phosphate and ammoniumaffect the balance between phosphorylated and non-phosphorylatedCtrA, which in turn determines whether GTA genes are transcribed; 2)cell population density-dependent accumulation of a homoserinelactone molecule stimulates transcription of GTA genes; 3) a decreasein the availability of carbon stimulates a pathway involving CckA,which is needed for release of GTA particles from cells.

PL-8.1

PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGENPRODUCTION.

Caroline S. Harwood, University of Washington, Seattle, WA USA.

In the world today, large quantities of hydrogen gas are used inpetroleum refining and ammonia production for fertilizer and in thefuture there is the potential for hydrogen to be used in huge amountsas a transportation fuel. Although still under development, there areseveral microbial routes for hydrogen production that depend on theenvironmentally benign use of biomass, solar energy and other readilyavailable resources. The development of an efficient process forhydrogen production by purple nonsulfur bacteria will require that thebiocatalyst for hydrogen production, the nitrogenase, be synthesizedand active under all growth conditions and it will be important todevelop strains that cannot recapture and dissipate the hydrogen thatthey produce. Beyond this, the metabolic modules ofphotophosphorylation, reductant generation and nitrogenase activitythat feed the process of hydrogen production, must each operate withmaximum efficiency for hydrogen generation as opposed to forammonia production. Obviously, the integrated functioning of thesemodules must also be maximally efficient. With this framework in mindthere are numerous strategies for strain development that can beexpected to lead to improvements and stabilization of the hydrogenproduction process. In addition to their practical usefulness, theapplication of such strategies will lead to an improved understandingof the hydrogen production process as it operates in the context ofwhole cells. In this talk I will discuss three strategies to improvehydrogen production. These are 1) Removal of regulatory constraintson nitrogenase, 2) Optimization of electron flow to nitrogenase, and 3)Design of efficient bioreactors.

PL-8.2

THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICALAPPLICATIONS

Paula Tamagnini1,2, 1IBMC - Instituto de Biologia Molecular e Celular;2Faculdade de Ciências, Departamento de Botânica; Universidade doPorto, Porto, Portugal.

Introduction: Cyanobacteria combine the ability to perform oxygenicphotosynthesis with typical prokaryotic features like the ability to fixnitrogen. Many strains produce exopolymers, mainly ofpolysaccharidic nature, named exopolysaccharides (EPS). Due to theircharacteristics, cyanobacteria have high potential for biotechnologicalapplications, such as the (i) production of biohydrogen and (ii) removalof heavy metals from contaminated waters.



(i) BioModularH2 uses a synthetic biology approach to designreusable, standardized molecular building blocks that integrated into achassis will produce a photosynthetic bacterium containingengineered chemical pathways for competitive, clean and sustainablehydrogen production [1].

(ii) The EPS project aims at optimizing the production ofcyanobacterial EPS to establish an effective system to remove metallicions from polluted waters.

Methods: (i) BioModularH2 - Generation of mutants without redundantparts (e.g. genes) and identification of neutral sites using standardizedvectors compatible with the BioBrick™; design and construction ofsynthetic parts and its assembly into modules/devices; characterizationof the devices in the cyanobacterial chassis.

(ii) EPS project - Metal removal assays, determination of EPSmonosaccharidic composition by ion-exchange chromatography,identification of the functional groups responsible for metal-bindingby potentiometric titration and Diffuse Reflectance Infrared FourierTransform spectrometry, and sequence comparisons usingbioinformatics tools.

Results: (i) Preparation of the chassis starting with the (i) deletion ofgenes encoding the bidirectional hydrogenase in Synechocystis sp.PCC 6803 (redundant part) and (ii) construction of mutants on putativeneutral sites (to integrate synthetic parts/devices such as an efficientheterologous hydrogenase). Concomitantly, several parts (e.g.promoters), and oxygen consuming devices (to provide a microaerobicenvironment required for an optimal heterologous hydrogenaseactivity) are being synthesized, characterized and tested.

(ii) Copper removal assays performed with Gloeothece sp. PCC 6909wild type and its sheathless mutant revealed that the mutant is moreefficient in the process. Subsequent studies showed that although themutant does not possess a sheath, it releases large amounts ofpolysaccharidic material (RPS) into the medium, and that its RPSpossess higher amount and/or more accessible functional groups (e.g.carboxyl and amide groups) [2]. An in silico analysis of cyanobacterialgenomes revealed the presence of genes encoding proteins that, inother organisms, are involved in the last steps of EPS production,although with a different physical organization. Based on thesefindings a putative mechanism for the biosynthesis and export ofcyanobacterial EPS was proposed [3].

Conclusions: (i) BiomodularH2 - The standardized parts, modules anddevices generated will be introduced to the chassis forphotobiological H2 production, as well as will be available for otherbiotechnological applications.

(ii) The future implementation of heavy metal removing systems basedon cyanobacterial EPS depends on the knowledge about thebiosynthetic pathways leading to their production.

References:

[1] http://www.biomodularh2.org

[2] Micheletti E et al (2008) Appl Environ Microbiol 74: 2797-2804. 19:139-185

[2] Pereira et al (2009) FEMS Microbiol Rev (accepted)

PL-8.3

CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES,ACTIVITIES AND BIOSYNTHESIS.

Kaarina Sivonen, Department of Applied Chemistry and Microbiology,Viikki Biocenter, University of Helsinki, Finland.

Introduction: Cyanobacteria are a prolific source of bioactivecompounds. These include potent toxins as well as biomedicallyinteresting molecules, drug leads or useful probes in cell biologystudies. Mass occurrences of neurotoxic and hepatotoxiccyanobacteria have caused number of animal poisonings and are a riskfor human health. The cyclic hepatotoxic heptapeptides microcystinsare most studied due to their worldwide occurrence.

Methods: The structures of cyanobacterial compounds weredetermined by LC-MS and NMR. Bioactivities were investigated byenzyme inhibition tests and cell assays. Molecular methods (PCR,qPCR, cloning, sequencing, heterologous expression, microarrays)were used to identify the biosynthetic gene clusters and to detecthepatotoxic cyanobacteria. Shotgun sequencing and bioinformaticanalyses were applied to genome of Anabaena strain 90.

Results: Microcystins and nodularins (pentapeptide hepatotoxinoccurring in brackish waters) are specific inhibitors of eukaryoticprotein phosphatases and act as tumour promoters. Cyanobacterialbioactive cyclic and linear peptides may also display variousbioactivities including serine protease inhibition and cytotoxicity.Novel bioactivities found recently include compounds acting asantidotes for microcystins and lipopeptides distroying the eukaryoticcell membrane. Microcystins and nodularin are products of a mixednon-ribosomal peptide and polyketide synthetase. The microcystinsand nodularin gene clusters encode peptide synthetases, polyketidesynthases and tailoring enzymes. This knowledge has been used todevelop number of molecular techniques to detect hepatotoxinproducing cyanobacteria. Studies on the evolution ofmicrocystin/nodularin synthetase genes suggest that these genes areancient and that present non-toxic strains have lost the genes andtoxin production. The cyclic anabenopeptilides, and anabaenopeptinsas well as linear spumigins are synthesised on non-ribosomal peptidesynthetases. Interestingly, in anabaenopeptin biosynthesis a new wayto create non-ribosomal peptide structural diversity was detected. Thecomplete genome of Anabaena sp. strain 90 showed that 5% of thegenome was dedicated to biosynthesis of bioactive compounds andthat all these gene clusters are carried on the chromosome. It alsorevealed ribosomal peptide synthesis of a novel family ofcyanobactins, anacycloamines. We demonstrated the widespread (48out of 132 strains) but sporadic occurrence of the cyanobactinbiosynthetic pathway among planktonic cyanobacteria.

Conclusions: Cyanobacteria are a rich source of bioactive compoundsfor drug leads and the number of new compounds identified fromcyanobacteria is increasing continuously. Cyanobacterial bioactivecompounds may prove useful in combating various diseases in thefuture. In addition, their biosynthetic machineries (ribosomal and non-ribosomal) provide enzymes to be used in combinatorial biosynthesisor chemoenzymatic synthesis to produce novel compounds.

References:

Sivonen, K. and T. Börner. 2008. Bioactive compounds produced bycyanobacteria. In: “The cyanobacteria: Molecular Biology, Genomicsand Evolution”, Herraro, A. & E. Flores (Eds.). p. 159-197. CaisterAcademic Press, Norfolk, U. K.

Sivonen, K. 2009. Cyanobacterial toxins. In: “Encyclopedia ofMicrobiology“, 3rd Edition, edited by M. Schaechter, pp. 290-307.Oxford:Elsevier.





Notes



OC-1.1

SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETICMARKERS FOR PURPLE SULFUR BACTERIA.

Marcus Tank, Vera Thiel, Johannes F. Imhoff

IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University),Kiel, Germany.

Introduction: Purple Sulfur Bacteria (PSB) are photoautotrophicbacteria phylogenetically grouped to the order Chromatiales withinthe Gammaproteobacteria. They perform anoxygenic photosynthesisunder anaerobic conditions generally using reduced sulfur compounds(e.g. H2S, S

2-, thiosulfate) as e--donator. PSB are ubiquitouslydistributed but mainly restricted to aquatic environments containingadequate light conditions, low/no oxygen tension and moderatesulfide concentrations. pufL and pufM are essential in photosynthesisof PSB and encode for polypeptides of the photosynthetic reactioncentres which are located in intracytoplasmic membranes (ICM).

The phylogenetic relationship of purple sulfur bacteria (PSB) of theChromatiales (Gammaproteobacteria) was analysed based onphotosynthetic gene sequences of the pufL and pufM genes and theresults compared to phylogenetic trees and grouping of the 16S rRNAgene.

Methods: Primers for pufL and pufM genes were constructed andused to successfully amplify the pufLM genes of members of 16genera of Chromatiales. pufLM and 16S rRNA gene sequences of 66PSB strains, including 29 type strains and 28 new isolates, weredetermined and phylogenetically analyzed. The phylogeneticrelationships based on the functional and ribosomal genes werecompared to each other as well as to current taxonomic classification.

Results: The inferred phylogenetic trees of pufLM and 16S rRNAgenes reflect a largely similar phylogenetic development suggestingcoevolution of these essential genes within the PSB. The twofunctional genes displayed similar phylogenetic relationship of thePSB regardless of the used gene (pufL, pufM or pufLM) and of weathernucleotides or deduced amino acid sequences were used. pufLMphylogeny is in good agreement to current taxonomic classification ofPSB.

Conclusion: It is concluded that horizontal gene transfer of pufLMgenes within the PSB is highly unlikely, which contrasts the situation inother groups of anoxygenic phototrophic bacteria belonging toAlphaproteobacteria and Betaproteobacteria. A phylogeneticclassification of PSB to the genus level is possible based on their pufLor pufM sequences, in many cases even to the species level. Inaddition, our data support a correlation between Puf protein structureand the type of internal photosynthetic membranes (vesicular, lamellaror tubular).

OC-1.2

BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTINGGENES IN MARINE PHYTOPLANKTON.

Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2.1School of Ocean and Earth Sciences, National Oceanography Centre,Southampton, United Kingdom; 2School of Biological Sciences,University of Sydney, Sydney, New South Wales, Australia.

Introduction: Photosynthetic light-harvesting proteins are themechanism by which energy enters the marine ecosystem. Thedominant prokaryotic photoautotrophs are the cyanobacterial generaProchlorococcus and Synechococcus that are defined by two distinct

light-harvesting systems, chlorophyll-bound protein complexes orphycobilin-bound protein complexes, respectively. Here, we use theGlobal Ocean Sampling (GOS) Project as a unique and powerful toolto analyze the environmental diversity of photosynthetic light-harvesting genes in relation to available metadata includinggeographical location and physical and chemical environmentalparameters.

Methods: All light-harvesting gene fragments and their metadatawere obtained from the GOS database, aligned using ClustalX andclassified phylogenetically. Each sequence has a name indicative of itsgeographic location; subsequent biogeographical analysis wasperformed by correlating light-harvesting gene budgets for each GOSstation with surface chlorophyll concentration.

Results: Using the GOS data, we have mapped the biogeography oflight-harvesting genes in marine cyanobacteria on ocean-basin scalesand show that an environmental gradient exists in which chlorophyllconcentration is correlated to diversity of light-harvesting systems.Three functionally distinct types of light-harvesting genes are defined:(1) the phycobilisome (PBS) genes of Synechococcus; (2) the pcbgenes of Prochlorococcus; and (3) the iron-stress-induced (isiA) genespresent in some marine Synechococcus. At low chlorophyllconcentrations, where nutrients are limited, the Pcb-type light-harvesting system shows greater genetic diversity; whereas at highchlorophyll concentrations, where nutrients are abundant, the PBS-type light-harvesting system shows higher genetic diversity

Conclusions: We interpret this as an environmental selection ofspecific photosynthetic strategy. Importantly, the unique light-harvesting system isiA is found in the iron-limited, high-nutrientlow-chlorophyll region of the equatorial Pacific. This observationdemonstrates the ecological importance of isiA genes in enablingmarine Synechococcus to acclimate to iron limitation and suggeststhat the presence of this gene can be a natural biomarker for ironlimitation in oceanic environments.

OC-1.3

PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THECYANOBACTERIAL GENERA ANABAENOPSIS AND CYANOSPIRA.

Stefano Ventura, Cristina Mascalchi, Claudio Sili

Institute of Ecosystem Study, National Research Council, Sesto,Fiorentino, Italy.

Introduction: Planktic heterocytous cyanobacteria from tropicalalkaline environments have been studied in our group since long ago.We described the genus Cyanospira, with its two species C. rippkaeand C. capsulata, that are among the few cyanobacterial taxa whichhold a formally valid taxonomic description under the BacteriologicalCode. The existence of the genus Cyanospira and of its two speciesand its separation from the genus Anabaenopsis has been repeatedlyquestioned. To contribute to clarify the relationships between the twogenera, we present a phylogenetic analysis of Anabaenopsis andCyanospira and a detailed study of the morphology of severalundescribed strains of Cyanospira.

Methods: Strains have been isolated from natron coming from Chadthat contained large numbers of dormant akinetes. Phylogeneticanalysis has been performed using the ARB and SILVA phylogenetictools and databases, which supply a constantly updated and aligneddatabase with quality evaluation of the sequences. Morphology hasbeen studied in laboratory cultures inoculated with dormantdesiccated akinetes. The entire life cycle has been studied anddocumented, from the akinete germination to the full development ofvegetative forms.



Results: Cultures of Cyanospira, obtained from germination ofdesiccated akinetes, were characterized by variable filamentdimensions and coiling degrees. Akinete development in vegetativefilaments was typically apoheterocytic; akinetes developed in chainsthat continuously expanded along the filament; in old cultures, all cellsin the filament can be transformed into akinetes. Filaments ofCyanospira were often surrounded with a thick mucilaginous capsule.The development pattern of akinetes in Cyanospira was definitelydifferent from that of Anabaenopsis.

After a selection of sequences based on length and quality, that isinherent to the SILVA database, all publicly available sequencesidentified as belonging to the genera Anabaenopsis and Cyanospira,plus new sequences of our strains have been compared with selectedentries of Nostoc, Thricormus, Nodularia, and Azolla cyanobionts. Thephylogenetic analysis produced several well defined clusters. Theassemblage of the two genera Anabaenopsis and Cyanospira formeda phylogenetically coherent and well defined unit, mostly related tothe genus Nodularia. Inside this unit, sequences of Anabaenopsiswere subdivided into three clusters, two housing freshwater strains,and one, more apart, housing representatives of the species A.abijatae which came from mineralized (alkaline) lakes in Ethiopia.Sequences of Cyanospira formed three distinct phylogenetic clustersthat were more related to the A. abijatae cluster.

Conclusions: As already demonstrated for hypersaline environments,alkaliphilic cyanobacteria stayed apart from their freshwatercounterparts. Among the alkaliphiles, the genus Cyanospira wassubdivided into three phylogenetic subunits. The relationships of twoof these units with A. abijatae should be investigated in more detailsby means of a precise morphological study. Our data support thetaxonomic robustness of the genus Cyanospira as the alkaliphiliccounterpart of Anabaenopsis, and the existence of more than onespecies inside it.

OC-1.4

CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATSOF A TROPICAL LAGOON, TIKEHAU ATOLL, TUAMOTUARCHIPELAGO.

Katarzyna A. Pali�ska1, Raeid M. M. Abed2†, Katja Wendt1, MariaŁotocka3, Loic Charpy4 & Stejpko Golubic5.1Institute of Chemistry and Biology of the Marine Environment,Geomicrobiology Dep.; CvO University of Oldenburg, Oldenburg,Germany; 2College of Science-Biology Department, Sultan QaboosUniversity, Al-Khod, Muscat, Sultanate of Oman; 3Institute ofOceanology, Polish Academy of Sciences, Powsta�ców Warszawy,Sopot, Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167(CYROCO), Traverse de la Batterie des Lions, Marseille, France;5Biological Science Center, Boston University, Boston, MA, USA.

Introduction: Cultivation of microorganisms is known tounderestimate the real diversity of bacterial communities thrivingunder natural conditions (Amann et al., 1995; Abed et al., 2008) andthe isolates are often selected by the supplied culture conditions.However, these minor bacterial populations in the field may becomedominant in response to environmental changes or in the case ofcertain perturbation events that favor their growth. While recentstudies in microbial ecology focuses on identifying dominant fieldbacterial populations using culture-independent molecular tools, withthe assumption that they are responsible for most bacterial activities,minor populations are often ignored. In this study, we investigated thediversity of opportunistic cyanobacteria that respond to the high loadof nutrients provided by the cultivation medium. We postulate that

these autochthonous assemblages of taxa are of ecologicalimportance.

Methods: Twelve strains of filamentous and unicellular cyanobacteriawere isolated from microbial mats of the lagoon of Tikahau atoll,Tuamotu Archipelago, the most oligotrophic Pacific waters; identifiedmorphologically and using 16S rRNA-based phylogenetic analysis.Taxonomic identity of the studied strains was further supported byobtaining CpcBA-IGS sequences. Cultures were isolated from naturalpopulations growing on the bottom of the lagoon formed ofcalcareous sand from the surface to a depth of ca. 20m. Weinvestigated strains with respect to their pigmentation, as well as theirpotential to perform chromatic adaptation, and nitrogen fixation.

Results: Using direct microscopy, 12 different cyanobacterialmorphotypes were identified. They were classified in sixmorphogenera sensu Geitler (1932) and Anagnostidis & Komarek(1988): Cyanocystis, Leptolyngbya, Aphanothece, Phormidium,Chlorogloea and Pseudanabaena. The genera Cyanocystis andChlorogloea were genotypically characterized for the first time Most ofthe strains posses’ phycoerythrin and have the ability to fix nitrogen..Non-heterocystous cyanobacteria with narrow trichomes wereclassified under Leptolyngbya and Phormidium following botanicalrecommendation, but homogeneity of their grouping was notgenotypically confirmed.

Conclusions: The strains represent autochthonous cryptic diversity,which dominated cultures but not the natural populations. Taxastudied here clearly responded to higher nutrient supply provided bythe standard artificial medium, which may simulate conditions innature following the impact of tropical storms, thus recruitingopportunistic taxa, which are normally present at low frequencies.They may be considered important members of microbial successionsduring restoration of the ecosystem following catastrophic impacts.With increasing man-made disturbances, such as eutrophication,pollution or dredging, it is expected that these taxa will increase inabundance.

While recent studies in microbial ecology focuses on identifyingdominant field bacterial populations using culture-independentmolecular tools, with the assumption that they are responsible formost bacterial activities, minor populations are often ignored. Wepresume that these less-dominant populations are equally importantand can only be identified by enrichment cultivation simulatingpossible environmental perturbations and changes.

OC-1.5

METAGENOMIC AND PHYLOGENETIC ANALYSES OFCYANOBACTERIAL MATS IN EXTREME HIGH ARCTICENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICALFUNCTION.

Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3,Warwick F. Vincent1.1Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté deMédecine, Université Laval; 3Québec-Océan, Département deBiologie, and Institut de biologie intégrative et des systèmes (IBIS);Université Laval, Québec, QC, Canada.

Introduction: Mat-forming cyanobacteria are among the oldest knownprokaryotes and are widely distributed in more extreme environments,and in environments where grazing pressure is reduced or absent. Thephysical form of the cyanobacteria living within the mats and the threedimensional structure of mats themselves is much conserved withfossils dating from more than 2 billion years. However given the wide



variety of habitats where mats tend to be abundant, a major questionis whether such morphologically similar structures harbour similarspecies and what particular genetic adaptations or metabolicpathways may be present in different environments.

Polar aquatic ecosystems are dominated by microbes and abundantmat-forming cyanobacteria are widely distributed in shallow lakes,streams and meltwater ponds on ice shelves of the High Arctic. Duringsummer, oxygenic photosynthesis by cyanobacteria is the primaryenergy source supporting a complex community within the mats. Incontrast to physically similar mats from other habitats, such as hotsprings, there is relatively little data on the taxonomic and functionaldiversity of these polar cyanobacteria-dominated mat communities.Without such information, questions on the evolutionary persistenceof cyanobacteria mats during both warming and glacial epochs, andon their global biogeography cannot be addressed. Therefore, wecharacterised High Arctic microbial mats using metagenomics touncover adaptive functional genes and targeted 16S rRNA genelibraries and phylogenetics to determine cyanobacterial diversity.

Methods: Microbial mats were investigated from freshwaterecosystems of different nutrient and physical regimes in the CanadianHigh Arctic. Microbial community analysis was performed usingpyrosequencing and bioinformatic analysis. The phototrophic diversitywas determined using signature pigment analysis. Cyanobacterialdiversity was targeted using strain isolation, and culture independentmethods including microscopic examination, and phylogeneticanalyses based on 16S rRNA gene clone-libraries.

Results: High Arctic microbial mats had chlorophyll a values rangingfrom 3.9 to 46.2 µg cm-2 with high concentrations of cyanobacterialpigments such as scytonemin. Metagenomic analyses of two microbialmat communities from ice shelves identified Cyanobacteria andProteobacteria as dominant bacterial groups. Other bacteria, Archaea,Eukaryota and viruses were also identified. A high proportion of geneswere implicated in metabolic pathways of major biogeochemicalcycles with evidence of specific molecular adaptations to thecryosphere.

Cyanobacterial diversity consisted of phylotypes withinChroococcales, Oscillatoriales, Nostocales and Stigonematales basedon morphological and 16S rRNA gene analyses. Phylotypes within theorders Chroococales and Nostocales had highest similarity totemperate environments, whereas the majority of oscillatorians hadclosest similarity (>99) to Antarctic and alpine sequences, including totaxa previously considered to be endemic to the Antarctica.

Conclusions: High Arctic microbial mats contain diverse assemblagesof microbes that perform an ensemble of biogeochemical cyclingprocesses, similar to cyanobacteria-dominated mats from otherclimate zones. Our results also imply that low-temperaturecyanobacterial ecotypes have a cosmopolitan distribution throughoutthe cold terrestrial biosphere.

OC-1.6

GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENONSPP. AND ANABAENA SPP. IN NORTHEAST GERMAN LAKES.

Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1.1Leibniz-Institute of Freshwater Ecology and Inland Fisheries,Department of Limnology of Stratified Lakes, Neuglobsow; 2FederalEnvironmental Agency, Berlin; 3Brandenburg Technical University,Department of Freshwater Conservation, Bad Saarow; Germany.

Introduction: In the last decades hepatotoxic cylindrospermopsin(CYN) and neurotoxic saxitoxins (STX) were detected in several lakes in

northeast Germany. CYN and STX are produced worldwide bymembers of the nostocalean genera Anabaena, Cylindrospermopsis,or Aphanizomenon. So far only Aphanizomenon sp. was confirmed asproducer of CYN in two German lakes, while the source of STX is notyet identified in German water bodies. In this study we attempted toidentify STX- and CYN producers in German water bodies andcharacterized them morphologically, genetically, and biochemically incomparison to non toxin producing strains.

Methods: In 2007 and 2008, 200 strains of Aphanizomenon spp.,Anabaena spp., and Anabaenopsis spp. from 8 selected lakes inBrandenburg, Germany were isolated and cultivated. The isolatedstrains were identified morphologically using light microscopy andgenetically using different genetic markers (cpcBA-IGS and RBCL).Primers were designed for a fast detection of the sxtA part of the STXgene cluster. For the detection of the CYN gene cluster primers toamplify the peptide synthetase (PS) gene were used. To confirmpotential STX and CYN producers ELISA as a biochemical method andliquid chromatographic tandem (LC-MS/MS) methods were applied.

Results: The isolated strains were assigned morphologically to fourdifferent species of Aphanizomenon and four different species ofAnabaena and one Anabaenopsis species. Our sequence data andphylogenetic analyses confirmed the formation of intermixed clustersby planktic Anabaena and Aphanizomenon strains. Aphanizomenongracile and Aphanizomenon flos-aquae could not be divided asseparate species. Using polymerase chain reaction (PCR), 14 strains ofAphanizomenon cf. gracile, three strains of Aphanizomenonissatschenkoi, and one strain of Anabaenopsis sp. were identified aspotential STX producers due to the possession of fragments of thesxtA gene. The ability to produce STX was confirmed with ELISA andLC-MS for the 14 strains of Aphanizomenon cf. gracile. Each strainproduced 6 STX variants in variable amounts The STX concentrationsfound were between 400 and 1300 µg/g cyanobacterial dry weight.No CYN producer was detected using genetical methods and ELISA.

Conclusions: This is the first study to confirm Aphanizomenon cf.gracile as saxitoxin producer in German water bodies. As strains ofAphanizomenon issatschenkoi and Anabaenopsis sp. also possessedfragments of the STX gene cluster other nostocalean producers of STXare very likely in German water bodies.

OC-2.1

MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS INPHOTOTROPHIC CONSORTIA.

Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller.

Section Microbiology, Department Biology I, University of Munich,Planegg-Martinsried, Germany.

Introduction: Phototrophic consortia represent the most highlydeveloped bacterial symbiosis and consist of green sulfur bacterialepibionts surrounding a central chemotrophic Betaproteobacterium. Arapid and specific exchange of multiple signals occurs between bothbacterial partners and allows tactic behaviour towards light andchemical stimuli. Laboratory cultures of the consortium“Chlorochromatium aggregatum“ as well as of the isolated epibionthave recently been established, providing the opportunity to elucidatethe molecular mechanisms underlying the symbiotic interaction.

Methods: Subcellular structures were studied by high resolutionanalytical scanning electron and transmission electron microscopy.Putative symbiosis genes of the epibiont were identified bysuppression subtractive hybridization of genomic DNA against that of16 relatives and bioinformatics. Transcription was studied by reverse



transcriptase-PCR. Cross-linking experiments were conducted withbis(sulfosuccinimidyl)suberate or 3,3´-dithiobis(sulfosuccinimidylpropionate).

Results: In “C. aggregatum”, numerous periplasmic tubules extendfrom the outer membrane of the central bacterium and are in directcontact to the epibiont. Our search for specific recognition and signaltransduction proteins identified a total of 189 genes of the epibiont of“C. aggregatum“ to be absent in all other green sulfur bacteria. Mostnoticable was the presence of four genes which were related tovirulence factors of human or plant pathogenic bacteria. These geneswere transcribed constitutively in the epibiont. The hemagglutinin-likeputative gene products of ORFs Cag0614 and Cag0616 probablyarose by gene duplication and represent the largest bacterial openreading frames so far documented for bacteria. Cag1920 codes for aputative hemolysin whereas the gene product of Cag1919 is aputative repeat in toxin (RTX)-like protein containing a Ca2+- bindingbeta roll motif. In fact, intact consortia disaggregated upon theaddition of EGTA. The RTX-type C-terminus coded by Cag1919exhibits a significant similarity to RTX modules of variousproteobacterial proteins, suggesting that this putative symbiosis genehas been acquired via horizontal gene transfer from aproteobacterium. This conclusion is further supported by the presenceof three genes encoding transposases which are located immediatelyupstream of ORF 1919. Most recently, cross-linking experiments ofintact consortia revealed the presence of a corresponding filamentoushemagglutinin/adhesin in the central bacterium which appears to beinvolved in maintaining the multicellular structure of the phototrophicconsortium.

Conclusions: While the adaptation of green sulfur bacteria to asymbiotic lifestyle in phototrophic consortia requires only acomparatively low number of niche-specific genes, it involves severalunique genes which most likely were acquired by lateral gene transferfrom a proteobacterium. Our study of the molecular mechanisms ofsymbiosis indicates that typical bacterial virulence factors have beenexploited by green sulfur bacteria to establish a mutualistic interactionand enabled these bacteria to occupy a novel ecological niche.

OC-2.2

LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBICANOXYGENIC PHOTOTROPHS IN A SEMIARID REALM.

J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov.

Department of Microbiology, University of Manitoba, Winnipeg, MB,Canada.

The discovery of aerobic anoxygenic phototrophs from biological soilcrusts (BSC) is reported. Living on an ecological knife-edge ofhabitability, BSC are drought-tolerant communities composedprimarily of microorganisms that form a cohesive, erosion-resistantveneer on the surface of soil. These communities inhabit semiarid toarid soils and are composed of cyanobacteria, bacteria, fungi andmosses. They are receiving increased attention from a landmanagement perspective because they reduce soil erosion andenhance moisture and nutrient status. From sandy soils (including sanddunes) in Manitoba and Alberta, Canada, 21 strains were isolated thatpossessed the diagnostic light harvesting pigment,bacteriochlorophyll. Anoxygenic phototrophs constituted 0.1 to 5.9%of the cultivable biological soil crust bacterial community. Sequencingthe 16S rRNA gene of 16 isolates revealed that nine strains wereclosely related to the genus Methylobacterium, while seven fell withinthe alpha-1- and alpha-4-proteobacteria, related to typical aerobicanoxygenic phototrophs such as Paracraurococcus, and to soil crust

heterotrophs such as Belnapia. This study is the first demonstration ofproteobacterial anoxygenic phototrophs from semiarid environments.The presence of anoxygenic phototrophs in BSC implies a higherefficiency of light harvesting by soil curst organisms than previouslyrealized. Utility of near-IR radiation not used by oxygenic phototrophsfacilitates light-accelerated turnover of soil organic carbon content.

OC-2.3

ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MAT-NITROGEN FIXATION.

Ina Severin, Lucas J. Stal.

Department of Marine Microbiology, NIOO-Center for Marine andEstuarine Ecology, Yerseke, The Netherlands.

Microbial mats on intertidal beaches and sand flats are often pioneersystems. Following their establishment, colonization by higher plantsoccurs and rigorously changes the morphodynamics of the ecosystem.One perquisite for this succession is the enrichment of the sedimentwith combined nitrogen as the result of N2 fixation. Cyanobacteria areusually the most conspicuous structural part of microbial mats. Theseorganisms have long been the focus of research on N2 fixation inmicrobial mats. However, there is growing evidence thatCyanobacteria are sometimes minor players in terms of N2 fixation.

We investigated a coastal microbial mat with regard to N2 fixationusing the acetylene reduction assay as well as with regard to thediversity of the 16S rRNA and nifH genes and transcripts. QuantitativeRT-PCR was used to access daily changes of nifH-gene expression ofthe dominant phylotypes and groups as well as the whole matcommunity.

The daily patterns of nitrogenase activity differed with respect to thepoint of time when the maximum was reached. The mat higher up inthe littoral zone (station I) showed several nitrogenase activity-maximabetween sunset and sunrise and harboured a variety of heterocystousand non-heterocystous Cyanobacteria. The mat situated close to thelow water mark (station II) was mainly composed of non-heterocystousCyanobacteria with Lyngbya aestuarii being the most dominantdiazotroph. Accordingly, a night time-maximum of nitrogenase activitywas recorded. A clear light dependency of nitrogenase activity impliedphototrophs as main diazotrophs. 16S rRNA gene clone librariesshowed that both microbial mats were dominated by Cyanobacteriaand Proteobacteria. Filamentous non-heterocystous Cyanobacteriaand Gammaproteobacteria were predominant at both stations but therelative contribution of taxa differed. Analysis of the nifH gene clonelibraries confirmed that Cyanobacteria were the dominant diazotrophsin both communities. In accordance with 16S rRNA gene clonelibraries, Gammaproteobacteria-related nifH sequences were mostfrequently retrieved at station I whereas the delta-subdivision made upfor almost all Proteobacteria-related nifH sequences at station II. Theanalysis of nifH transcripts of station I revealed a dynamic communitywith changing relative abundances of nifH transcripts of members ofthe community over time. While Cyanobacteria-related sequenceswere predominant from midday to midnight, Proteobacteria-relatednifH transcripts were found to become more important from midnighton. A high proportion of these proteobacterial sequences were mostclosely related to known anoxygenic phototrophs. Relativecontribution of sequences to cDNA clone libraries differedconsiderably from those observed for DNA clone libraries,highlighting active diazotrophs. Gene expression levels yielded furtherinsight into diel nifH gene expression patterns of key diazotrophs incomparison to whole mat community nitrogenase activity. The resultssuggest that species composition affects the maximum attainable



nitrogenase activity and its diel pattern. Oxygenic as well asanoxygenic phototrophs were found to be the key players in N2

fixation in the investigated microbial mats.

OC-2.4

BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCEDPHOTOSYNTHETIC MICROBES.

Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer.

SRI International, Menlo Park, CA, USA.

The BioCyc [1] collection of 409 Pathway/Genome Databases (PGDBs)is available at URL BioCyc.org. BioCyc includes the genomes, andpredicted metabolic networks and operons of eleven photosyntheticmicrobes. BioCyc also includes the EcoCyc [2] database (DB), whichdescribes the metabolic and genetic regulatory network of Escherichiacoli; and the MetaCyc DB, which describes more than 1200 metabolicpathways that were experimentally elucidated in more than 1,600organisms.

BioCyc contains PGDBs for most organisms with completelysequenced genomes. Each BioCyc PGDB combines the annotatedgenome sequenced with predicted metabolic pathways, predictions ofpathway hole fillers, and operon predictions. BioCyc is generated ona regular ongoing basis using the MetaCyc reference database ofexperimentally elucidated pathways.

BioCyc offers many capabilities to researchers for analysis andinvestigation of genomes, metabolic networks, and regulatorynetworks, and for analysis of high-throughput datasets. A genomebrowser allows exploration of a microbial genomes, and comparisonof conserved genome regions.

The predicted metabolic network of each organism can be queriedand visualized using a variety of bioinformatics tools that displayinformation about metabolites, enzymes, reactions, and individualpathways. Computational tools for analysis of metabolic networksinclude tools for finding dead-end metabolites, and for performingreachability analysis of metabolic networks. In addition, each PGDBprovides a diagram of the complete metabolic map of the organism,and of the complete regulatory network when it has been curated forthat organism. These tools support analysis of large-scale omicsdatasets [3], such as gene-expression and metabolomics data, bypainting those data onto the full metabolic map diagram andregulatory network diagram to place omics data within a biologicalcontext. A toolkit of comparative analysis tools permits comparisonsof the genome and biochemical networks of multiplePathway/Genome Databases.

BioCyc can be accessed at BioCyc.org, as a set of downloadable datafiles, and as an installable software distribution. The BioCyc.org Website and data files are freely available to all; the downloadablesoftware is freely available to research institutions.

[1] R. Caspi et al, “The MetaCyc Database of Metabolic Pathways andEnzymes and the BioCyc Collection of Pathway/GenomeDatabases,” Nucleic Acids Research 36:D623-31 2008.

[2] Karp, P.D. et al, “Multidimensional annotation of the Escherichiacoli K-12 genome,” Nucleic Acids Research 35:7577:90 2007.

[3] Paley, S.M. et al, “The Pathway Tools Cellular Overview Diagramand Omics Viewer,” Nucleic Acids Research 34:3771-8, 2006.

OC-2.5

DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACEWATERS OF MARINE AND FRESHWATER ENVIRONMENTS WITHAN EMPHASIS ON CYANOBACTERIA.

Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, TorstenStaller2, Rüdiger Schulz2, Jens Appel1.1School of Life Sciences, Arizona State University, Tempe, AZ, USA;2Botanisches Institut, Universität Kiel, Kiel, Germany.

Introduction: After methane hydrogen is the second most abundanttrace gas in the atmosphere, making up around 0.5 ppm to 0.6 ppm.Surface waters of different aquatic environments have been shown toevolve hydrogen. The ocean is estimated to be the major naturalsource of H2. Still its origin and the proportion of biological and non-biological production and consumption processes are unknown.

Hydrogen concentrations of surface waters above the expectedequilibrium prompted us to investigate the presence and distributionof all known hydrogenases in marine and fresh water environmentswith a special emphasis on cyanobacteria.

Methods: DNA was isolated from samples from the North Sea, theBaltic Sea, the North Atlantic, the Mediterranean Sea and two Germanlakes (Westensee and Selenter See). Degenerated primers were usedto amplify part of the gene of the large subunit of the bidirectionalNAD(P)-linked hydrogenase (hoxH) from these samples. In additionthe available genomes of cyanobacteria, marine bacteria and the GOSdatabase were searched for the presence of all the knownhydrogenase genes.

Results: The search of the cyanobacterial genomes confirms that theiruptake hydrogenase (hupL) is strictly linked to the nitrogenase genes.This is not surprising since it is used to recycle the hydrogen inevitablyproduced by the nitrogenase in each catalytic cycle. But neverthelessthere are some strains that do have the nitrogenase genes but nouptake hydrogenase indicating that the hydrogenase is not essentialto compete and might not be necessary under all environmentalconditions.

The distribution of the hoxH sequences in the complete genomes aswell as in the different samples analyzed indicates a strong bias tofreshwater environments and coastal waters. It seems to be absent inthe open ocean, which is confirmed by the database searches also inother marine bacterial genomes. Therefore, the presence of thishydrogenase is especially linked to the probability to encounteranaerobiosis.

In approximately 20 % of all marine genomes hydrogenases werefound. The presence of H2-sensing hydrogenases in photosyntheticbacteria such as Roseovarius strains and other marine bacteriasuggests that H2 triggers the expression of the structural genes of theiruptake hydrogenases.

Conclusions: Our results show that in marine waters all the geneticcomplement is present for biological hydrogen production as well asuptake. Therefore, biological processes alone might be responsible forthe measured H2 turnover in these habitats. The sequences foundsuggest that hydrogen might be used as an additional energy sourceby marine bacteria in nutrient-limited environments. In addition, ourdata supports the conclusion that the bidirectional NAD(P) linkedhydrogenase is especially needed for adaptation to rapidly changingredox conditions and unnecessary in the open ocean.



OC-2.6

HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRINGMICROBIAL MATS DOMINATED BY THE FILAMENTOUSANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUSAGGREGANS.

H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1.1Department of Biology, Tokyo Metropolitan University, Tokyo;2Institute for Biological Resources and Functions, National Institute ofAdvanced Industrial Science and Technology (AIST), Ibaraki; Japan.

Introduction: The filamentous anoxygenic phototrophic bacteriaChloroflexus aggregans and C. aurantiacus are often present in hotsprings dominated by cyanobacteria. In some hot springs however,there are distinctive microbial mats that lack cyanobacteria and areinstead dominated by Chloroflexus spp. At Nakabusa hot spring(Nagano, Japan) at 65°C, C. aggregans develops mats by growingautotrophically using hydrogen sulfide as an electron donor forphotosynthesis. These mats also contain a thermophilic sulfate-reducing bacterium, Thermodesulfobacterium sp., which produceshydrogen sulfide. It is known that this sulfate-reducing bacterium canutilize hydrogen, but it is still unknown whether this bacteriumconsumes hydrogen in the mats or whether biological hydrogenproduction even occurs in this community. In this study, weinvestigated net hydrogen production in the 65°C microbial mats atNakabusa.

Methods: Samples of these mats, mainly composed of C. aggregans,were collected and hydrogen production was measured in artificialhot-spring water at 65°C under various treatments in anaerobicbottles.

Results: In the absence of any treatment, hydrogen was not detected,but significant production was observed in the presence of molybdate,an inhibitor of sulfate reduction or with a homogenization treatment ofthe mat. This net hydrogen production largely decreased underillumination. These results suggest that the metabolism resulting inhydrogen production was not photosynthetic; we hypothesize thathydrogen was produced by fermentative bacteria breaking downorganic compounds supplied by anoxygenic photosynthetic bacteria.This hydrogen was then consumed by sulfate-reducing bacteria asthey reduced sulfate to sulfide. The decrease of net hydrogenproduction under illumination can be explained by the anoxygenicphotosynthetic bacteria using hydrogen as an electron donor orcompeting with fermentative bacteria for organic compounds.

Conclusions: Hydrogen was shown to be produced and consumedwithin the mats, and net hydrogen production could not be detectedwithout the inhibition of sulfate reduction or physical blocking ofhydrogen consumption. In the 65°C mats at Nakabusa, anoxygenicphotosynthetic bacteria produce organic compounds using hydrogensulfide as an electron donor. Fermentative bacteria use this carbon asan energy source and release hydrogen as a byproduct. This hydrogenis then used by sulfate-reducing bacteria that produce hydrogensulfide. Thus, electrons cycle via organic compounds, hydrogen andhydrogen sulfide in the community.

OC-3.1

CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROMTHE HALOCLINE OF THE SALINE MEROMICTIC LAKE, LAKESUIGETSU, JAPAN.

Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya.

Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui,Japan.

Introduction: The surface layer of Lake Suigetsu (35�35’N, 135�52’E,coast of the Japan Sea in Fukui Prefecture, Japan) has O2-containingfresh water, while the deeper layer is anoxic salt water where apermanent halocline (salinity is ca.0.4 to 1.4%) is created betweenthese two water layers. The concentration of O2 decreases to zero atabout the middle of the halocline; and H2S, a potent inhibitor ofoxygenic photosynthesis, is detected deeper below the oxic-anoxicboundary layer (OABL). Only weak green light (less than 1% of surfaceintensity) penetrates to the OABL. We found small size cyanobacteria(<2 μm, PCy) were distributed through the year within the halocline asthe dominant phytoplankton (0.5 to 5×105 cells/ml) during four years2005 - 2008. The attempt to determine the adaptation mechanism(s)of PCy to their habitat, phylogenetic and physiologic properties ofPCy were studied using isolated strains from the halocline of LakeSuigetsu.

Methods: Water samples were collected from the different depthswithin the halocline of Lake Suigetsu during the summer of 2005-6.Unicyanobacterial isolates were obtained by isolating colonies on agarplates using various salinities in artificial media (0.4 to 1.6 %).Morphology observations were performed using epifluorescence andelectron microscope. Phycobiliprotein (PB) compositions weredetermined spectrophotometrically using intact cells and/or isolatedphycobilisomes. The most part of the 16S rRNA and the internaltranscribed spacer (ITS) between 16S and 23S rRNA genes weresequenced from the genomic DNA of the isolated PCy; and molecularphylogenetic trees were constructed using the neighbor-joiningmethod.

Results: C-phycoerythrin (R-type) or C-phycocyanin (G-type) was themajor PBs. The isolated clones (about 100) were grouped into six sub-groups using the PB composition and sequence homology of the ITSbetween the 16S and 23S rRNA genes. Further analyses wereperformed using six clones from the different sub-groups (three R-types and three G-types). The six clones were included within thefreshwater Synechococcus-Cyanobium clade, but they were notmonophyletic in the 16S rRNA-based tree. Cells were spherical orellipsoidal at 0.5 to 1.5 μm in diameter or width and reproduced usingtransverse binary fission in a single plane. The thylakoids wereperipheral and were oriented parallel to the cytoplasmic membrane.They were able to grow in a wide range of salinities (0.2 to 2 % ormore). Significant growth was observed under weak green light (2μmol·m-2·sec-1, ca. 460 to 600 nm). Whereas, the cells were bleachedirreversibly under the white light at relatively high intensities (≥15μmol·m-2·sec-1 for the R-type; ≥25 μmol·m-2·sec-1 for the G-type). Atleast four clones survived in medium containing sulfide (0.3 to 1.2 mMas S2-, pH7.6) for more than a week.

Conclusion: The physiological properties found in this study show PCysurvive under unique physicochemical environments in the halocline ofthe saline meromictic lake, Lake Suigetsu. The ability to use weakgreen light for photosynthesis and sulfide tolerance may provide anadvantage over other phytoplankton under sulfide-rich environments.The present phylogenetic analyses suggested this ability was acquiredby PCy in Lake Suigetsu more than once.



OC-3.2

THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIAPROCHLOROCOCCUS.

Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA.

The marine cyanobacteria Prochlorococcus is thought to be the mostnumerically abundant phototropic prokaryote in the global oceans andas such it plays a critical role in the ecology and biogeochemistry ofmarine ecosystems. Recently we have completed several global oceansurveys covering vast oceanic regions where we measured theabundance and photosynthesis (primary production) of this importantphototrophic organism. We find that over these regions, theintegrated water column abundance of Prochlorococcus is remarkablyconserved, in spite of significant variability in the vertical distributions.Further, although these surveys covered many oceanographicbiogeochemical provinces, each with unique nutrient, temperatureand other environmental variables, the contribution ofProchlorococcus to primary production remains highly conserved andranges between 25 – 50% of the total production rates. Quantitativephylogenetic analyses using clade specific qPCR shows that while thetaxonomic composition of Prochlorococcus can change dramatically inresponse to these environmental variables (and in particulartemperature), the integrated abundance and production of the genusremains relatively consistent. Together, these global abundance andprimary production estimates suggest that the Prochlorococcus nicheis more consistent and larger than previously thought.

OC-3.3

THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTALCYANOBACTERIA COMMUNITY AND ITS CONTRIBUTION TOTOXICITY IN A TROPICAL BRAZILIAN RESERVOIR.

Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos.

Department of Botany, Institute of Biological Sciences, UniversidadeFederal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.

Introduction: Autotrophic picoplankton (APP) represents an importantpart of the planktonic communities in aquatic systems and may beresponsible for a large amount of carbon fixation. Even though theseorganisms are widespread and abundant, in the past their populationswere often not recognized because of their diminutive size (0.2-2 µm).A large part of the picoplanktonic community is represented bycyanobacteria. The importance of picocyanobacteria for globalprimary production and biomass has received less attention infreshwater than in marine systems. The development of moleculartechniques opened new possibilities for the study of the diversity ofthese organisms and even their quantification. In the present work wepresent results obtained from a large mostly oligotrophic reservoirlocated in Southeastern Brazil, Furnas Reservoir.

Methods: Samples were taken regularly over a two years period,concentrated on glass fibre filters and frozen until extraction. Samplesfor phytoplankton counting were preserved unfiltered with a Lugol’siodine solution. Extraction for molecular analyses were performed bythe phenol-chloroform technique. Extracts were analyzed byconventional PCR and real-time quantitative PCR (Step One AppliedBiosystem). Primers were designed aimed at the detection of the cpcBregion (total cyanobacteria) and mcyD (toxic cyanobacteria) genes innatural populations. New primers were designed for the q-PCR. Somesamples were selected for the investigation of cyanobacterial diversityby the technique of “Amplified Ribosomal DNA Restriction Analysis”(ARDRA) using primers for amplification of part of the 16S rRNA geneand the entire internal transcribed spacer (ITS 16S-23S), and the

analyses of sequences obtained from part of the 16S rRNA gene.

Results: Results from q-PCR revelead the importance and thenumerical dominance of picocyanobacteria in the overallcyanobacteria community in the reservoir but not a sgnificantcontribution in its toxicity. The percent of mcyD genes copies relativeto cpc-B varied between 0.1 to 1.6 % but increased up to 55% whenpicoplanktonic cells were eliminated. The analyses of ARDRA patternsshowed that although eighteen patterns were found, fifteen of themwere related with the sequences of just two picoplanktonic genera(Cyanobium and Synechococcus).

Conclusions: Our results suggest that the cyanobacterial APPcommunity can be extremely important in tropical freshwater systemsand may represent a significant contribution in the plankton diversity.Further development of the genomic approach to photosyntheticpicoplankton will lead to new understanding of their roles in warmoligotrophic systems.

OC-4.1

ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUMTEPIDUM: GENES, METABOLITES AND LINKAGES TO LIGHTHARVESTING.

Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan,Rachael Morgan-Kiss.

College of Marine and Earth Studies and DBI, Newark, DE, USA.

Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is amodel green sulfur bacterium for studying light harvesting inphototrophs that utilize chlorosomes as the major antenna complexand pathways of anaerobic sulfur oxidation. Current studies arefocused on delineating pathways of anaerobic sulfur oxidation,including required gene products and metabolites, and how thesepathways interact with global physiology in this organism.

Methods: C. tepidum is capable of phototrophic growth with sulfideor thiosulfate as the sole electron donor or with both substrates incombination. This flexibility enabled the genetic dissection of sulfuroxidation pathways by targeted transposon mutagenesis, randomtransposon mutagenesis, and the addition of His-tags to specificgenes in the C. tepidum chromosome. The resulting mutant strainsand wild type grown under various sulfur, light and temperatureregimes were analyzed by techniques of photobiology and a methodfor detecting and characterizing thiol metabolites.

Results: C. tepidum mutant strains were recovered that implicatedpreviously unsuspected gene products in thiosulfate oxidation andclearly showed that two of three sulfide:quinone oxidoreductase (SQR)homologs display in vitro SQR activity and are required for C. tepidumto grow on high concentrations of sulfide. Furthermore, mutant strainsand the wild type grown under different conditions displayed alteredchlorosome properties and altered energy transfer between thechlorosome and reaction center. Analysis of thiol metabolites revealedthat C. tepidum contains structurally novel thiols and that thiol poolsizes are significantly altered in mutant strains defective for sulfuroxidation and in the wild type grown under a variety of conditions.

Conclusions: Taken together, the results suggest that C. tepidum iscapable of a range of responses to altered environmental andphysiological states. These responses presumably allow C. tepidum tocoordinately regulate and balance fluxes of light energy and reducingpower in order to grow under a wider variety of conditions than wasappreciated when it was first isolated. Furthermore, it appears thateven a small genome can encode metabolic surprises as evidenced bynovel thiol metabolites that we hypothesize are integral to bothregulatory and anaerobic sulfur oxidation pathways.



OC-4.2

CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS.

Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E.Blankenship1.1Departments of Biology and Chemistry; 2Department of Energy,Environment and Chemical Engineering; Washington University, St.Louis, MO, USA.

Introduction: Aerobic anoxygenic phototrophs (AAPs) are the onlyknown organisms performing photosynthesis requiring oxygen but notproducing oxygen. The AAPs are potentially major contributors toglobal carbon metabolism as they make up at least 10% of themicrobial community in some euphotic upper ocean. Previous geneticanalysis indicated the lack of the key genes required for the Calvincycle, and physiological characterization suggested the requirement oforganic carbon for Roseobacter denitrificans (R. denitrificans) growth.It is not clear how the R. denitirificans (and other AAPs) can assimilatecarbon for the formation of the cellular components withoutautotrophic growth.

Methods: We optimized the growth conditions of R. denitrificans withdifferent defined carbon sources, and used metabolomic approaches,biochemical studies, and gene expression profiles to investigate theorganic carbon metabolism, organic carbon and CO2 incorporationrate, and the carbon assimilation pathway(s).

Results:We have investigated the R. denitrificans growth with variousdefined carbon sources in different growth conditions. The optimizedgrowth conditions without non-specific carbon sources (such as yeastextract) are critical to minimize the background for the followingmetabolomic studies. The spectra of the LHII antenna complex andLHI-RC complex for the cultures with either pyruvate or glucose as thesole carbon source are very similar to those of the cultures grown inthe rich growth medium. Metabolomic studies to probe the proposedcarbon assimilation pathway in R. denitrificans with [1-13C]-labeledpyruvate, [1-13C]-labeled glucose, or 13C-labeled sodium bicarbonatewere carried out. The N-(tert-butyldimethylsilyl)-N-methyl-trifluoro-acetamide was used to derivatize amino acids for gaschromatography/mass spectrometry analysis. We have detected verylow 13C-incorporation level of leucine and isoleucine and identified theCO2 assimilation to glutamate and proline. About 70% of alanine andvaline are 13C-labeled. The incorporation rate of pyruvate, glucose, orCO2 was estimated with various biochemical approaches. Moreover,we used gene expression studies to identify the transcriptional level ofgenes encoding four anaplerotic carbon fixation enzymes in R.denitrificans under various growth conditions. In all of the conditionswe tested, the transcript level is higher for malic enzyme (RD1_0421)and PEP carboxykinase (RD1_1376) than for pyruvate carboxylase(RD1_3376) and PEP carboxylase (RD1_4248), and different geneexpression profiles for the anaplerotic carbon fixation enzymes areobserved.

Conclusions: We have successfully grown the R. denitrificans witheither pyruvate or glucose as the sole carbon source, without theaddition of yeast extract as previous literature reported. Ourmetabolomic studies confirm the biosynthesis of leucine andisoleucine, in which the citramalate pathway is favorable compare toderive from theronine for isoleucine biosynthesis, and incorporation ofCO2 to oxaloacetate is suggested. The incorporation of pyruvate ismore efficient than that of CO2. With the studies of the metabolomic(relatively low 13C-incorporation on several amino acids), biochemicaland gene expression profiling, a working hypothesis on the carbonassimilation pathway in R. denitrificans is presented herein.

OC-4.3

STRUCTURAL AND FUNCTIONAL STUDIES OF APHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGHCOMPARATIVE METATRANSCRIPTOME ANALYSIS.

Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt,Lynn Tomsho, Stephan C. Schuster, David M. Ward, Donald A. Bryant.

The Pennsylvania State University, University Park, PA, USA.

Introduction: The microbial mats in alkaline siliceous hot springs inYellowstone National Park are model systems for microbial communitystudies, especially communities constructed by photosyntheticprokaryotes. Recently developed metatranscriptome analyses usinghigh-throughput sequencing technology have revealed valuableinformation about the composition, in situ gene expression activitiesand gene regulation behavior of members of this microbialcommunity.

Methods: Samples of the photosynthetic microbial mat at MushroomSpring in Yellowstone National Park at 61-64 °C were collected on 10-11 July 2008 at four different time points in one 24-h period: at sunset(2100 h), prior to sunrise (0515 h), in dim light after sunrise (0640 h)and shortly after full illumination of the mat (0840 h). Total RNAs wereextracted from these samples and were reverse transcribed intocDNAs. Reads generated from 454-pyrosequencing of the cDNAswere searched by blastN against reference genomes and databases.Taxonomic composition information was obtained by analysis of rRNAreads while analyses of mRNA reads revealed functional characteristicsand regulatory patterns.

Results: Pyrosequencing generated 409,567 reads, totaling94,692,205 basepairs for the four samples. For each sample, from 90to 95 percent of the reads were derived from rRNA, most of whichcould be assigned to different taxa. Ribosomal RNAs related to sixchlorophototrophic microbial sources, Roseiflexus sp. RS-1,Synechococcus sp. JA-2-3B’a, Synechococcus sp. JA-3-3Ab, one ormore Chlorobi species, Chloroacidobacterium thermophilum and aChloroflexus species, accounted for nearly 90% of the total rRNAreads. The fact that the composition of this microbial community is sowell defined, and that reference genomes of the major communitymembers are available, allowed an in-depth survey of the 3.5 to 8percent of reads corresponding to mRNA reads. Comparative analysesuncovered not only which genes of the major species are most activelytranscribed, but also how they are regulated at light transition periods.This information sheds new light on the metabolism of thechlorophototrophic microbes in the community and their interactions.Insights on other components of the mat will also be discussed.

Conclusions: This study vastly improves our understanding of a modelphotosynthetic microbial community because it provides acomprehensive view of the physiology of major mat phototrophiccommunity members. It should also serve as a model for future studiesof this and other microbial communities.

OC-5.1

HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOCPUNCTIFORME DESTINED TO BECOME HORMOGONIA.

H. Christman, E.L. Campbell, J.C. Meeks.

Section of Microbiology, University of California, Davis, CA, USA.

Introduction: Nostoc punctiforme is a developmentally diversefilamentous cyanobacterium capable of differentiating three distinctcellular forms- heterocysts, hormogonia and akinetes. Differentsignals give rise to these different states, however there is some



overlap. Cultures growing with ammonium can be induced to makehormogonia or heterocysts upon removal of combined nitrogen.

Methods: Our DNA microarray of 6,893 genes predicted from the9.05 Mb annotated genome sequence has been used to generate aglobal transcription data set consisting of seven time points over a 24hour period of nitrogen deprivation that results in heterocystformation and a similar profile that results in hormogonia formation.

Results: 1 hour after nitrogen deprivation leading to heterocystdifferentiation, there is enhanced expression of nitrate utilization andurea transport genes, and the nitrogen response regulator nrrA, whichis required for timely development of heterocysts. Within 3 hours, thepositive acting heterocyst master regulator hetR shows enhancedexpression. From 6 to 12 h, enhanced levels of heterocyst structuralgenes, such as polysaccharide and glycolipid synthesis genes, andfunctional genes, such as those encoding two additional sets ofcytochrome oxidase specific to heterocysts were observed. At 18 to24 h the nif genes encoding nitrogenase are transcribed. In thenitrogen starvation induced (NSI) hormogonia time course, the nitrateutilization genes are enhanced at 1 h and nrrA is up regulated from 3to 18 hours. hetR is maintained at a repressed level throughout thetime course. The urea transport and cytochrome oxidase genes areenhanced transiently at the 12 hour time point and there is no changein expression of the heterocyst structural genes. At 24 h, the nif genesshow up to 8-fold enhanced expression.

Conclusion: The heterocyst time course is consistent with previousdata, demonstrating a sequence of events required to form a fullyfunctional heterocyst. NSI hormogonia show an early nitrogenresponse, but lack enhanced expression of heterocyst regulatorygenes that would be expected to elicit transcription of nif genes. Thisis in contrast to previous work that has shown nif gene transcription tobe under heterocyst developmental control. These data indicate alink in the regulatory decision to become either hormogonia orheterocysts. In order to sort out this regulation, examination ofexpression profiles on heterocyst deficient mutants is underway.

OC-5.2

PLANT CELL WALL EPITOPES ARE EXPRESSED BYCYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS.

Owen Jackson, J. Paul Knox, David G. Adams.

University of Leeds, West Yorkshire, United Kingdom.

Introduction: Gunnera is the only angiosperm to host acyanobacterial symbiont. Uniquely for plant-cyanobacteria symbiosesthe cyanobiont, Nostoc punctiforme, is intracellular. The plantbenefits from the nitrogen-fixing properties of the bacterium, and inreturn Nostoc receives physical protection, an uncompetitiveecological niche and nutrients. The Gunnera-Nostoc system haspotential uses in developing new, beneficial symbioses betweennitrogen-fixing cyanobacteria and crop plants. However, whencompared to the highly characterised relationship betweenleguminous plants and bacteria of the genus Rhizobium, the Nostoc-Gunnera relationship is not well understood. We present resultsshowing the presence of arabinogalactan-protein (AGP) and pectinepitopes to be associated with both partners in the symbiosis. AGPsand pectic polysaccharides are important plant cell wall componentsimplicated in cell growth, cell development and cell interactions.

Methods: We have used a variety of immunochemical techniques(including ELISAs and epifluorescence microscopy) with anti-AGP andanti-pectin monoclonal antibodies, along with β-glycosyl Yariv reagent(known to specifically interact with AGPs), to investigate the presenceand localisation of these polymers.

Results: Use of anti-AGP antibodies indicated the presence of AGPsassociated with cyanobacterial strains grown in liquid media. Thesame set of antibodies also indicated the presence of AGPs in thegland mucilage of uninfected Gunnera glands, and associated withthe walls of plant cells surrounding symbiotic Nostoc colonies ininfected Gunnera glands. Anti-pectin antibodies revealed thepresence of pectins on the surfaces of symbiotic cyanobacterialcolonies.

Conclusions: Cyanobacterial strains are capable of producing andexpressing AGPs on their surfaces independent of their plantsymbiotic partner, and are associated with pectins when in a symbioticrelationship with Gunnera. Both AGPs and pectin are present at thesymbiotic surface at different times during the symbiotic relationship,and hence we discuss their potential roles in the initiation andcontinuation of a stable symbiosis

OC-5.3

REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE INHETEROCYST-FORMING CYANOBACTERIA.

Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, EnriqueFlores2, David G. Adams3.1School of Biological and Chemical Sciences, Queen Mary, Universityof London, London, United Kingdom; 2Instituto de Bioquímica Vegetaly Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty ofBiological Sciences, Institute of Integrative and Comparative Biology,University of Leeds, Leeds, United Kingdom.

Introduction: When heterocyst-forming filamentous cyanobacteriasuch as Anabaena sp PCC7120 are growing diazotrophically, theymust exchange metabolites between cells in the filament. Amino acidssynthesised in the heterocysts must be transferred to the vegetativecells, and sugars synthesised in the vegetative cells must betransferred to the heterocysts. Recently we showed that intercellularmolecular diffusion in Anabaena could be visualised by loading thecytoplasm with calcein, a fluorescent tracer dye. FluorescenceRecovery after Photobleaching (FRAP) was used to quantify rates ofexchange of the dye among vegetative cells and heterocysts. Wefurther showed that FraG, a protein with a DME permease domainlocated at the cell-cell interface, is essential for the diffusion ofmolecules from cell to cell (Mullineaux et al, 2008, EMBO J. 27, 1299-1308). FraG is probably a central component of pore structures(“microplasmodesmata”) that link the cells in the filament. Here wetest the possibility that the activity of the microplasmodesmata isregulated according to the requirement for exchange of metabolitesbetween cells.

Methods: We used confocal FRAP to quantify rates of exchange ofcalcein at vegetative-vegetative and heterocyst-vegetative celljunctions, and GFP-tagging and confocal microscopy to quantify levelsof FraG at cell junctions. Changes in both parameters were measuredfollowing nitrate step-down and heterocyst formation, and then afteradding nitrate back to the cultures.

Results: FraG is constitutively present at cell junctions, even whenfilaments are grown on nitrate. This is consistent with EM studiesshowing that the microplasmodesmata are always present at celljunctions. However, intercellular molecular exchange is slow in nitrate-grown filaments. The rate of exchange between vegetative cellsincreases by more than a factor of 10 following nitrate step-down.When nitrate is added back to diazotrophically-growing cultures,molecular exchange becomes slower again.

Conclusions: 1. Rates of intercellular diffusion are controlled accordingto the requirement for metabolite exchange. When nitrate is added



back to differentiated, diazotrophic filaments, exchange becomesmuch slower. We suggest that the reduced supply of sugars to theheterocysts is a major factor leading to their subsequent death anddisappearance. Under these conditions the vegetative cells maybenefit by minimising the loss of sugars to the heterocysts.

2. The intercellular channel structures are constitutively present, evenwhen molecular exchange is slow. Therefore there must be post-translational mechanisms that regulate the opening and closing of thechannels. We argue that the channels are constitutively presentbecause they can only be formed during cytokinesis. Channels aresynthesised in undifferentiated filaments so they are ready and waitingto be activated when the supply of combined nitrogen runs out.

OC-5.4

SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS INANABAENA SP. STRAIN PCC 7120.

Jinjie Liu, C. Peter Wolk.

MSU-DOE Plant Research Laboratory, Michigan State University, EastLansing, MI, USA.

Introduction: In wild type Anabaena sp., heterocysts form with anaverage interval of ca. 10 intervening vegetative cells. patA (all0521)affects the regulation of the pattern of heterocyst differentiation:heterocysts of a patA mutant form nearly exclusively at the ends offilaments (1). PatA has also been reported to control the level of HetR(2). We have sought to identify the protein(s) with which PatA interactsand what their roles may be in the process of pattern formation.

Methods: We used PatA as bait to identify interacting proteins in ayeast two-hybrid (Y2H) library. The library was constructed by partiallydigesting Anabaena sp. DNA separately with seven 4-bp restrictionendonucleases whose products match 3 different cloning sites in theprey vectors. Different size ranges of the digested genomic DNA wererecovered separately and ligated into appropriately digested preyvectors. Products of ligation were electro-transformed into Stratagenecompetent cells of E. coli XL10 Gold, isolated, and re-transferred toSaccharomyces cerevisiae strain AH109. Bait vector was transferred toS. cerevisiae strain Y187. The two yeast strains were mated on platesof SD/-Ade-His-Leu-Trp with X-alpha-galactosidase. Blue colonieswere selected, purified, their inserts amplified by colony PCR, and thePCR products of several hundred clones were sequenced andanalyzed.

Because PatA appears to be a two-component response regulator thatlacks an intact DNA-binding domain, we focused our attention on 11prey genes that presumptively encode kinases and on genes for whichmultiple, independent clones showed interaction with PatA. Preygenes that survived those tests were mutated in Anabaena sp. bydouble reciprocal recombination with insertion of the Omegainterposon (4). Mutations in six of those genes have been fullysegregated.

Results: The 11 candidate prey clones, all of which when re-tested byY2H showed interaction with PatA, were tested for interaction with theREC, the PATAN, or both domains of PatA. According to Y2H analysis,All3305 interacted with the PATAN, but not with the REC, domain.

Double-recombinant mutants of the prey genes alr3304, all3305,all5210, all2699, alr3442 and all0192, respectively, have been fullysegregated.

The all3305 mutant showed a phenotype very similar to that of a patAmutant.

Preliminary observations and statistical analyses with the Wilcoxonrank-sum test suggest that mutants of alr3304, all5210, and all2699,

but not of alr3442 and all0192, form heterocysts closer together thandoes the wild type strain, and that over-expression of patA resulted incloser heterocysts than in the wild-type strain.

Conclusions: Yeast two-hybrid analysis provided evidence of proteinsthat interact with PatA. Six genes that consistently showed interactionwith PatA were tested by mutation in Anabaena sp., looking for apossible alteration in pattern. Because the phenotype of an all3305mutant has a phenotype like that of patA, there is a substantiallikelihood that they interact in vivo. We are trying to test whether theyinteract in vitro. Over-expression of patA by use of PpetE elicitedcloser spacing of heterocysts.

1. Liang J et al. PNAS 89: 5655 (1992); 2. Risser DD, Callahan SM. JBacteriol 190: 7645 (2008); 3. Makarova KS et al. Bioinformatics 22:1297 (2006); 4. Prentki P et al. Gene 103: 17 (1991).

OC-5.5

THE INVOLVEMENT OF TWO SIGMA FACTORS INCYANOBACTERIAL AKINETE FORMATION.

Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers.

Department of Biology, California State University, Northridge, CA,USA.

Introduction: Akinetes are desiccation and cold-resistant spore-likeresting cells formed from vegetative cells in certain heterocyst formingcyanobacteria. In Nostoc punctiforme, akinetes first appear midwaybetween heterocysts, or randomly along a filament lackingheterocysts, following phosphate or potassium limitation and lowlight. A previous DNA microarray study comparing vegetative cells toakinetes differentiated in a zwf metabolic mutant indicated that twoputative sigma factors (NpR4091 and NpF4153) were up-regulatedduring this process. Almost nothing is known about the geneticregulation involved in the morphogenesis of vegetative cells intoakinetes. To elucidate if these sigma factors were involved in akinetedifferentiation, each was over-expressed to test the hypothesis thatover-expression of the sigma factor would lead to increased akineteformation.

Methods: Random amplification of cDNA ends (RACE) was used tomap the transcriptional start sites for each gene. DNA fragmentsbearing promoter regions were cloned into the green fluorescentprotein (GFP) vector pSUN119, electroporated into N. punctiformeand reporter strains visualzed by epifluorescence microscopy followinginduction of akinetes by phosphate starvation. PCR fragmentscontaining the entire reading frame of both sigma factors wereamplified from the N. punctiforme genome and cloned into theBamHI site of pRL490 in the correct orientation to drive constitutiveexpression from the strong tac promoter. The expression plasmidswere electroporated into the wild-type strain and neomycin selectionused to obtain over-expression strains. DNA microarray analysis of theover-expression strain expression relative to wild-type is in progress todetermine the regulon of each sigma factor.

Results: NpR4091 encodes for an alternate group 2 sigma factorsimilar to SigB2 in Anabaena sp. strain 7120. Two promoters wereidentified, and a GFP reporter containing both was more highlyexpressed in akinetes relative to neighboring vegetative cells.Electroporants overexpressing NpR4091 exhibited large numbers ofakinetes in filaments growing on non-inducing plates containing A&Amedium containing Mops and ammonium. Over-expression strains inA&A/4 Mops and ammonium liquid culture grew slowly relative towild-type cultures. Microscopic examination revealed large numbersof abnormally large cells that had the characteristics of akinetes whengrowing in non-inducing conditions. NpF4153 encodes for anExtracytoplasmic Function (ECF) sigma factor similar to SigG in



Anabaena sp. strain 7120. Two promoters were identified; theupstream promoter conferred akinete-specific expression of a GFPreporter, and the downstream promoter did not, although it wasrequired for the higher-level gene expression in akinetes when bothwere present. The over-expression strain exhibited large numbers ofakinetes under non-inducing conditions, and slow growth in liquidculture, similar to that of NpR4091.

Conclusions: Cell-type specific gene expression or transcriptionalreporters and altered cell morphology of over-expression strainssupport the hypothesis that the sigma factors encoded by NpR4091and NpF4153 are involved in akinete formation.

OC-5.6

SEQUENCES REGULATING NITROGENASE GENE EXPRESSION INTHE CYANOBACTERIUM ANABAENA VARIABILIS.

Teresa Thiel, Justin L. Ungerer.

Department of Biology, University of Missouri St. Louis, St. Louis, MO,USA.

Introduction: The goal of this research is to identify and describe thefunction of key genetic elements that govern expression of thenitrogenase structural genes in heterocysts of cyanobacteria. Oncewe understand how specific sequences affect high-level, heterocyst-specific expression of nitrogenase, we can use these regulatoryelements to provide heterocyst specific expression of other oxygen-sensitive genes such as hydrogenases or alternative nitrogenases. ThenifHDK regulatory elements would also be useful for expressing genesfor bio-diesel production in heterocysts; an environment rich in lipidsthat may serve as the precursors to bio-diesel.

Methods: Transcriptional lacZ fusions of various sized nifH1 promoterfragments, either wild type or containing specific mutations, wereused to identify important nifH1 promoter sequences.

Activity of the promoter fragments was determined using a standardβ-galactosidase assay. A fluorescent substrate for β-galactosidase wasused to visualize localization of expression of the reporter. The resultsfrom the lacZ expression study were confirmed by reconstructing themost interesting mutations or deletions directly in the upstreamnifHDK region on the chromosome.

Results: We have determined that the first 100 bp upstream of thenifH transcription start site are necessary but insufficient to providenormal regulated expression of nifH. Deletions in this region severelylimit expression of nifHDK, whereas upstream sequences up to thestart codon of nifB can be deleted from the chromosome withoutaffecting expression. Conversely, the same required region and/orupstream sequences are not capable of driving high-level expressionof nifH or the lacZ reporter in trans, though the limited expression thatis detected is localized to heterocysts. This nifH1 upstream regioncontains a non-canonical NtcA binding site, however mutations of theNtcA binding site do not affect expression of nifHDK, therefore NtcAdoes not directly regulate nifHDK.

Conclusions: The 100 bp upstream of the nifH transcription start siteare sufficient to determine heterocyst specific expression of thenifHDK transcript; however, it is insufficient to provide high-levelexpression. Our data suggest that transcriptional activation of nifHmay be under control of the nifB promoter. No region between thenifH start codon and the nifB promoter can drive high-level expressionof lacZ; therefore, expression must result from the nifB promoter. It ispossible that the known nifH1 transcription start site is a result ofprocessing of a larger nifBSUHDK transcript, as there is evidence of niftranscript processing at stem-loop structures in other species 1. A

similar stem-loop structure exists in the nifU-nifH intergenic region.We hypothesize that the required sequences upstream thetranscription start site of nifH act to prevent transcriptional terminationof nifBSU or function in transcript processing. NtcA activatesexpression of various genes under conditions of nitrogen deprivation.We show that NtcA does not directly regulate nifHDK, however it islikely that NtcA regulates nifBSU because this promoter contains anNtcA-binding site. This supports the hypothesis that nifHDKexpression results from nifBSU promoter activity.1Wilson, J., Pierrard, J., Hubner, P (1993) Gene, 133: 39-46

OC-6.1

THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES ATTHE MONOMER-MONOMER INTERFACE OF PHOTOSYSTEM II INTHE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.

Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley.

Department of Biochemistry, University of Otago, Dunedin, NewZealand.

Introduction: X-ray diffraction data of Photosystem II (PS II) crystalsfrom Thermosynechococcus elongatus have revealed a dimericstructure of PS II at 2.9 – 3.5 Å resolution [Ferreira et al. (2004) Science303: 1831-1838; Guskov et al. (2009) Nat. Struct. Mol. Biol. 16: 334-342]. Three low-molecule-weight polypeptides, PsbL, PsbM and PsbThave been identified at the monomer-monomer interface. We haveused the model organism Synechocystis sp. PCC 6803 to investigatethe role of these polypeptides in PS II dimer formation, assembly,turnover and electron transport.

Methods: Mutagenesis, biochemical characterization andphysiological measurements were carried out as described in [Hao etal. (2008) Photosynth. Res. 98: 337-347]. Chlorophyll a fluorescenceinduction and decay kinetics were measured with a fluorometersupplied by PSI Instruments, Brno, Czech Republic. Polysomes fromthylakoid and cytosolic fractions were isolated according to [Tyystjärviet al. (2001) Mol. Microbiol. 40: 476-484].

Results: Mutations in the cytosolic N-terminal extension of PsbLdestabilized PS II dimers, although the monomeric complexes retainedtheir ability to split water. Mutations in this region of PsbL alsoactivated diuron-insensitive cyclic electron transfer around PS II.Additionally, truncation of 4 residues at the C-terminus was sufficientto prevent association of PsbL with a subcomplex of the photosystemthereby blocking assembly of active PS II complexes and creating anobligate photoheterotrophic strain. The absence of PsbM decreasedthe number of assembled PS II centers by one third and the remainingcenters exhibited an increased susceptibility to high-light-inducedphotodamage suggesting turnover and assembly were slowed whencompared with PS II centers in wild type. Similarly, absence of PsbTresulted in a strain that was highly susceptible to high-light-inducedstress. ∆PsbT cells were able to recover from photodamage whentransferred from high- to low-light conditions. Recovery was observedto be dependent on protein synthesis and light, and was prevented inthe absence of Psb27. Moreover, evidence for a specific role for PsbTin PS II turnover following photodamage was found sinceaccumulation of psbA transcripts with thylakoid-associated polysomeswas observed in wild type but not seen in ∆PsbT cells. Conversely,following exposure to high light, elevated levels of psbA transcriptswith polysomes isolated from the cytosolic fraction were onlyobserved in ∆PsbT cells. Electron transfer between the PS II primaryand secondary plastoquinone acceptors, QA and QB, was alsoimpaired in the ∆PsbT strain. Unexpectedly, loss of oxygen-evolvingactivity under high light was reversed by addition of bicarbonate in



∆PsbT cells and oxygen evolution was abolished by addition offormate under ambient CO2 levels in the presence of the artificialelectron acceptors 2,5-dimethyl-p-benzoquinone (DMBQ) andK3Fe(CN)6. Our results suggest that DMBQ may bind and inhibit at theQA binding site in ∆PsbT cells and in some PsbL mutants.

Conclusions: C-terminal mutations of PsbL prevent association with PSII blocking assembly at the CP43-less inactive monomer. N-terminalmutations within PsbL alter the acceptor side and stability of PS II, asdoes the absence of PsbM and PsbT. The absence of PsbT may alsodestabilize the association of polysomes with thylakoid membranesfollowing high light stress and therefore PsbT could play a key role inefficient repair of PS II following photodamage.

OC-6.2

PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO INSYNECHOCYSTIS.

Danny Yao, Dan Brune, Wim Vermaas.

School of Life Sciences and Center for Bioenergy and Photosynthesis,Arizona State University, Tempe, AZ, USA.

Introduction: The lifetime of at least one of the photosystem IIprotein components, PsbA (D1), is short (less than an hour at high lightintensity) but little information is available on the lifetimes of mostother photosystem II subunits. As the subunits all form a complex inthe membrane, different lifetimes of components raise interestingquestions regarding mechanisms of repair. The lifetime of chlorophylls(determined by in vivo labeling with 13C or 15N) in Synechocystis ismore than a week, suggesting that chlorophylls are recycled efficientlyif photosystem II breaks down. Small Cab-like proteins (SCPs) havebeen shown to play a role in the stability of the chlorophyll, with thechlorophyll lifetime decreasing in the absence of SCPs. The questionsaddressed in this research are what the range of lifetimes ofphotosystem II proteins is, and whether SCPs also affect the stability ofproteins in the photosystems.

Methods: Stable isotope labeling and mass spectrometry haveproven to be a powerful combination to determine lifetimes. Using15N-ammonium nitrate labeling that is added to growing cultures at aspecific time, both chlorophyll and protein are labeled, and thedisappearance of unlabeled (old) compounds can be followed overtime after isolation of photosystem II that carries a His-tagged CP47.For these experiments we used photosystem I-less strains that weregrown at a light intensity of 4 �mol photons m-2 s-1.

Results: The lifetimes of specific photosystem II proteins (PsbA (D1),PsbB (CP47), PsbC (CP43), PsbD (D2), PsbE and PsbF (cytochromeb559), PsbH, PsbO and Psb27) in assembled photosystem II complexeswere followed in living Synechocystis cells under standard growthconditions by monitoring the amount of unlabeled protein that couldbe extracted from membranes using the His tag on CP47. The half-lifetime of D1 was short as expected (about 2 hours at low lightintensity), but particularly PsbE and PsbO first showed a ~25%increase in unlabeled protein integrated into photosystem IIcomplexes in the first 9 hours after the start of labeling beforeunlabeled protein amounts decreased and labeled protein becamemore prevalent. This suggests that a significant amount of PsbE (andPsbO) is present in cells that is not incorporated into photosystem IIcomplexes, and that may serve as an anchor for photosystem IIassembly. The role of SCPs in photosystem protein stability will bepresented as well.

Conclusion: Stable-isotope labeling in combination with massspectrometry is a powerful method for determining protein lifetimes,

and –in contrast to radioactive labeling- can detect both “old”(unlabeled) and newly synthesized protein. The lifetime ofpolypeptides in photosystem II complexes varies greatly, implying thatthe polypeptides are replaced independent of each other. Also, thepresence of a pool of photosystem II proteins that are not yetincorporated into a mature photosystem II complex but that may formthe nucleus of a new photosystem II complex is demonstrated.

OC-6.3

IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FORTHE OXYGEN DEPENDANCE OF THE PHOTOSYNTHETICACTIVITY OF ROSEOBACTER DENITRIFICANS.

Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and AndreasLabahn1.1Institut für Physikalische Chemie, Universität Freiburg, Freiburg,Germany; 2Department of Cell Biology, Harvard Medical School,Boston MA, USA.

Introduction: Roseobacter denitrificans belongs to the group ofobligate aerobic photosynthetic bacteria. The typical bacterialphotosynthesis is an anaerobic process where the formation of thephotosynthetic apparatus is down-regulated in the presence ofoxygen. Roseobacter produces bacteriochlorophyll a as well but isincapable of performing photosynthetic energy transduction withoutoxygen. Only under aerobic conditions, the protein components ofthe photosynthetic apparatus are synthesized and photoinducedelectron transport is operative.

Methods: Core complexes, consisting of reaction center and lightharvesting complex 1 (RC-LH1) were solubilized from thephotosynthetic membranes of Roseobacter denitrificans withdecylmaltoside and purified by anion exchange chromatography andsucrose density gradient centrifugation.

Single particle electron microscopy was used to perform a firststructural characterization of the core complex . 30,000 particles wereselected from micrographs of negatively stained samples, aligned toeach other and classified to produce class averages.

Results: The class averages showed different views of the corecomplex which could thus be combined to calculate a three-dimensional reconstruction. The density map at 30 Å resolution showsthat the LH1 complex surrounding the RC forms a closed circle.

Although the resolution of the reconstruction is not yet sufficient toresolve possible gaps in the LH1 ring, this tight ring around thereaction center may prevent the exchange of dihydroquinone and thuslight-induced cyclic electron transfer.

Conclusion: With the RC-LH1 complex from R. denitrificans we haveisolated and structurally characterized a core complex from anobligate aerobic photosynthetic bacterium for the first time.

A tight LH1 ring surrounding the reaction center was found which mayinhibit the exchange of dihydroubiquinone with the quinone pool.Thus, we conclude that light-driven energy production is insufficientfor bacterial growth and that oxygen is not only required for enablingphotosynthetic electron transport but mainly for alternative metabolicpathways.

We will now 2D-crystallize the core complex for cryo-electronmicroscopy to obtain improved resolution and a detailedreconstruction of its 3D structure.



OC-6.4

VARIABLE FLUORESCENCE IN HELIOBACTERIUMMODESTICALDUM CELLS: OBSERVATION AND EXPLANATION.

Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, StefanoSantabarbara1,2, Robert Blankenship3.

1Arizona State University, Dept. of Chemistry and Biochemistry,Tempe, AZ, USA; 2Institut de Biologie Physico-Chimique, Paris, France;3Washington University, Departments of Biology and Chemistry, StLouis, MO, USA.

Introduction: Heliobacteria have the simplest photosyntheticapparatus of all known phototrophs. They possess a single type Ihomodimeric reaction centre (RC), which harvests light using a pool of22-34 bacteriochloropyll g (Bch g) as part of each RC’s intrinsicantenna. There is no extrinsic antennae. The immediate electrondonor is a membrane-attached cytochrome c, which is re-reduced by adiheme-containing cytochrome bc complex, and the immediateelectron acceptor is a di-cluster ferredoxin. At this point, it is not clearhow the cycle is closed to allow light-driven proton pumping. Alsounclear is the role of the pool quinone (menaquinone-9) – each RCcontains 2 of these molecules, but there is conflicting evidence as totheir role in electron transfer. Although heliobacteria arephotoheterotrophs and do not fix CO2, all species examined so far fixN2, and nitrogenase is expected to be a large consumer of low-potential electrons under N2-fixing conditions.

Methods: We have examined in vivo kinetics of electron transferwithin living cells of Heliobacterium modesticaldum using acombination of transient absorption and fluorescence spectroscopy.

Results: We found that H. modesticaldum cells exhibit thephenomenon of variable fluorescence, although the characteristics arequite different from those seen in oxygenic phototrophs. It usuallytook ~100 ms of illumination with actinic light before fluorescencebegan to rise. Variable fluorescence could be abolished by blockingre-reduction of P800 + (oxidized primary electron donor), either byaddition of ferricyanide (oxidant) or stigmatellin (Qo inhibitor).Contrariwise, reduction of intracellular electron acceptors by additionof dithionite and PMS (as mediator) increased variable fluorescence, asdid inhibition of nitrogenase by addition of ammonia. Underconditions that led to high variable fluorescence, it was observed thatthe yield of photo-oxidizable P800 (in the µs timescale) was lower. Wecan explain all of these data by a model in which saturation of theelectron acceptor pool leads to reduction of the FX Fe4S4 cluster inthe RC, provoking a back-reaction from the P800 + A0 state, whichproduces delayed fluorescence. We tested this model by using pump-probe spectroscopy in the ns timescale and found that a ~20-nsback-reaction from the P800 + A0 state was indeed observed underconditions that produce high variable fluorescence. Moreover, we canquantitatively account for almost all of the variable fluorescence asbeing due to back-reaction. The effects of QB-type inhibitors uponvariable fluorescence provide evidence in favor of the participation ofmenaquinone in forward electron transfer within the RC.

Conclusions: These results demonstrate a new phenomenon inheliobacterial physiology – variable fluorescence – and explain it as aconsequence of delayed fluorescence stemming from a fast back-reaction within the heliobacterial RC. This demonstrates that theintracellular electron acceptor pool can easily become saturated understrong illumination conditions, and indicates the importance ofelectron donor/acceptor dynamics. These facts likely explain why thisorganism does not require a large antenna (as it is not required)

OC-6.5

NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM IIPROTECTS THE CYANOBACTERIUM MICROCOLEUS SP. AGAINSTEXCESS LIGHT STRESS.

Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2.

Departments of 1Biological Chemistry, 2Plant and EnvironmentalSciences and 3The Interuniversity Marine Institute, Eilat, The HebrewUniversity of Jerusalem, Israel.

Photosynthetic organisms are exposed to light-induced oxidativestress due to photoinactivation of the oxygen evolving photosystem II(PSII). We investigated the persistence of PSII activity of thedesiccation-tolerant Microcoleus vaginatus, a cyanobacteriuminhabiting biological desert crusts where it is exposed to high lightintensities. Surprisingly, CO2-dependent oxygen evolution persisted atlight intensities 2-3 times higher than saturation. In contrast, lightintensities close to or higher than required to saturate oxygenevolution triggered extensive loss (85-90%) of radiative PSII chargerecombination measured as variable fluorescence orthermoluminescence (TL) emission. Radiative charge recombination,which involves the generation of singlet oxygen, recovered slowlyfollowing exposure to low light intensity but did not recover indarkness. Light induced loss of fluorescence and its recovery are notinhibited by herbicides that bind to the PSII-QB site, indicating thatreduction of plastoquinone or O2 is not involved. The temperaturerequired for maximum TL emissions resulting from QA

-/S2 chargerecombination (Q band, 22oC) indicated a significant upshift of thePSII-QA redox potential as compared to “model” photosyntheticorganisms (Q band, 10-15 oC). This property of Microcoleus mayreduce harmful radiative charge recombination as a function of lightintensity, thereby lowering the generation of 1O2 and related oxidativestress. On the basis of our findings we present a novel model wherebya non-radiative charge recombination within PSII lowersphotoinactivation damage under excess illumination and a possiblereason why this effective photo-protective mechanism was apparentlylost during the evolution from the ancestor cyanobacterium to thehigher plant chloroplast.

OC-6.6

THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THECYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.

Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-MariAro.

Department of Biology, Plant Physiology and Molecular Biology,University of Turku, Turku, Finland.

Introduction: The prokaryotic photoautotrophic cyanobacteria (blue-green algae) are favored model organisms to investigatephotosynthetic mechanisms. Stress conditions like high light orlimitation in inorganic carbon often lead to restriction in the availabilityof the terminal electron acceptors of the photosynthetic electrontransfer chain. To ensure the delicate balance of energy input andconsumption, thus preventing the generation of dangerous reactiveoxygen species, photosynthetic organisms developed differentstrategies. It is suggested that flavodiiron proteins (Flv) are involved inthose adaptation strategies. Though quiet well studied in (facultative)anaerobic Bacteria and Archaea, knowledge on Flvs in cyanobacteriais very poor. However, the modular character (a β-lactamase-likedomain containing a diiron center, a flavodoxin domain with FMNbinding site and an additional C-terminal flavin reductase domain in



cyanobacterial Flvs) of those proteins strongly suggests theirparticipation in electron transfer processes [1].

Methods: Detailed material and methods are described in [2].

Results: The genome of the cyanobacterial model strain Synechocystissp. PCC6803 comprises four genes, encoding the hypotheticalflavodiiron proteins Flv1, Flv2, Flv3 and Flv4. While Flv1 and Flv3 wereshown to function in the Mehler reaction as NAD(P)H:oxygenoxidoreductases [3], the physiological function of Flv2 and Flv4remains open. RT-PCR and Western Blot analyses show that theexpression of flv2 and flv4 is high under LC (air level of CO2) andnegligible at HC (3% CO2). Moreover, the rate of accumulation of flv2and flv4 transcripts upon shift of cells from HC to LC is stronglydependent on light intensity. Characterization of Flv-mutants inSynechocystis revealed a specific decline in PSII centers and impairedtranslation of the D1 protein in the mutants ∆flv2 and ∆flv4 whengrown at LC, whereas at HC the Flvs were dispensable. ∆flv2 and ∆flv4were also more susceptible to high light induced inhibition of PSII thanWT or ∆flv1 and ∆flv3. In fact, ∆flv1 and ∆flv3 seemed to have morefunctional PSII centers, and in both mutants the photoinhibition of PSIIwas slightly less severe than in the WT.

Analysis of published amino acid sequences revealed thatcyanobacterial genomes always contain two up to six genes encodingdistinct Flvs within the same organism. Furthermore we could findcyanobacteria-like Flvs also in some oxygenic photosyntheticeukaryotes like green algae, mosses and lycophytes. The occurrenceof Flvs in pairs seems to be a special feature for oxygenicphotosynthetic organisms.

Conclusions: We demonstrate a novel and crucial function offlavodiiron proteins, particularly Flv2 and Flv4, for oxygenicphotosynthesis in Synechocystis cells at LC. There seems to be anevolutionary trend in the diversity and function of the flavodiironproteins: In anaerobic microbes the activity of Flvs is directed againstO2/NO toxicity. In oxygenic photosynthetic organisms Flv1 and Flv3reduce oxygen to water while Flv2 and Flv4 protect the oxygenevolving PSII complex against photoinhibition. Higher plants lack Flvsand distinctly different mechanisms have evolved for photoprotectionof PSII.

[1] Vicente JB, Justino MC, Gonçalves VL, Saraiva LM, Teixeira M(2008) Methods Enzymol 437: 21-45

[2] Zhang P, Allahverdiyeva Y, Eisenhut M, Aro EM (2009) PLoS ONE

[3] Helman Y, Tchernov D, Reinhold L, Shibata M, Ogawa T et al.(2003) Curr Biol 13: 230-235

OC-7.1

PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA(PRRA), TRANSCRIPTIONAL REGULATORS OF THE CBB OPERONS(CO2 FIXATION) IN RHODOBACTER SPHAEROIDES.

Andrew W. Dangel, F. Robert Tabita.

Department of Microbiology and Plant MolecularBiology/Biotechnology Program, The Ohio State University,Columbus, OH, USA.

Introduction: CbbR and RegA (PrrA) are transcriptional regulators ofthe cbbI and cbbII (Calvin-Benson-Bassham CO2 fixation pathway)operons in Rhodobacter sphaeroides. CbbR is a LysR typetranscriptional regulator (LTTR) which is the most commonly utilizedprotein family for gene regulation in prokaryotes. RegA is part of atwo-component signal transduction system also involving themembrane-bound histidine kinase, RegB (PrrB). RegA isphosphorylated by RegB to activate its regulatory function. Both CbbR

and RegA are DNA binding proteins with helix-turn-helix motifs forDNA interaction. DNA binding sites for both proteins are found in theregulatory regions of the cbbI and cbbII operons in Rhodobactersphaeroides. RegA has four DNA binding sites in the cbbI promoter.The CbbR binding site and RegA binding site 1 overlap each otherupstream of the cbbI operon in R. sphaeroides, as demonstrated byfootprint analysis.

Results: CbbR and RegA interact and CbbR must be bound to DNAfor this protein-protein interaction to occur. Conversely, cbbI promoterbinding by RegA is not required for interaction with CbbR. Thepresence of both CbbR and RegA in cbbI promoter/protein complexeswas confirmed. In addition, the presence of RegA enhances the abilityof CbbR to bind the cbbI promoter. Gel mobility shift analysisdemonstrates that RegA binds to cbbIpromoter DNA and formsincrementally larger multimeric complexes with DNA as theconcentration of RegA increases, referred to as oligomerization. WhenRegA binding sites 1, 2 and 3 are present in the cbbI promoter, RegAforms a complex with the DNA at a significantly lower concentrationand a greater mobility compared to complex formation with RegAbinding sites 1/2, 3 or 3/4, separately. Specific site-directed mutants ofRegA have been generated that cannot bind CbbR and also havereduced oligomerization function. These mutations of the RegAprotein are located in the N-terminus receiver domain as well as in theDNA binding domain.

Methods: Methods include gel mobility shift experiments using 32P-labeled DNA probes and chemical cross-linking analysis usingdimethylpimelimidate. Isolation of the protein/DNA complexes fromnative acrylamide gels and immunoblot analysis using antibodiesspecific to CbbR and RegA was employed to determine thecomposition of promoter/protein complexes.

Conclusions: There is strong interaction between RegA and CbbR,and the presence of RegA increases the quantity of CbbR that bindsthe cbbI promoter. Possibly, RegA lowers the activation energyrequired for CbbR to bind DNA, thereby increasing the DNA bindingaffinity of CbbR. CbbR must be bound to DNA to interact with RegA,possibly ensuring that RegA only binds transcriptional regulators suchas CbbR at the appropriate promoter site. RegA sites 1, 2 and 3 arenecessary for optimal binding of RegA to the cbbIpromoter. There iscommunication or cooperation between RegA binding site 1/2 andsite 3 that allows RegA/DNA complex formation at a lowerconcentration of RegA. An intermediate loop structure could beformed to facilitate the communication between RegA site 1/2 andsite 3. In addition, several specific regions of the RegA protein areimportant for interaction with CbbR.

OC-7.2

PpsR AS A NEW TYPE OF HEMIN SENSOR.

Liang Yin, Vladimira Dragnea, Carl Bauer.

Indiana University, Bloomington, IN, USA.

My research focuses on PpsR-AppA system. To date, this regulatorysystem is the only cascade identified that coordinates light- andredox- signaling in transcription control. PpsR was identified as aDNA-binding transcription factor in R. sphaeroides. Under aerobicconditions, PpsR blocks the transcription of the tetrapyrrolebiosynthetic and the photosynthetic genes, which are responsible forbiosynthesis of the pigments and the light harvesting II complex (1).AppA, an antirepressor of PpsR, is able to inactivate PpsR by forminga PpsR2-AppA complex under anaerobic and dark conditions (1). In thepast year, our results indicate that hemin provides another level ofcontrol in this system, showing the direct link between free hemin pool



and photosynthesis for the first time.

We demonstrated that PpsR is a hemin-binding protein withstoichoimetry of 1:1 ([PpsR]/[hemin]). The presence of hemin inhibitedthe DNA-binding ability of PpsR significantly, while other tetrapyrroleproducts did not show any inhibition. Experiments with truncatedPpsR suggested that it bound to hemin via its C-terminal region,possibly through the Cys located in its DNA-binding domain. Insummary, our results suggest that PpsR is likely to be a hemin sensor.Most of well characterized hemin-binding proteins have tightly-coordinated hemin to perform many different tasks, such as electrontransfer, oxygen storage and gas molecule sensing (2). Only in recentyears people started to appreciate the hemin sensors, which are ableto sense the free hemin in vivo, from bacteria (3) to mammal (4). Yet allthe identified hemin sensors to date contain the conserved -Cys-Pro-motif, which PpsR does not have. Our research will provide us newinsights about hemin sensors, which seem to be more diverse than weused to think.

1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623

2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L.,Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073

3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A96, 13056-13061

4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc NatlAcad Sci U S A 105, 76-81

OC-7.3

ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN:AN EXPERIMENTAL SCENARIO FOR A STRICT ANAEROBICPHOTOTROPH.

Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg,Françoise Reiss-Husson, Chantal Astier, Soufian Ouchane.

CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette;Université Paris-Sud, Orsay; Université Pierre et Marie Curie, Paris;France.

Introduction: The appearance of oxygen in Earth’s atmosphere as aresult of oxygenic photosynthesis required strict anaerobes andobligate-phototrophs to cope with the presence of this potentiallytoxic molecule. Nitrogen-fixing organisms, for example, have evolveda number of strategies to protect nitrogenase from environmentaloxygen. Among these strategies, oxygen depletion by specificoxidases was shown to play an important role.

We have previously detailed the AcsF/BchE enzyme replacement andsuggested a similar HemF to HemN replacement during the shift fromaerobic to anaerobic environment to emphasize the O2 dependentand independent metabolic networks. Here, we further investigate theproblem of O2 and photosynthesis (PS) in the anoxygenic phototrophbacterium Rubrivivax (R) gelatinosus and show how the terminaloxidases (cbb3, bd and caa3) expand the physiological range of O2

environments under which this bacterium and probably many other“aerobic” phototrophs can carry out photosynthesis.

Methods: PCR-based cloning allowed the identification of fouroxidase encoding operons in R. gelatinosus. Mutations weregenerated to disrupt each operon to study the respiratory chain of thisbacterium. Mutants were further analyzed to check howphotosynthesis and respiration or O2 detoxification are functionallyrelated in this microorganism.

Results: We have shown that:

1. Only two terminal oxidases are functional in wild type R.gelatinosus: the cbb3 cytochrome c oxidase, and the bd type quinoloxidase.

2. Unlike the wild type and the single mutants, the cbb3--bd- deficient

double mutant can carry out photosynthesis only under strictanaerobicity.

3. In oxygenated environments, this mutant survives non-photosynthetically until the O2 tension drops. Co-inoculation withaerobes (E. coli) and PS deficient strains restores PS growth of thismutant even in oxygenated medium.

4. Spontaneous suppressor mutations especially in caa3 cytochrome coxidase that restore respiration simultaneously restorephotosynthesis in oxygenated environment.

Conclusions: The cbb3, bd and caa3 oxidases are therefore not onlyrequired for respiratory growth but also play an essential role in thereduction of the environmental O2 pressure during anaerobicphotosynthesis. These results demonstrate that photosynthetic andother anaerobic metabolisms in environments which are (at leasttransitorily) exposed to oxygen are critically dependent on the O2-detoxifying action of terminal oxidases.

OC-8.1

CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BYFERREDOXIN:THIOREDOXIN REDUCTASE, FERREDOXIN ANDTHIOREDOXIN M.

David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, MasakazuHirasawa, Marcellus Ubbink.

Texas Tech University, Lubbock, TX, USA.

Introduction: Ferredoxin:thioredoxin reductase (FTR), catalyzes thetwo-electron reduction of thioredoxins in chloroplasts and incyanobacteria (thioredoxins f and m and perhaps other thioredoxinsare substrates), using reduced ferredoxin as the electron donor.Reduced thioredoxins then play important roles in redox regulation.FTR, a heterodimer with a unique [4Fe-4S] cluster as its sole prostheticgroup, has a single binding site for ferredoxin and a separate singlebinding site for thioredoxin. The binding sites for FTR from thecyanobacterium Synechocystis sp. PCC 6803 on ferredoxin and onthioredoxin in the 1:1 complexes of the enzyme with its two proteinsubstrates have been mapped and the ternary complex between allthree proteins has been characterized.

Methods: Perturbations of the two-dimensional NMR spectra of bothferredoxin and thioredoxin arising from complex formation with FTRhave been used to identify specific amino acids on the two proteinsinvolved in complex formation with FTR.

Results: A mono-gallium analog of the [2Fe-2S] Synechocystis sp. PCC6803 ferredoxin was obtained by reconstituting apo-ferredoxin in agallium-containing refolding buffer. The use of this diamagnetic Gastructural analog eliminates the paramagnetic broadening of NMRresonances of amino acids in the vicinity of the [2Fe-2S] cluster innative ferredoxin. This has allowed the first complete mapping of theinteraction interface of a [2Fe-2S] ferredoxin for a target enzyme. NMRhas also been used to contain a three-dimensional structure of the Ga-ferredoxin in solution, confirming that its structure is almost identicalto that of the native, iron-containing protein. NMR spectroscopy wasalso used to map the interaction domain for FTR on thioredoxin m in a1:1 complex of the two proteins. Both similarities and differences areseen in the thioredoxin m interaction domain for FTR in the non-covalent complex examined by NMR and in a disulfide-linked covalentcomplex of FTR and thioredoxin m for which an X-ray crystal structure



has been obtained. NMR has also been used to characterize a ternarycomplex between ferredoxin, FTR and thioredoxin m in solution.

Conclusions: The interaction domains on both ferredoxin andthioredoxin for FTR appear to consist largely of hydrophobic aminoacids. The fact that FTR can form a ternary complex with ferredoxinand thioredoxin simultaneously confirms the presence of separatebinding sites on FTR for its two substrates. Furthermore, Ga-substituted ferredoxin has been established as a valuable,non-paramagnetic analog for ferredoxin for NMR investigations of theinteraction of ferredoxin with its target enzymes.

OC-8.2

CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA.

Shinichi Takaichi1, Mari Mochimaru2.

1Department of Biology, Nippon Medical School, Kawasaki;2Department of Natural Sciences, Komazawa University, Setagaya,Tokyo; Japan.

Introduction: Cyanobacteria grow by photosynthesis, and essentiallycontain chlorophyll and various carotenoids, whose main functions arelight-harvesting and photoprotection. We have summarizedcarotenoids, characteristics of carotenogenesis enzymes and genes,and carotenogenesis pathways in some cyanobacteria, whose bothcarotenoids and genome DNA sequences have been determined(Takaichi & Mochimaru (2007) Cell. Mol. Life Sci. 64: 2607-2619).

Methods: Carotenoids were extracted from cyanobacterial cells andpurified. They were identified based on absorption spectra, retentiontimes on C18-HPLC, MS,

1H-NMR and CD analyses (Iwai et al. (2008)Plant Cell Physiol. 49: 1678-1687). Knockout mutants ofcarotenogenesis genes were constructed for characterization ofenzymes (Mochimaru et al. (2008) J. Bacteriol. 190: 6726-6733).

Results & Discussion: Cyanobacteria contain various carotenoids; β-carotene, its hydroxyl derivatives, such as zeaxanthin andnostoxanthin, or keto derivatives, such as echinenone andcanthaxanthin, and carotenoid glycosides, such as myxol glycosidesand oscillol diglycoside. Both ketocarotenoids, such as echinenoneand 4-ketomyxol, and the carotenoid glycosides are the uniquecarotenoids among phototrophic organisms. Some cyanobacteria, e.g.Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, containboth unique carotenoids, while others, e.g. Synechococcus elongatusPCC 7942 and Prochlorococcus marinua, do not contain suchcarotenoids. From these findings and characteristics ofcarotenogenesis enzymes, certain carotenogenesis pathways can beproposed. The different compositions of carotenoids might be due tothe presence or absence of certain gene(s), or to different enzymecharacteristics. For instance, two distinct β-carotene hydroxylases, CrtRand CrtG, are bifunctional enzymes; CrtR catalyzes β-carotene tozeaxanthin and deoxymyxol to myxol, CrtG catalyzes zeaxanthin tonostoxanthin and myxol to 2-hydroxymyxol, and substrate specificitiesof CrtR vary across species. Two distinct b-carotene ketolases, CrtOand CrtW, are found only in the first group, and properly used in twopathways, β-carotene to echinenone and myxol to 4-ketomyxol,depending on the species. Lycopene cyclases of CrtL fromSynechococcus elongatus PCC 7942, CruA and CruP fromSynechococcus sp. PCC 7002, and CrtL-b and CrtL-e fromProchlorococcus marinus have been functionally confirmed, whilethose in other species are not found yet. In Synechococcus sp. PCC7002, 1’,2’-hydratase (CruF) for myxol synthesis is functionallyconfirmed, and novel unique carotenoid of synechoxanthin is found.At present, the number of functionally confirmed genes is limited, and

only a few species are examined. Therefore, further studies ofcarotenoids, characteristics of carotenogenesis enzymes and genes,and carotenogenesis pathways are needed.

OC-8.3

EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROMPURPLE BACTERIA AND CYANOBACTERIA.

Gerhard Sandmann.

Biosynthesis Group, Molecular Biosciences 213, J. W. GoetheUniversität, Frankfurt, Germany.

Introduction: Carotenoids are essential pigments for photosyntheticorganisms. Their predominant function is protection of photosynthesisfrom photooxidation. This antioxidative property depends on theconjugatd double bond system of the carotenoid molecule which isformed in a series of desaturation steps. During evolution ofcarotenogenesis, two basically different desaturation pathways wereacquired. The “ancient” pathway exists in archeae, bacteria and fungiinvolving a single CrtI type enzyme which can catalyze up to sixdesaturations and isomerizes cis to trans double bonds. Among thepurple bacteria, CrtI desaturases exist either as 4-step desaturasesstarting the spirilloxanthin pathway as in Rhodospirillum rubrum or as3-step desaturases as in Rhodobacter species synthesizingspheroidene derivatives. In contrast, double bonds are formed bystructurally and mechanistically unrelated CrtP type desaturases incyanobacteria, Chlorobaculum species and photosynthetic eukaryotes.The most likely evolutionary root of CrtP is a CrtU desaturase which isresponsible for aromatization of b-ionone rings in several groups ofbacteria including Chlorobiaceae.

Methods: Carotenogenic genes were cloned from different species.They were heterologously expressed and the resulting enzymes usedto carry out enzyme kinetic studies including determinations ofsubstrate and product specificities

Results: Within cyanobacteria, one finds among the existing species arelic of the CrtI desaturation pathway, Gloeobacter violaceous, whichis the only known cyanobacterium with a CrtI-type desaturase. Incontrast to the CrtI-catalyzed desaturations, the desaturation productsof CrtP-related reactions are poly-cis carotenes. Consequently, theresulting tetra-cis lycopene has to be isomerized to all-trans by anadditional isomerase CrtH which developed from CrtI by loosing itsentire desaturation function but retaining its isomerization function.There is an enzyme resembling an evolutionary link between CrtI andCrtH in the cyanobacterium Nostoc PCC7120. It resembles anadditional CrtI-related enzyme named CrtQa which is a z-carotenedesaturase producing all-trans products. Its participation in the poly-cis desaturation pathway of Nostoc PCC7120 could be specified.

The evolutionary modification of the product specificity of the CrtIdesaturases was studied in Rubrivivax gelatinosous in which CrtIresembles a transitional state yielding 3-fold and 4-fold desaturatedproducts, simultaneously. The in vitro and in vivo results werecompared to a 3-step desaturase from Rhodobacter. We could findthat the kinetic property of this phytoene desaturase is one factor todetermine the type of reaction product. However, structural propertiescan prevent that the last desaturation step is catalyzed. The reactionstops at the level of neurosporene which is no longer a substrate forthe desaturase.

Conclusion: Among the prokaryotes, two basically differentcarotenoid desaturation pathways were acquired. Our enzyme kineticinvestigations were focussed on two desaturases, a missing linkbetween both pathways and one desaturase comprising all



characteristics of a 3- and of a 4-step desaturase. The results will helpto understand the evolutionary origin and the divergence of carotenedesaturation.

OC-9.1

THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM– A STRUCTURAL MODEL BASED ON SOLID-STATE NMR DATACOMBINED WITH MOLECULAR SIMULATION STUDIES OF CDAND ABSORPTION SPECTRA.

Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1,Peter Højrup2, Anders Giessing2, Juha Linnanto3, Niels-Ulrik Frigaard4,Mette Miller2 and Niels Chr. Nielsen1.1Center for Insoluble Protein Structures (inSPIN), InterdisciplinaryNanoscience Center (iNANO) and Department of Chemistry,University of Aarhus, Århus, Denmark; 2Department of Biochemistryand Molecular Biology, University of Southern Denmark, Odense,Denmark; 3Department of Chemistry, University of Jyväskylä, Finland;4Department of Biology, University of Copenhagen, Copenhagen,Denmark.

Introduction: Green sulfur bacteria possess two external light-harvesting antenna systems, the chlorosome and theFenna-Matthews-Olson (FMO) protein. A small 6.1 kDa chlorosomeprotein, CsmA, plays a major role by connecting these two antennaephysically and functionally. CsmA is located in the chlorosomebaseplate - a paracrystalline structure observed by freeze fractureelectron microscopy. Each CsmA coordinates one molecule ofbacteriochlorophyll (BChl) a, and reconstitution experiments haveshown that the CsmA-BChl a complex forms dimers and higheraggregates (1).

The CsmA baseplate is one of the simplest known antenna systems.Thus, a structural characterization might provide new insights into howphotosynthesis evolved, as well as serve as inspiration to thedevelopment of new photosynthetic devices.

Methods:We have deduced a solution NMR structure of monomericCsmA in a mixed solvent system, which shows CsmA to be an α-helical protein (2). To find the native organization of the CsmA-BChl acomplex in the baseplate, we have studied isolated BChl c-lesschlorosomes (so-called carotenosomes (3)) by solid-state NMR. In thissample, CsmA is the dominating protein so it can be investigateddirectly without further purification. The native state of the pigment-protein complex was investigated by absorption and CD spectroscopyand used for molecular simulation studies.

Results and Conclusions. Based on NMR and CD data, we present amodel of the chlorosome baseplate in which CsmA is arrangedsymmetrically in long rows of dimers with BChl a molecules in ahydrophobic pocket between the dimers.

Literature:

(1) Pedersen, M.Ø.; Pham, L.; Steensgaard, D.B.; Miller, M. (2008). “Areconstituted light-harvesting complex from the green sulfurbacterium Chlorobium tepidum containing CsmA andbacteriochlorophyll a.” Biochemistry 47(5): 1435-1441.

(2) Pedersen, M. Ø.; Underhaug, J.; Dittmer, J.; Miller, M.; Nielsen,N.C. (2008). “The three-dimensional structure of CsmA: A smallantenna protein from the green sulfur bacterium Chlorobiumtepidum.” FEBS Letters 582(19): 2869-2874.

(3) Frigaard, N. U., Li, H.; Martinsson; P., Das, S.K.; Frank, H.A.;Aartsma, T.J.; Bryant, D.A. (2005). “Isolation and characterization ofcarotenosomes from a bacteriochlorophyll c-less mutant ofChlorobium tepidum.” Photosynthesis Research 86(1-2): 101-111.

OC-9.2

THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLSIN CHLOROSOMES OF CHLOROBACULUM TEPIDUM.

Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, MichaelReus, Aline Gomez Gomez Maqueo Chew, Alfred R. Holzwarth, EgbertJ. Boekema, Huub J. M. de Groot.

Department of Biochemistry and Molecular Biology, The PennsylvaniaState University, University Park, PA, USA.

Introduction: Chlorosomes are remarkably efficient light-harvestingstructures that allow green sulfur bacteria to live at light intensities atwhich no other phototrophs can survive. Each chlorosome can containup to 250,000 bacteriochlorophyll (BChl) c/d/e molecules, and cellscan contain up to 50 million or more BChl c/d/e molecules. Becausethese BChls are not bound to proteins and are self-organized, thestructural basis for BChl organization has been of intense interest asmodels for biomimetic systems for solar energy applications.

Methods: Chlorosomes were isolated from wild type or triple-mutantstrain bchQ bchR bchU of Chlorobaculum tepidum (1) by standardprocedures using 2M sodium isothiocyanate. Cryo-electronmicroscopy and negative stain microscopy were performed aspreviously described (2). Magic-angle spinning NMR studies wereperformed with uniformly 13C labeled chlorosomes as described (3).

Results: The NMR assignments of the 13C and 1H signals of BChl dmolecules in chlorosomes from the bchQRU mutant were obtainedfrom two-dimensional 13C-13C and 1H-13C magic-angle spinning (MAS),solid-state NMR dipolar correlation spectra collected from 13Cenriched chlorosome preparations. The observed interstackcorrelations provided convincing evidence that syn-anti monomerstacking is the basic building block in the bchQRU chlorosomestructure. Additional structural constraints were obtained from Fouriertransforms of cryoEM images of chlorosomes. Supramolecular modelswere constructed for different orientations of the stacks relative to thetube axis. With stacks running perpendicular to the tube axis along thecircumference of a cylinder in rings, the simulated image and itsFourier transform reproduced the strong periodicity of 0.83 nm andthe distinct striped appearance with a spacing of 2.1 nm that isobserved in the cryoEM images of the chlorosomes of the bchQRUmutant. Wild-type chlorosomes also had BChl c nanotubes with thesame 2.1-nm lamellar spacing. However, indicating that the stacks aredifferently oriented relative to the long-axis of the chlorosome, theyalso exhibited a weak layer line at 1.25 nm, which corresponds to thedistance between repeating syn-anti pair units in the direction of thestacks.

Conclusions: Computational integration of two different bio-imagingtechniques, solid-state NMR and cryoEM, revealed a previouslyundescribed syn-anti stacking mode and showed how ligated BChl cand d self-assemble into coaxial cylinders to form concentric tubular-shaped suprastructures. The close packing of BChls via π-π-stackingand the helical H-bonding networks present in both the mutant andwild-type chlorosomes forms the basis for ultrafast, long-distancetransmission of excitation energy. The structural framework is robustand can accommodate extensive chemical heterogeneity in the BChlside chains for adaptive optimization of the light-harvestingfunctionality in low-light environments. In addition, syn-anti BChlstacks form sheets that allow for strong exciton overlap in twodimensions, which enables triplet exciton formation for efficientphotoprotection.



References

Gomez Maqueo Chew, A, Frigaard, NU & Bryant, DA (2007) JBacteriol 189: 6176-6184.

Oostergetel, GT, et al. (2007) FEBS Lett 581: 5435-5439.

Ganapathy, S, et al. (2009) Proc. Natl. Acad. Sci. USA, in review.

OC-9.3

MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANESIN CYANOBACTERIA.

Sawsan Hamad, Wim Vermaas.

School of Life Sciences and the Center for Bioenergy andPhotosynthesis, Arizona State University, Tempe, AZ, USA.

Introduction: The process of biosynthesis of photosyntheticmembranes in oxygenic phototrophs remains elusive. The first proteinthat was linked directly to thylakoid membrane biosynthesis wasVipp1, a membrane-associated polypeptide. The vipp1 gene (sll0617)could not be fully deleted from the cyanobacterial genome. In orderto determine the mechanism of Vipp1 function we have nowoverexpressed this protein in Synechocystis.

Methods: A second vipp1 gene, identical in sequence to the nativeone, was placed under the control of the strong psbA3 promoter. Inaddition, a YFP or His tag sequence was fused to the second copy ofthe vipp1 gene as appropriate for localization or for identification ofco-isolating proteins.

Results: Overexpression of vipp1 resulted in a significantly enhancedcontent of thylakoid membranes and of vesicles inside cyanobacterialcells, and in an increased amount of photosystem II. Moreover, largeinclusions with membranous content were frequently observed inVipp1 overexpresser cells, often near the constriction site for the nextcell division; these inclusions may be used to provide the buildingblocks for membrane biosynthesis. The Vipp1-YFP fusion protein inVipp1-overexpressing Synechocystis was localized at specific locationsnear the periphery of the cell, again often near the constriction site forthe next cell division, suggesting that Vipp1 is associated with thelarge membranous inclusions in the cell that may be used for thylakoidbiogenesis. Slr1641, which is one of the ClpB chaperones inSynechocystis, was found to be the only protein that co-isolated withHis-tagged, overexpressed Vipp1 from a cyanobacterial proteinextract.

Conclusion: Our results suggest Vipp1 to be critical for and toregulate thylakoid membrane biogenesis, and Vipp1 appears to beassociated with inclusions at specific membrane biogenesis sites in thecell. This work also demonstrates the value of overexpression ofnative proteins if corresponding segregated deletion mutants cannotbe obtained.

OC-9.4

PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OFFUNCTION IN THE CARBOXYSOME.

Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C.Bagby3, Sabine Heinhorst4, Fei Cai4, Gordon Cannon4, Sallie W.Chisholm3.1US Department of Energy, Joint Genome Institute, Walnut Creek, CA;2Department of Plant and Microbial Biology, University of California,Berkeley, CA; 3Department of Biology, Massachusetts Institute ofTechnology, Cambridge, MA; 4Department of Biochemistry, Universityof Southern Mississippi, Hattiesburg, MS; USA.

Introduction: Cyanobacteria and many chemoautotrophic microbeshave evolved a special mechanism for dramatically enhancing CO2

fixation by encapsulating RuBisCO and carbonic anhydrase intosubcellular microcompartments called carboxysomes.

Methods: Bioinformatic and Structural Studies

Results: Our recent structural studies revealed the basic architecturalprinciples underlying construction of the carboxysome shell.Expansion of these studies into the Prochlorococcus model system hasled to the identification and elucidation of the structures of a new typeof building block for the carboxysome shell, one with the potential forgated transport across the shell. The carboxysome is the best studiedbacterial microcompartment; bioinformatic surveys of genomicsequence reveal that the tendency to form bacterialmicrocompartments is widespread. For example, structurally relatedmicrcompartments are found in Rhodopseudomas palustris andRhodospirillum rubrum that appear to be involved in functions otherthan CO2 fixation.

Conclusions: The carboxysome provides general insights into asurprisingly widespread strategy for biological compartmentalization;recent data provide an increasingly sophisticated view of its functionand regulation.

OC-9.5

PROTEOMIC ANALYSIS OF THE DEVELOPINGINTRACYTOPLASMIC MEMBRANE DURING CHROMATICADAPTATION IN RHODOBACTER SPHAEROIDES.

Kamil Woronowicz, Robert A. Niederman.

Department of Molecular Biology and Biochemistry, RutgersUniversity, Piscataway, NJ, USA.

Introduction: Although the primary photochemical events and energytransduction processes in Rba. sphaeroides are well understood, muchless is known about mechanisms involved in the formation of theparticipating light-harvesting (LH) and photochemical reaction center(RC) complexes, or the interactions of their apoproteins with numerouscomplex-specific and general assembly factors in the site-specificassembly of functional photosynthetic units in the growingintracytoplasmic membrane (ICM). Here, we report a detailedstructural and functional proteomic analysis of the ICM assemblyprocess during a downshift in a light intensity.

Methods: Proteomic approaches have focused upon identifyingproteins temporally expressed during ICM development and spatiallylocalized in both membrane growth initiation sites, isolated as anupper pigmented band (UPB) after rate-zone sedimentation in sucrosedensity gradients, and in the mature ICM, isolated as the main band(chromatophores). The isolated membrane fractions have been furtherpurified by a two-phase partitioning procedure and subjected to non-denaturing clear gel electrophoresis (CNE). Bands are excised fromthe gels and subjected to LC-MS/MS analysis.

Results: CNE gives rise to four distinct bands: a top band containingthe LH1 (B875)-RC core complex; a bottom band containing the LH2(B800-850) peripheral antenna complex; and two bands ofintermediate migration in which the LH2 and core complexes remainassociated. Proteomic analysis yielded a large array of proteinsassociated with the gel bands and the resulting profiles can becorrelated with the local organization of ICM as revealed by atomicforce microscopy (AFM). The PuhA RC-H subunit was the mostabundant protein, while surprisingly, the Puc2A LH2-α polypeptide,encoded by puc2BA operon, was the second most abundant proteinwith PucB LH2-β and Puc2B LH2-β comprising the other detectable



LH2 subunits. The spectral counts of several of the F1FO– ATP synthasesubunits were unexpectedly high, given the low abundance of the ATPsynthase in chromatophores and the inability to detect this couplingfactor, as well as the more abundant cytochrome bc1 complex in AFM.Moreover, the high levels of ATP synthase and subunit IV of the bc1complex found associated with the LH2-enriched fractions isconsistent with their localization at ICM vesicle edges, thought tocontain LH2-only domains, which are outside the flat ICM vesicleregions imaged by AFM. Significant levels of a preprotein translocaseYidC homolog were found, along with lesser amounts of other generalmembrane assembly factors such as TatA, the twin-arg translocationsystem subunit, the SecY preprotein translocase subunit and thebacterial type 1 signal peptidase. In contrast, no complex-specificassembly factors were found in any of the bands. Forty-five proteins ofunknown function were associated with the gel bands. Onehypothetical protein, RSP6124, was observed in high abundance andis associated with the LH2 band, with its appearance preceding theincrease of LH2 levels during chromatic adaptation.

Conclusions: Proteomic analysis of bands containing the LH1-RC andLH2 complexes obtained by CNE of chromatophore from low lightadapting cells, yielded a variety of proteins that could be correlatedwith their localization determined in AFM topographs and thepossible roles of general membrane assembly factors that may beinvolved in the insertion of their nascent polypeptides into the ICM.(Supported by DOE Grant No. DE-FG02-08ER15957).

OC-9.6

BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODINGTHE MEMBRANE-ASSOCIATED THIOREDOXIN PROTEIN PLAYSAN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OFPS I.

Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1.1Department of Biochemistry and Molecular Biology; 2Department ofChemistry; The Pennsylvania State University, University Park, PA, USA.

Introduction: The biogenesis of PS is a complex, multi-step process inwhich many factors are involved, mostly in assembly of the proteincomponent and insertion of cofactors. In recent years, advances ingenome sequencing of several cyanobacteria (includingSynechococccus sp. PCC 7002) and functional genomics ofphotosynthetic genes have provided much new information forexploring assembly and regulation of the PS I reaction center. In thisstudy, we describe a novel gene (Syn7002_A0564) encoding a proteindisulfide isomerase (PDI)-like protein that is found be involved inassembly and/or regulation of PS I

Methods: DNA and protein sequence analyses were performed withMacVector software. Homologous sequences of the DPI-likeSyn7002_A0564 were retrieved from the IMG and Cyanobasedatabases. A Syn7002_A0564 mutant was generated through insertionof a kanamycin-resistance cartridge at the unique EcoRV site withinSyn7002_A0564 coding sequence. Growth rates of the wild type andmutant were measured at different conditions. The low-temperaturefluorescence emission spectroscopy was applied to compare changesof PS I and PS II complexes in cells. PS I complexes were isolated foranalysis of subunit composition. The cell-permeant indicator CM-H2DCFDA was used for probing intracellular reactive oxygen species(ROS).

Results: Sequence analyses indicate that Syn7002_A0564 encodes aPDI-like protein that is composed of two thioredoxin-like domains andthat it is predicted to be membrane-associated. Homologs ofSyn7002_A0564 can be found in all oxygenic photosynthetic

organisms. Inactivation of the Syn7002_A0564 gene in Synechococcussp. PCC 7002 leads to about 40% reduction of chlorophyll content,which is due to impairment of PS I accumulation as demonstrated bymeasurements of the low temperature fluorescence emissionspectroscopy and PS I analysis. However, no obvious difference couldbe observed in the composition of PS I, as shown in the results ofSDS-PAGE analysis of isolated PS I complexes. Underphotoautotrophic growth conditions the mutant exhibited a slowergrowth rate and higher sensitivity to high light. The stress response ofthe mutant is possibly related to an increase of ROS in cells. Asmeasured by using a cell-permeant ROS probe and compared to thewild type, the ROS content is two-times higher in the mutant cellseven when grown in reduced light. A 30-min treatment in high lightled to a 6-fold increase in ROS content. As shown by RT-PCR analysis,expression of the sodB gene is significantly up-regulated in the mutantcells grown under the reduced light conditions. An enhancedphotoautotrophic growth rate was observed for the mutant when cellswere grown under semi-anaerobic growth conditions by bubbling with99% N2 and 1% CO2.

Conclusion: Our results demonstrate that the PDI-like proteinSyn7002_A0564 is involved in biogenesis of PS I reaction center. Itmay function as a membrane-associated dithiol/disulfideoxidoreductase or may play an important role in maintaining thebalance of oxidizing/reducing equivalents of thiol redox in PS Iassembly and regulation, especially in facilitating cofactor insertionand maturation.

OC-10.1

A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THEAPPARENT PHOTOSYNTHETIC AFFINITY FOR CO2 BYMODULATING LOW-CO2-INDUCED GENE EXPRESSION.

Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2,Yukako Hihara2, Ariel Gaathon3, Mario Lebendiker4, Aaron Kaplan1.1Dept. of Plant and Environmental Sciences, Hebrew University ofJerusalem, Jerusalem, Israel; 2 Dept. of Biochemistry and MolecularBiology, Saitama University, Saitama, Japan; 3Bletterman Laboratory,Interdepartmental Equipment Unit, Hebrew University of Jerusalem,Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre, HebrewUniversity of Jerusalem, Jerusalem, Israel.

In Synechocystis sp. strain PCC 6803, over 450 genes are up-regulatedfollowing transfer of the cells from a high (1-5% CO2 in air, HC) to alow level of CO2 (as in air or lower, LC). This includes sbtA, ndhF3 andcmpA involved in inorganic carbon (Ci) uptake. Earlier studiesimplicated NdhR in the regulation of LC-induced genes but there areindications that additional components are involved. Followingextraction of proteins from cells grown under HC and (NH4)2SO4

fractionation, we have identified LexA and two AbrB-like proteins,Sll0359 and Sll0822 that bind to a fragment of the sbtA promoter.Using extracts prepared from LC-grown cells, Sll0822 did not bind tothe sbtA promoter despite its presence in the cells, suggesting that itmay serve as a repressor of LC-induced genes. This is supported bythe fact that sbtA, ndhF3, and cmpA normally expressed only underLC in the wild type are transcribed under both HC and LC in a∆sll0822 mutant. When grown under HC this mutant exhibits anelevated apparent photosynthetic affinity to Ci, typically observed inthe wild type only under LC. Clearly, expression of genes essential forCi uptake was sufficient to raise the apparent photosynthetic affinityfor external Ci.



OC-10.2

THE EFFECTS OF TEMPERATURE AND OXYGEN ONNITROGENASE ACTIVITY IN THE THERMOPHILICCYANOBACTERIUM FISCHERELLA SP.

Lucas J. Stal, Department of Marine Microbiology, NetherlandsInstitute of Ecology, Yerseke, The Netherlands.

Introduction: Nitrogenase – the enzyme complex responsible for thefixation of dinitrogen in diazotrophic prokaryotes - is irreversiblyinactivated by oxygen. Cyanobacteria evolved strategies that allowedthe coexistence of the two incompatible processes of dinitrogenfixation and oxygen-evolving photosynthesis. Heterocysts aredifferentiated cells that contain nitrogenase but have lost the oxygenicphotosystem. The heterocyst possesses a thick glycolipid cellenvelope that serves as a gas diffusion barrier. Any oxygen that entersthe heterocyst is respired thereby guaranteeing an anoxic interior ofthe heterocyst. However, a too efficient glycolipid cell envelope wouldalso limit dinitrogen from entering the heterocyst. Hence, the gasdiffusion barrier must be a trade-off between the entrée of dinitrogenand a limitation of the entrée of oxygen. Ideally, the amount of gasthat diffuses into the heterocyst should be as high as possible so thatthe respiratory system can still keep it to sufficient low intracellularconcentrations. This would require a dynamic gas diffusion system ofthe heterocyst in order to adequately respond to changes in theenvironmental conditions.

Methods: I have selected the thermophilic heterocystouscyanobacterium Fischerella sp. to investigate the question of thedynamic response of gas diffusion in the heterocyst by measuringnitrogenase activity by an on-line, near real-time acetylene reductionassay (ARA) using a laser photoacoustic ethylene detector. Fischerellasp. was grown under continuous light at 27oC.

Results: I measured ARA at temperatures from 18-39oC at 3oCintervals. At each temperature light response curves of ARA wererecorded. The light response curves were fitted to the rectangularhyperbola function yielding the following parameters. Nd and Nm werethe dark- and maximum light-dependent acetylene reduction rates,respectively and Ntot was the sum of these and represented the actualnitrogenase activity measured at light saturation. The light saturationconstant Ik equals Km in Michaelis-Menten kinetics and is the lightintensity at which 0.5Nm. Ik equals Nm/α. The light affinity constant α isthe initial slope of the light response curve at zero light. I found thatnitrogenase activity increased with temperature with a Q10 of 6, threetimes higher than what one would expect from enzyme kinetics. Thedimensionless factor Ntot/Nd equaled ~2 and was constant over thetemperature range. Nd increased with temperature and this is onlypossible when the amount of oxygen that enters the heterocystincreased. This would indicate a possible dynamic regulation of theoxygen (gas) diffusion into the heterocyst. In order to investigate thisfurther, I measured ARA at a range of oxygen concentrations from 0-90%. I found that Ntot remained constant in the range of 5-30%oxygen. This was the result of an increasing Nd and simultaneouslydecreasing Nm. Above 30% oxygen acetylene reduction decreased,obviously because an increasing part of the reducing equivalents werenecessary for respiration and nitrogenase activity became electronlimited.

Conclusions: I conclude that the heterocyst did not dynamicallyregulate the entrée of oxygen at a constant temperature but that ithappened as a function of temperature. The changes of thephotosynthetic parameters Ik or α were fully attributed to changes in Nm

and therefore photosynthesis itself did not affect dinitrogen fixation.

OC-10.3

PHOSPHATE SCAVENGING IN AN UNPREDICTABLEENVIRONMENT, HOW DO CYANOBACTERIA MEET THECHALLENGE?

Frances D. Pitt, David J. Scanlan.

Department of Biological Sciences, University of Warwick, Gibbet HillRoad, Coventry, United Kingdom.

Introduction: Cyanobacteria are ubiquitous in aquatic and marineenvironments where they play a key role as primary producers. Theirgrowth is limited by the availability of nutrients, particularlyphosphorus (P), and many species persist and flourish in environmentswith an unpredictable and constantly fluctuating supply of P. Whilstgenome-wide analyses have revealed many details of the P uptakeand regulatory machinery there are still very few reports thatdemonstrate the functional significance of individual, and apparentlyredundant, components of the cyanobacterial Pho regulon. Forexample, Synechocystis sp. PCC6803 encodes duplicate ABCtransporters (i.e. two PstCAB operons) with at least three associatedperiplasmic binding proteins (two of the PstS type and one of theSphX type). This contrasts with virtually all other known bacteria whichencode a single ABC transport system. Current research into thespecific functional attributes of the multiple phosphate transporterarrangement in Synechocystis sp. PCC6803 aims to elucidate theirinterplay with other proteins involved in phosphate acquisition andregulation. By answering the question of evolutionary adaptation bybiochemical or regulatory means, we aim to reveal new strategies forthe acquisition of phosphate during conditions of environmentalstress.

Methods: In order to assess the role of multiple ABC transporterelements in Synechocystis sp. PCC6803 we created gene deletions ofthe two pstCAB operons as well as single knock-outs of the associatedpstS / sphX genes. Gene expression profiles were generated usingreal time quantitative PCR to compare mutants with WT underphosphate replete and deplete conditions. Phosphate uptake assaysusing radiolabelled 32P were then performed to compare the kineticsof phosphate uptake in the various mutants.

Results: Disruption of one of the PBPs in Synechocystis sp. PCC6803,pstS1 (sll0680) led to an impairment in the expression of specificgenes of the pho regulon during P-deplete growth. In addition, themutant was unable to alter the expression of various genes normallyinduced in WT in response to changes in external P concentration.This phenotype was not observed when the remaining phosphatebinding proteins were disrupted suggesting that only one of the twotransporters is involved in sensing and regulation of the pho regulon.Preliminary P uptake assays suggest potential differences in thebinding affinities of the two transporters. Further uptake work usingspecific gene knock out mutants should clarify their contribution tophosphate uptake under different growth conditions.

Conclusion: Differential expression of P binding protein genes in WTSynechocystis sp. PCC6803 during P replete and deplete conditionssuggests that each ABC transporter performs a distinct functional role.This conclusion is supported by the phenotype of the pstS1 deletionmutant which displays impaired ability to respond to changes inexternal P. Preliminary results from P uptake assays go further andsuggest that there may be differences in the P binding protein’s affinityfor P, thus leading to the development of a novel strategy for P uptakein a freshwater cyanobacterium.



OC-10.4

REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY INCYANOBACTERIA. IDENTIFICATION OF THE REGIONS INVOLVEDIN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS.

Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J.Florencio.

Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla-CSIC, Sevilla, Spain.

Glutamine synthetase plays a central role in the regulation of nitrogenassimilation in cyanobacteria via the control of the carbon/nitrogenmetabolic flow. Thus, this enzyme is tuned up depending on thenitrogen source and the carbon disponibility. The intracellularconcentration of 2-oxoglutarate defines the carbon/nitrogen status ofthe cell and it is the key metabolite in the control of the GS activity,being high level of GS activity when 2-oxoglutarate concentration ishigh and low GS activity at low 2-oxoglutarate level. The regulation ofcyanobacterial GS activity occurs by a original system consisting in theprotein-protein interaction between the GS protein and twoinactivating factors (IFs) named IF7 and IF17. This interaction provokesthe inactivation of the enzyme when the carbon/nitrogen ratio is low,for example using ammonium as nitrogen source, but if it is nitrate,the enzyme is active and the IFs are absents. The expression of the IFsare controlled at the level of transcription by the global nitrogenregulator NtcA. Here, we have investigated the molecular interactionbetween GS and the IFs, we try to know which are the regions in bothGS and IFs implicated in the protein-protein interaction by using twodifferent approaches. The first one was to construct different GSchimeras using Synechocystis and Anabaena GSs, it is worth to notethat both cyanobacteria possess the same system to inactivate GS, butthe IF are different in the way that IF7 from Anabaena is able toinactivate both GS in vitro, but IF7 or IF17 are unable to inactivate theAnabaena GS. Since IFs from Synechocystis are unable to inactivateAnabaena GS, we expect to find a region responsible for theinteraction in Synechocystis GS. The second method was to constructdifferent site-directed mutants of IFs from Synechocystis in order tostudy their capacity to inactivate or not GS.

We will present results indicating that the region required for theinteraction resides in the carboxy-terminal part of the GS. We alsofound that a change of single residue is this region is sufficient todisrupt the interaction between GS and IFs in Synechocystis. Withrespect to the IFs interaction studies, we have found that at least 3basic amino acid residues are essential to maintain the interaction withthe GS and that these residues are localized in the central part ofthese proteins. The relevance of these results will be discussed inrelation to a possible model that explain how these GS-IFs interactionpromotes the GS inactivation.

OC-10.5

BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS.

Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A.Bryant1.1Department of Biochemistry and Molecular Biology, The PennsylvaniaState University, University Park, PA; 2 Department of BiologicalSciences, University of New Orleans, New Orleans, LA; USA.

Introduction: Cyanobacteria employ a number of differenttetrapyrrole/biliprotein combinations both as accessory pigments toaugment their ability to utilize wavelengths of light which are notefficiently captured by chlorophyll and as sensors to perceive and

respond to light conditions in their environment. Up to four differentlinear tetrapyrroles may be incorporated into variousphycobiliproteins: phycocyanobilin, phycoerythrobilin, phycoviolobilinand phycourobilin. When bound to its cognate protein, each of thesebilins confers unique spectral properties, which enhance the ability ofthe cyanobacteria to exploit the light available in its environment. Onthe other hand, plants are only known to utilize biliproteins known asthe phytochromes as sensors. Plant phytochromes uniquelyincorporate the bilin, phytochromobilin. In the last several years thecomponents involved in the assembly of biliproteins have beencharacterized. This study was conducted to determine the factorsinvolved in maintaining the specificity between phycobiliproteins andtheir cognate chromophores and to determine the effects on acyanobacterium when chromophores are misincorporated into a non-cognate apoprotein.

Methods: A dual-plasmid E. coli expression system was used to studythe attachment of chromophores to phycobiliproteins in isolation. Useof this heterologous system allowed the co-expression of the minimalcomponents necessary to assemble a holophycobiliprotein and madeit possible to assay the effects of substitutions of various components,specifically, bilin lyases and bilin biosynthetic enzymes, on the finalholophycobiliprotein made in E. coli. In addition, we studied theeffects of potential mis-pairings of chromophores and apoproteinsinside Synechococcus sp. PCC 7002 using site-specific integrationsinto the genome.

Results: With various lyase and chromophore synthesis combinations,CpcA from Synechococcus sp. PCC 6803 can bind six different lineartetrapyrrole chromophores, including the plant-specific chromophorephytochromobilin and an A-ring isomerized version ofphytochromobilin not known to occur naturally. Additionally, theheterologous over-expression of the phycoerythrobilin orphytochromobilin biosynthetic enzymes in Synechococcus sp. PCC7002, a cyanobacterium that utilizes only phycocyanobilin as achromophore, results in strains with readily apparent alteredabsorbance profiles.

Conclusions: Different chromophore/apoprotein/lyase combinationscan be used to yield several different colored versions of a singlechromophore binding protein. The specificity of attachment of alinear tetrapyrrole chromophore to its cognate apoprotein is moregeneral than once believed. Although in nature the misincorporationof chromophores is believed to occur only rarely, if at all, it can bemade to occur quite readily in genetically modified E. coli andcyanobacteria. These studies shed new light on the assembly andevolution of phycobiliproteins, and the results have implications forthe production of recombinant proteins with properties not found innatural proteins.

OC-10.6

ACCUMULATION OF TREHALOSE IN RESPONSE TODESICCATION AND CONTROL OF TREHALASE IN THETERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE.

Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima.

Graduate School of Natural Science and Technology, KanazawaUniversity, Ishikawa, Japan.

Introduction: Desiccated organisms have little to no metabolic activityand rapidly resume metabolism upon rehydration. This phenomenonis termed “anhydrobiosis”. The terrestrial cyanobacterium Nostoccommune sustains the capacity for cell growth for over 100 years in a



desiccated state; thus, N. commune is considered an anhydrobioticmicroorganism with oxygenic photosynthetic capabilities. Since N.commune does not differentiate into akinetes (spores), themechanisms of its extreme desiccation tolerance most likely involvemultiple processes. Anhydrobiotic organisms accumulate trehalose,which protects biological membranes and proteins against thedestructive effects of water removal by replacing the primary waters ofhydration, as well as through the formation of amorphous glasses(vitrification). In this study, we investigated changes in photosyntheticactivity and trehalose levels in the terrestrial cyanobacterium Nostoccommune responding to desiccation and the biochemical propertiesof the enzymes involved in trehalose metabolism to elucidate themechanisms of trehalose accumulation.

Methods: Trehalose was determined by gas-liquid chromatography(GLC) and by high performance liquid chromatography (HPLC).Photosynthetic O2 evolution during desiccation was measured using agas-phase Clark-type oxygen electrode. The three genes for trehalosemetabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY(maltooligosyltrehalose synthase, Mts), and treH (trehalase), werefound as a gene cluster, and the mRNAs for these genes weredetected by RT-PCR. The activity of trehalose synthesis, consisting ofMts and Mth, was measured by the production of trehalose fromsoluble starch. The treH gene was heterologously expressed in E. colicells in an active form with a molecular mass of 52 kDa and the activityof trehalase was measured by the production of glucose fromtrehalose.

Results: As the water content decreased in N. commune coloniesduring desiccation, photosynthetic O2-evolving activity decreased andno activity was detected in desiccated colonies. No detectabletrehalose was found in fully hydrated colonies; however, trehaloseaccumulation occurred in response to water loss during desiccationand high levels of trehalose were detected in the air-dried colonies.Moreover, NaCl treatment also induced trehalose accumulation andthe levels of trehalose induced were equivalent to that in thedesiccated colonies. The transcripts for treZ, treY and treH genes weredetectable at similar levels during desiccation. Trehalase activity wasstrongly inhibited in the presence of 10 mM NaCl while trehalosesynthesis activity remained active in the presence of salt.

Conclusions: Trehalose accumulation and cessation of photosynthesisoccurs during desiccation. Since transcriptional regulation of the genecluster for trehalose synthesis and trehalose hydrolysis cannot fullyexplain trehalose accumulation in response to desiccation, post-transcriptional regulation most likely controls the cellular level oftrehalose. Under water-stress conditions characterized by increasedcellular solute concentrations, the rate of trehalose productionexceeds that of trehalose hydrolysis because of the inhibition oftrehalase. Control of trehalase plays an important role in trehaloseaccumulation under conditions of extreme desiccation.

OC-11.1

FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAININ RHODOSPIRILLUM CENTENUM PPR.

J.A. Kyndt, T.E. Meyer, M.A. Cusanovich.

Biochemistry Dept., University of Arizona, Tucson, AZ, USA.

Introduction: Ppr is unique in that it has a blue light photoreceptor(PYP) N-terminally fused to a bacteriophytochrome (Bph) domain.Photoactive Yellow Protein (PYP) is related to the PAS domainsuperfamily of signalling proteins. It generally occurs as a free-standing cytoplasmic protein and has been found in 18 species ofbacteria of diverse lifestyles. It contains a p-hydroxy-cinnamic acid

chromophore that typically absorbs 446 nm light, although there issome variation amongst species. The Ppr from the purplephotosynthetic bacterium, Rhodospirillum centenum is a chimera ofPYP, bacteriophytochrome (Bph), and histidine kinase. The function ofPpr is to regulate a polyketide synthase. When both photoreceptorsare activated, the PYP domain appears to hasten the recovery of Bphto the dark state. However, the cause of this phenomenon is unclear.We have now analyzed the Ppr recovery kinetics and kinase activity ingreater detail under three different light conditions.

Methods: Ppr was produced in E. coli using a two plasmid basedsystem. Purification was performed on a Q-sepharose column followedby a TALON His-tag column. Absorption spectra and kinetics wereobtained using a Cary 300 spectrophotometer. Recovery kinetics weremeasured for up to 300 minutes and the data were fit using Sigmaplot8.0 (Systat Software). Autophosphorylation was measured by adding1mM ATP (containing ~ 2.5 μCi P32 ATP) and keeping the sampleilluminated for 30 min. Six time points were taken and analyzed onSDS-page gels. The resulting autoradiogram was digitized using aTyphoon Imager (GE Healthcare) and intensities were quantified byvolume using ImageQuant TL.

Results: Bph normally absorbs 700 nm light in the Pr state and whenilluminated with red light, it is red-shifted in most species to thephotoreversable Pfr state absorbing near 750 nm. However, the Bphdomain within Ppr bleaches upon red-light illumination to a formwhich weakly absorbs near 650 nm and cannot be photoreversed bynear red light. Dark reversion normally takes from 20 to 60 minutes.However, recovery of the Bph 701 nm absorbance is accelerated inpart to about 1 minute following white light illumination. White andblue light bleaches PYP, which initially recovers with kinetics similar tothat of the PYP construct, however, only about half the absorbance at434 nm is recovered in ~ 4 minutes, the remainder takes several daysto recover due to formation of an intramolecular complex with Bphwhose absorption is red-shifted. The Bph within the metastablecomplex can no longer be bleached by red light. The PYP remainslocked in its bleached state which absorbs near 355 nm, but it can bephotoreversed by UV light, in which case the Bph also returns to itsinitial 701 nm dark state. Autokinase activity is turned on by both redand white light, but is inactivated when photoreversed by UV light.

Conclusions: The kinase activity of Bph is normally turned off in thedark and activated in red light. It is switched off again by far-red lightwith simultaneous return of the 700 nm absorbance. Because the Bphwithin Ppr does not form the Pfr state but is instead bleached by redlight, it cannot be photoreversed in the usual way. Thus, the role ofPYP is threefold, to block activation of Bph in blue light throughformation of the metastable complex, to accelerate recovery of Bphabsorbance in white light, and to photoreverse the effects of blue andwhite light by subsequent illumination in the UV. We thereforepostulate that Ppr functions as a UV-red light sensor.

OC-11.2

SHORT-TERM LIGHT ADAPTATION STRATEGIES OFPHYCOBILISOME-CONTAINING PHOTOSYNTHETICS,CYANOBACTERIA AND RED ALGAE.

Igor Stadnichuk.

A.N.Bakh Institute of Biochemistry, Russian academy of Sciences,Moscow, Russia.

Phycobilisome (PBS)-containing photosynthetics, the cyanobacteriaand red algae, have evolved several short-term reversible adaptationmechanisms to survive under different light quality and quantityconditions. Realized on a timescale of minutes, all mechanisms do not



involve alterations in gene expression or photoinhibition of thepigment apparatus and are revealed as non-photochemicalfluorescence quenching (NPQ) that includes alterations of thefluorescence emission from PBS. The appropriate phenomenachanging rapidly the cross-sections of the photosystems I (PSI) and II(PSII) are: state transitions realized in cyanobacteria and red algaewhen light is normal to growth (≤ 150 microE m-2s-1); orange caroten-protein (OCP)-induced PBS quenching realized in cyanobacteria forrelatively high light intensities (≥ 500 microE m-2s-1); induced by weak(~10 microE m-2s-1) far-red light increase of PBS emission, in the redalgae. State transitions are well-known phenomenon of the adaptationof pigment apparatus to unbalanced light absorption by PSI and PSIIfirst described 40 years ago ([1] and ref. there). Contrary to elucidationof the effect in green plants, the cause of state transitions incyanobacteria and red algae is still in debate and different modelshave been proposed. In the spillover model, the excitation energy istransferred directly from PSII to PSI core chlorophylls. The partialdissociation of PBS from PSII and its reassociation without docking toPSI is the main event of the detachment model. In the mobile model,the PBS could reversibly migrate in the plane of the thylakoidmembrane from the surface of PSII to PSI. It could be possible thatnone of the existed models would stay correct after additionalinvestigations [2]. In cyanobacteria, most probably, themonomerisation of the PSII dimers is the main event of transition tostate 1 [3].

In 2004, due to the registered action spectrum of the process, wehave described [4] an additional to state transitions short-termmechanism of energy dissipation in PBS of cyanobacteria involving acarotenoid identified as OCP [5]. Light-activated OCP was found tocause reversible NPQ via interception the energy transfer from PBS tochlorophyll a of PSII as well as of PSI cores [2, 5].

Recently [5], we have demonstrated quite new display of short-termlight adaptation of the pigment apparatus in the extremophilicmicroalga, Galdieria sulphuraria. Comparing to the dark-adapted cells,weak far-red light primarily absorbed by photosystem I (PSI) causes anincrease in the amplitude of the PBS low-temperature fluorescenceemission. Contrary to emission, the PBS peak in fluorescenceexcitation spectrum of PSI increases simultaneously with PBSquenching while excitation spectrum of PSII stays invariable.Therefore, in G. sulphuraria, the registered changes of 77 Kfluorescence emission correspond to the reversible detachment of thePBS from the core complexes of PSI. The data indicates the adaptivedecrease of cross-section and the diminishing of the PSI activity whenit is not accompanied by PSII function. This kind of NPQ is not knownfor cyanobacteria where PBS docking to PSI has to be faster than thedocking to monomeric PSI in the red algae.

References

1. Murata N. (2009) Photosynth. Res. 99, 155-160.

2. Stadnichuk I. et al. (2009) Photosynth. Res. 99, 227-241.

3. Stadnichuk I. et al. (2009) FEBS Lett. in press.

4. Rakhimberdieva M. et al. (2004) FEBS Lett. 574, 85-88.

5. Wilson A. et al. (2006) Plant Cell 18, 992-107.

OC-11.3

REGULATORY RNAS IN CYANOBACTERIA AND BEYOND.

W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1,J. Vogel2, A. Wilde3.1Freiburg Initiative in Systems Biology and Faculty of Biology,University Freiburg; 2Max-Planck-Institute for Infection Biology Berlin;3Justus-Liebig University Giessen, Institute of Microbiology andMolecular Biology; Germany.

Regulatory RNA has been discovered in all three domains of life.However, transcriptional units that give rise to non-coding RNAs(ncRNAs) or antisense RNAs (asRNAs) are not identified during normalgenome annotation, and in phototrophic bacteria only a small numberof ncRNAs has been described. We have used 5 different methods forthe identification of novel non-coding and antisense RNAs in variouscyanobacteria, with a focus on model cyanobacteria (1-4). Thereliability of computational predictions for the detection of ncRNAsand asRNAs in Synechocystis sp. PCC 6803 and for marineSynechococcus species was scrutinized in microarrays, complementedby deep sequencing of the small RNA population and validated by anextended set of Northern hybridizations and 5’ RACE experiments.

The total number of asRNAs in Synechocystis sp. PCC 6803 alone ismore than 350 and of classical trans-acting ncRNAs about 100. Thus,regulatory RNAs play a much more important role in this modelorganism and probably also in most other bacteria. Although functionsare still unknown for the vast majority of regulatory RNAs, we willprovide functional evidence for cyanobacterial ncRNAs acting againstinvading phages, controlling the expression of key photosyntheticgenes, or serving as signals for RNA maturation, processing anddegradation.

References

(1) Axmann et al. (2005), Genome Biol. 6, R73: 1-16.(2) Voss et al. (2007), BMC Genomics 8:375.(3) Steglich et al. (2008), PLoS Genetics 4 (8) e10000173.(4) Voss et al. (2009), BMC Genomics 10, 123.

OC-11.4

FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUMPROCHLOROCOCCUS: WHAT CAN WE LEARN?

C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, JoergVogel3, Wolfgang Hess1.1University of Freiburg, Freiburg, Germany; 2University of Algarve,Faro, Portugal; 3Max Planck Institute for Infection Biology, Berlin,Germany.

Small non-coding RNAs (ncRNAs) are functional RNA molecules,mostly without a protein-coding function, that have been found in alldomains of life. In bacteria these functional RNA molecules range insize between 50 – 400 nt and frequently play a crucial role inregulatory networks particularly in response to environmental stress.ncRNAs are also known to control plasmid and viral replication,bacterial virulence and quorum sensing, while the function of othershas remained unknown. A detailed survey for ncRNA was performedin the ecologically important marine cyanobacterium Prochlorococcus,which it is the most abundant phototroph in the vast nutrient-poorareas of the ocean. Genome analyses of several isolates revealedcompact streamlined genomes that contain a core set of about 1,200genes augmented with a variable number of ‘flexible’ genes. Allsequenced Prochlorococcus isolates possess only a small number of



genes coding for regulatory proteins. Applying different methods forthe detection of ncRNAs in Prochlorococcus MED4, the total numberof ncRNAs, which have been experimentally confirmed by twodifferent methods, rises to 26 (including Yfr1-6, 6S RNA, SRP RNA,tmRNA, RNase P RNA). Thus, unlike its reduced suite of regulatoryproteins, the number of ncRNAs relative to genome size inProchlorococcus is comparable to that found in other bacteria,suggesting that RNA regulators likely play a major role in regulation inthis group. Moreover, the ncRNA genes are concentrated in previouslyidentified genomic islands, which carry genes of significance to theecology of this organism. Of the 12 completely sequencedProchlorococcus strains only 2 possess the RNA chaperone Hfq. A 454deep sequencing approach was conducted to compare the number ofregulatory RNAs between the high light-adapted hfq-lacking strainMED4 and MIT9313 who is adapted to low light and contains an hfqhomolog.

OC-11.5

REGULATION BETWEEN PHOTOAUTOTROPHIC ANDPHOTOMIXOTROPHIC GROWTH AND ITS DEPENDENCE ON THECO2 LEVEL IN SYNECHOCYSTIS PCC 6803.

Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy Lieman-Hurwitz1, Aaron Kaplan1.1Plant and Environmental Sciences, The Hebrew University ofJerusalem; Israel; 2Department of Biochemistry and Molecular Biology,Saitama University, Japan.

Cyanobacteria represents a unique case were carbohydrates formationin photosynthesis and their breakdown in respiration occurs in thesame compartment at the same time, unlike eukaryotes where thecatabolic and anabolic activities are spatially separated. Themechanisms whereby these activities are regulated in the context ofthe environment is poorly understood. Glucose sensitive mutants ofthe cyanobacterium Synechocystis sp. PCC 6803 were isolated. Manyof them are far more resistant to the presence of glucose whenexposed to low level of CO2. Analyses of the expression of certainCO2-dependent genes in the wild type and the mutants as affected bythe ambient CO2 concentration and presence of glucose revealed anintriguing but complex regulation between these processes. Ourresults shows for the first time the interconnectivity between CO2

availability and organic carbon supply and provide some insight intothe molecular mechanisms involved.

OC-11.6

CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY INOSCILLATORIA.

Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson,Dave Adams.

University of Leeds, Leeds, West Yorkshire, United Kingdom.

Introduction: When attached to a surface many filamentouscyanobacteria move by a process known as gliding. The mechanismof gliding is unknown, although two main theories, slime extrusion andsurface waves, have been proposed to explain the generation ofthrust. In the first, filaments are pushed along by the extrusion ofslime from rows of junctional pores that are known to encircle each cellseptum. The second theory proposes that gliding results from therhythmical progression of waves along the cell surface and theirinteraction with the substrate to which the filament is attached. It hasbeen suggested that such waves are created by protein fibrils beneaththe outer membrane. We have been using Atomic Force Microscopy

(AFM) and Field Emission Gun Scanning Electron Microscopy(FEGSEM) to study the cell walls and cell surfaces of Oscillatoria strainsto identify structures that may be associated with motility.

Methods: For FEGSEM, samples were fixed in 2.5% (vol/vol)glutaraldehyde and post-fixed with 1.0% (wt/vol) osmium tetroxide.Samples were then dehydrated in a graded ethanol series, critical-point dried and mounted on aluminum pin stubs. Finally, sampleswere coated with either gold or platinum-palladium and examinedwith a LEO 1530 series FEGSEM instrument operating at 3 kV.

For AFM imaging of cells under liquid, cyanobacterial filaments werefirst immobilized by partial embedding in dental wax which had beensoftened by warming in an oven.

Results: AFM scanning under liquid of filaments of Oscillatoria sp.strain A2 immobilised in wax confirmed the presence of an array ofparallel 40 nm wide corrugations on the surface. These surfacecorrugations are a consequence of the presence of protein fibrilsbeneath the outer membrane. By increasing pressure on the AFM tipit was possible to remove the fibrillar array from a defined area of thecell surface. By contrast, FEGSEM images of the very large filamentsof O. princeps revealed an irregular array of corrugations on thesurface. These proved difficult to visualise by AFM under liquid,possibly because of a slime layer obscuring the finer surface details.However, the O. princeps surface is covered by raised structures whichcorrespond in spacing to large pores, approximately 150 nm indiameter, seen in FEGSEM images to penetrate the extremely thickpeptidoglycan layer. The function of these pores seems to be to bringthe cytoplasmic and outer membranes into closer proximity. However,they may also have a function in slime production. The significance ofthese cell wall and surface features, in terms of the mechanism ofgliding motility, will be discussed.

Conclusions: It is possible to immobilise cyanobacterial filaments indental wax in a way that preserves the filaments in a fully hydratedstate and allows AFM scanning under liquid. The AFM tip can beused to remove surface layers, such as the slime layer, outermembrane and fibrillar array, and by repeat scans of the same area, itis possible to visualise surface changes. We hope in the near future toemploy these techniques to visualise the putative surface waves thatdrive gliding motility.

OC-12.1

GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGENCONDITIONS AND THE ROLE OF THE HISTIDINE KINASE, HIK31,IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.

Tina C. Summerfield1, Louis A. Sherman2.1Botany Department, University of Otago, PO Box 56, Dunedin, NewZealand; 2Purdue University, Department of Biological Sciences, WestLafayette, IN, USA.

Introduction: In habitats such as hot springs, soils (e.g. rice paddies orestuarine mud), and eutrophic lakes, cyanobacteria experience lowoxygen conditions. These conditions are associated with the ability ofcyanobacteria to produce ethanol and H2 indicating decreased O2

may play an important role in this energy production.

Methods: Using DNA microarrays, we examined gene expression inSynechocystis sp. PCC 6803 on transition from aerobic growth to lowoxygen conditions. We observed up-regulation of the gene encodingthe histidine kinase Hik31 under low oxygen conditions. To examinethe role of this histidine kinase we compared gene expression in thewild type and a ∆Hik31 strain that lacked Hik31. Additionally, wemeasured growth, oxygen evolution and whole cell spectra during



incubation under low oxygen conditions.

Results: In the wild type strain, expression of >13% of chromosomalgenes were altered within 1 hour under low oxygen conditions.Similar numbers of genes showed increased and decreased transcriptabundance. This included down-regulation of genes encodingphotosynthesis proteins, regulatory proteins and chaperones. Up-regulated genes included the hox genes encoding the bidirectionalhydrogenase and the LexA-like transcription regulator (sll1626) thatbinds the hox promoter. Genes encoding proteins involved intransport, regulation and energy metabolism were up-regulated underlow oxygen conditions, including an operon encoding Hik31 andresponse regulator (Rre34). The transition from aerobic to low oxygenconditions resulted in differential regulation of ~8% of chromosomalgenes in the ∆Hik31 strain. The majority of these genes (~90%)showed increased transcript abundance under low oxygen conditions.Almost half these genes, including the hox operon, were up-regulatedin the wild type under the same conditions. Genes up-regulated inthe ∆Hik31 strain but not the wild type under low oxygen conditionsencoded proteins involved in photosynthesis, such as phycobilisomecomponents, and ribosomal proteins. Consistent with this, extendedincubation under low oxygen conditions resulted in cells withincreased phycobilisome content relative to chlorophyll content in the∆Hik31 strain but not the wild type. Furthermore, unlike the wild type,the ∆Hik31 strain did not down-regulate genes encoding chaperonesand ATP synthase.

Conclusions. These data suggest that Synechocystis sp. PCC 6803responds to low oxygen conditions by down-regulating genesinvolved in key processes such as photosynthesis and translation andthat the histidine kinase, Hik31, plays a role in this negative regulation.

OC-12.2

INCREASING HYDROGEN PRODUCTION BY PERTURBINGFERMENTATIVE METABOLISM IN THE MARINE, UNICELLULARCYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002.

Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G.Charles Dismukes.

Princeton University, Princeton, NJ, USA.

Introduction: Certain strains of cyanobacteria contain a bidirectionalhydrogenase and make hydrogen as a byproduct of sugar catabolismduring fermentation via the reaction: NADH + H+ � NAD+ + H2. Inthe cyanobacterium Synechococcus 7002, nitrate reduction andlactate production are two pathways that utilize reductant duringfermentation and therefore compete with hydrogenase. Thesepathways have been eliminated in this study. Lactate dehydrogenaseutilizes one equivalent of NADH to reduce pyruvate to lactate duringfermentation. Nitrate metabolism is largely unexplored duringfermentative metabolism, but is expected to be analogous tophotoautotrophic nitrogen metabolism in which nitrate is reduced tonitrate with 2 electrons from ferredoxin. Nitrite is further reduced toammonium with 6 electrons.

Methods: Cultures were grown photoautotrophically to stationaryphase in medium A sparged with 2% CO2. Cultures were prepared forfermentation experiments by washing cells in media lacking nitrateand then resuspending cells media lacking nitrate or with notedconcentration of nitrate added. Anoxic conditions were induced in10mL vials with 3mL headspace by wrapping the vials in foil andpurging with argon. The hydrogen concentration in the headspacewas measured using gas chromatography. The metaboliteconcentrations in the extracellular media were quantified using protonNMR spectroscopy. Nitrate/nitrite concentrations in the media and

sugar concentrations in the cells were assayed chemically. The ldhAgene was insertionally inactivated to create the mutant ldhA.

Results: In order to examine the effect of nitrate limitation underfermentative conditions, cells were resuspended in fresh medium Acontaining 0 µM, 200 µM, or 1 mM sodium nitrate. Eliminating nitratefrom the media prior to fermentation gives a 10-fold increase in therate of hydrogen production as compared to media containing 200µM or 1 mM nitrate. The cells assimilate 100% of the extracellularnitrate added within one day of fermentation. Nitrite is detected inthe extracellular media after four days of fermentation. The cells thatassimilate 200 µM nitrate excrete 70 µM nitrite; the cells thatassimilate 1 mM nitrate excrete 700 µM nitrite. The presence of nitratein the fermentation media acts as an outlet for reductant duringfermentation. Nitrate competes for reductant with hydrogenase asevidenced by the 10-fold increase in in vivo hydrogen when nitrate isabsent.

The primary product excreted by Synechococcus 7002 underfermentative conditions when nitrate is not present is lactate, at aconstant rate of 21.6 mol*day-1 per 1017 cells over the 4 daysexperiment. To redistribute reductant flux away from lactate andtowards hydrogen, a mutant lacking D-lactate dehydrogenase wasexamined under fermentative conditions. The ldhA mutant excretesno lactate, while all other metabolites (hydrogen, acetate, alanine, andsuccinate) are excreted in higher amounts compared to WT.Hydrogen production increases 5-fold in ldhA cultures, to a rate of14.1 mol*day-1 per 1017 cells.

Conclusions: We have gained insight into carbon and nitrogenmetabolism of these photoautotrophs during fermentativemetabolism, a branch of metabolism largely unexplored incyanobacteria. We show increases of 10-fold, with removal of nitrate,and 5-fold, with removal of pyruvate reduction to lactate. Engineeringphotoautotrophs gives promise for obtaining substantial increases forproducing biofuels.

OC-12.3

PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATIONTRANSFER TO PHOTOSYSTEM I IN THE FILAMENTOUSCYANOBACTERIUM NOSTOC PUNCTIFORME.

Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring,Ann Magnuson.

Department of Photochemistry and Molecular Science, UppsalaUniversity, Uppsala, Sweden.

Cyanobacteria as well as eukaryotic algae and plants have the capacityto adapt to varying light conditions by controlling the amount ofexcitation energy distributed to the photosystems. On the minute timescale this control leads to so-called state transitions, that incyanobacteria involve the movement of the phycobilisomes (PBS) aswell as non-photochemical quenching. On longer time scales,cyanobacteria regulate the size and protein composition of PBS as aresponse to light conditions (‘chromatic adaptation’) on the geneticlevel. State transitions have been suggested to be dependent on theredox state of the plastoquinone pool, which in part results fromelectron transfer between the photosystems, but in spite of decadesof research, the molecular mechanisms behind the observedphenomena are still not fully understood.

In the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC29133 ca 5-10% of the vegetative cells differentiate into heterocysts.The process brings extensive structural reorganization of the thylakoidmembranes, and due to oxygen sensistivity of the nitrogenase,



Photosystem II is inactivated in the heterocyst. In the heterocystthylakoids, cyclic electron transfer around Photosystem I (PSI) isessential for nitrogen fixation, but linear electron transfer between thephotosystems does not take place. Nevertheless, isolated heterocystsdisplay changes in the excitation transfer to PSI reminiscent of a statetransition.

Here we present a characterization of the composition andfunctionality of thylakoid membranes of the heterocysts. We haveinvestigated the ability of the heterocyst to regulate excitation transferto PSI, and present the outline of a mechanistic model for excitationre-distribution that does not depend on linear electron transfer.

OC-12.4

GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC7942.

Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, YasunoriNakamura.

Akita Prefectural University, Akita, Japan.

Introduction Our previous analysis of the mutants of Synechococcuselongatus PCC 7942 defective in glycogen biosynthesis indicated thatthey were highly susceptible to salt and oxidative stresses. It istherefore suggested that the storage of the polysaccharide and itsefficient mobilization are very important for cyanobacteria to copewith various environmental stresses. It is postulated that thedegradation of storage β-glucans in bacteria (glycogen or starch) ismediated by phosphorylase (PHO), debranching enzyme (DBE) and �-1,4-glucanotransferase (disproportionating enzyme, DPE), encoded byglgP, glgX and malQ, respectively. However, their roles incarbohydrate metabolism of cyanobacteria have been poorlyinvestigated. In the present study, mutants of glgP, glgX and malQhave been constructed and characterized for the catabolic activity ofglycogen accumulated in the cell. S. elongatus PCC 7942 is suitablefor the functional analysis of the genes involved in the glycogencatobolism, as the organism contains just one each of these genes.

Methods The three genes of S. elongatus PCC 7942 were disruptedindependently by insertional inactivation using antibiotic resistantmarkers. Inactivation of the targeted enzyme was confirmed by in vitroassays as well as non-denaturating electrophoresis using amylopectin-containing polyacrylamide gels, followed by iodine staining.Distribution of chain length (as expressed by degree ofpolymerization, or DP) in the debranched glycogen and malto-oligosaccharides was determined by capillary electrophoresis.

Results Comparison of the electrophoretic patterns of crude extractsbetween wild type (WT) and the mutants led us to the identification ofthe specific bands on the zymograms, corresponding to the activitiesof all three enzymes. When grown under continuous illumination, thephenotype of the mutants was not clearly distinguished from that ofWT, except for the increased accumulation of glycogen and relativeincrease of short chains in the glycogen molecule (DP 2 through 6) inglgX mutant. The cellular content of glycogen was therefore examinedin the continuous darkness, where the turnover of the polysaccharidewas stimulated. After 72 hours of dark treatment, the glycogencontent in WT cells was decreased to 27% of the initial level. Theaddition of 0.2 M NaCl to the culture further enhanced the glycogenbreakdown (to 6% of the original level after 72 hours) in WT cells.Under the same conditions, the activity of glycogen degradation inthe glgP, glgX and malQ mutants was markedly reduced as comparedto WT. In addition, accumulation of malto-oligosaccharides wasobserved specifically in malQ mutant, irrespective of the growthconditions. The molecular species of the malto-oligosaccharides was

determined as DP 3 through 6 (maltotriose to maltohexaose).

Conclusions These results demonstrated the pivotal roles of the threeenzymes PHO, DBE and DPE in the glycogen catabolism ofcyanobacteria.

OC-12.5

IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTISAERUGINOSA UNDER IRON LIMITATION.

Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1.1School of Biotechnology and Biomolecular Sciences; 2School of Civiland Environmental Engineering; University of New South Wales,Sydney, Australia.

Introduction: The production of toxins during cyanobacterial bloomsposes a significant public health threat in water bodies globally andrequires the urgent development of effective bloom managementstrategies. Microcystin is a hepatotoxin synthesised non-ribosomallyby members of several cyanobacterial genera. Although themicrocystin biosynthesis pathway is now characterised, thecontribution of toxicity to the adaptation of cyanobacteria toenvironmental stresses, such as changing light intensity and nutrientlimitation, is still unclear. Previously, microcystin synthesis has beenproposed to be regulated by iron availability. The aim of this study wasto compare the transcription of genes involved in iron uptake andredox control in toxic and non-toxic Microcystis aeruginosa subjectedto moderate and severe iron stress.

Methods: Three strains of M. aeruginosa, the toxic PCC7806, the non-toxic PCC7005 and the mutant mcyH-, were grown in Fraquil mediacontaining 10-1000 nM Fe. The transcription of a number of genesinvolved in iron uptake, oxidative stress response and toxin synthesiswas accessed by quantitative real-time PCR (qRT-PCR). The possibleinvolvement of the ferric uptake regulator homologues FurA, FurB andFurC in regulating toxicity and survival under redox stress was alsoassessed.

Results: Both non-toxic strains (PCC7005 and mcyH-) used in thisstudy showed pronounced bleaching by the end of the experiment,with the growth of mcyH- being significantly retarded under ironlimitation. Toxin production in PCC7806 was increased in an iron-dependent manner and appeared to be regulated by FurA. Thetranscription of the genes studied here showed a high degree ofvariability between strains and the growth stage at which the cellswere harvested. The inability to produce microcystin, either due tonatural mutations in the mcy gene cluster or insertional inactivation ofmcyH, affected the remodeling of the photosynthetic machinery iniron-stressed cells and the transport of Fe3+ in early exponentialgrowth.

Conclusion: This is the first comprehensive study on the effects of ironstarvation in toxic and non-toxic M. aeruginosa. Our results show thatthe process of adaptation to iron stress is strain-specific and highlydynamic. Toxicity appears to protect microcystin-producingcyanobacteria in the early stages of exposure to severe iron stress andmay protect the cell from reactive oxygen species-induced damage.We have initiated proteome studies to confirm this hypothesis.



OC-12.6

THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITEWITH ECOPHYSIOLOGICAL ROLES.

S.B. Watson1, F. Juttner2.1Environment Canada, Canada Centre for Inland Waters, Burlington,ON, Canada; 2Limnological Station, University of Zurich, Switzerland.

Introduction: Microcystis species produce sulphides, a sometimessignificant source of malodour. Although well documented in marinesystems, little is known about freshwater photoautotrophic productionof sulphides, which to date has been generally regarded asinsignificant. However, the apparent increasing number of Microcystisblooms may in fact represent a significant source of these compoundsin many inland waters.

Methods: We investigated the physiology and cellular/ecologicalfunction of sulphide production by four axenic and non-axenicMicrocystis aeruginosa strains. Using short-term incubations and anadapted Headspace-GC-MS technique and more focussed studieswith the axenic strain using different light regimes, metabolicinhibitors (sodium azide, DCMU), antioxidant enzymes (superoxidedismutase, catalase) and isotopically labelled precursors(hydrogencarbonate, acetate and sulphate)

Results: We found that isopropyl sulphides were of cyanobacterial inorigin. Our work demonstrated that i) isopropyl thiol (ISH) is the initialcell product, with subsequent extracellular conversion to diisopropyldisulphide and trisulphide); iv) ISH is actively produced over thegrowth cycle, iii) is continuously excreted with no internal storage orpost-lysis catalytic generation; iii) production occurs in both light anddark, increases up to an optimal light level and decreases significantlyunder very high irradiance; iv) labelling patterns indicate ISH issynthesized via the acetate pathway.

Conclusions: These experiments and related work using the twoaquatic invertebrates Thamnocephalus and Daphnia indicate thatisopropyl thiol plays several important physiological and ecologicalroles. It acts as an effective antioxidant against high levels of reactiveoxygen species, particularly in surface blooms; it elicits avoidance-related behavioural responses in grazer communities and at highlevels, can be toxic to some invertebrates.

OC-13.1

COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFURBACTERIA AND PURPLE SULFUR BACTERIA.

N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3.1Department of Biology, University of Copenhagen, Copenhagen,Denmark; 2Plant Biology Department, University of Geneva, Geneva,Switzerland; 3Department of Biochemistry and Molecular Biology,Pennsylvania State University, State College, Pennsylvania, USA.

Introduction: Green sulfur bacteria (GSB) and purple sulfur bacteria(PSB) are photoautotrophs that oxidize reduced sulfur compounds forgrowth. Whereas some aspects of their ecology are similar, otheraspects of their physiology and evolution are rather different. We areinterested in the evolution of these lineages and their photosynthetic,sulfur, and carbon metabolisms. Genome sequence data are availablefor >12 strains of GSB, but not for any PSB of the Chromatiaceae. Wehave therefore sequenced the genome of the PSB Thiodictyon sp.Cad16, which grows both photolithoautotrophically andchemolithoautotrophically.

Methods: Genome sequence data of Thiodictyon sp. Cad16 wasgenerated by pyrosequencing and annotated using software packagesfrom Softberry Inc. (www.softberry.com).

Results: The core genes of all analyzed GSB strains largely exhibitcongruent phylogeny. Photosynthesis genes are largely in agreementwith this core phylogeny. However, sulfur metabolism genes are oftennot congruent and are scattered across the GSB lineages suggestingextensive horizontal gene transfer events. The PSB Thiodictyon sp.Cad16 has an unexpectedly large genome (~8 Mbp), which reflectsthe large physiological diversity of this strain. CO2 fixation metabolismis supplemented by carboxysomes, previously not known to occur inPSB. Genes encoding sulfur metabolism exhibit unexpectedcombinations and similarities with both GSB and other PSB.

Conclusions: GSB have relatively small genomes (2–3 Mbp) reflectingtheir limited physiological capabilities. Some PSB have very largegenomes (~8 Mbp), whereas other PSB have much smaller genomes(3–4 Mbp), probably reflecting large variations in their environmentaladaptation. Finally, whereas the cellular core and key metabolismssuch as photosynthesis and carbon assimilation have rather differentorigins in GSB and PSB, their sulfur metabolism appears to share acommon origin.

OC-13.2

THE GENOME OF THE TOXIC BLOOM-FORMINGCYANOBACTERIUM ANABAENA SP. 90.

David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2,Kaarina Sivonen1.1Department of Microbiology and Applied Chemistry, University ofHelsinki, FIN-00014, Helsinki, Finland; 2Beijing Institute of Genomicsof the Chinese Academy of Sciences, Beijing Genomics Institute,Beijing, China.

Introduction: Toxic cyanobacterial blooms are linked to the deaths ofwild and domestic animals and are a health risk for human beings viarecreational or drinking water. Strains of the genus Anabaena areimportant primary producers in freshwater bodies throughout theworld and contribute to the formation of noxious cyanobacterialblooms through the production of a range of hepatotoxins andneurotoxins. Here we report the genome sequence of a hepatotoxicbloom-forming cyanobacterium Anabaena sp. 90.

Methods: We constructed a whole-genome shotgun assembly from119,316 sequencing reads from 2 kb, 6 kb and 40 kb libraries. Thesewere organized in pairs by virtue of end-sequencing 6-kb and 50-kbinserts from shotgun clone libraries. The quality-trimmed readscovered the genome 12.5 times.

Results: The length of the genome amounts to 5,305,674 bp and ispartitioned into two circular chromosomes and three plasmids.Essential house keeping genes were distributed across bothchromosomes while the plasmids consisted largely of hypotheticalopen reading frames. A total of 5,750 putative ORFs were annotatedfrom the genome with just over 20% of the predicted ORFs beingunique to Anabaena sp. 90. This strain is known to produce toxins andenzyme inhibitors and over 5% of the genome is dedicated to theproduction of bioactive compounds. During the assembly wedocumented insertions, deletions and single nucleotidepolymorphisms which in some cases has lead to the inactivation ofbiosynthetic pathways. Interestingly, approximately 7% of the genomeis composed of repetitive autonomous and non-autonomous mobileDNA which have inactivated a number of genes. Together thissuggests a mechanism by which Anabaena can tailor and customize its



genomic repertoire through genomic rearrangements. Analysis of thegenome revealed an investment in restriction modification systemsexplaining in part difficulties in genetic manipulation of this organism.Bioinformatic analysis revealed strong similarities to other heterocystforming cyanobacteria and preliminary evidence that phage infectionhas shaped the genome of this bloom forming cyanobacterium.

Conclusions: Further research will target a system biologicalinvestigation of this organism and is likely to reveal in-depthinformation of this ecologically relevant, bioactive compoundproducing cyanobacterium.

OC-13.3

CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS INDEVELOPMENT AND BEHAVIOR OF HORMOGONIA OF NOSTOCPUNCTIFORME.

Jack Meeks, Elsie Campbell, Rui Chen.

Department of Microbiology, University of California, Davis, CA, USA.

Introduction: The genome of the symbiotic nitrogen-fixingcyanobacterium Nostoc punctiforme contains five loci of genes thatencode core complex proteins analogous to the chemotaxis proteinsCheY, CheW, MCP and CheA of Escherichia coli. Genes in four of theloci appear to have evolved in the cyanobacterial lineage, while thosein the fifth locus have roots in the proteobacteria. We have set out tocharacterize these genetic loci in terms of organization andphysiological role.

Methods: A DNA microarray of N. punctiforme was used to examineglobal transcription profiles during nitrogen starvation induction ofheterocysts and hormogonia, and plant produced exudate containinga hormogonium inducing factor. The data were analyzed in the Rstatistical platform and normalized log2 of reference/experimentalvalues were k-means-clustered using the program Genesis. Insertionmutants were constructed by gene replacement, and cellulardifferentiation and behavior phenotypes analyzed by standardtechniques.

Results: Global transcriptional analyses indicate that 13 out of 35chemotaxis-like genes are expressed at a low level in ammonium-grown cultures; these include 3 CheB methylesterase and 2 CheRmethyltransferase-like adaptive proteins, all of which lack the responseregulator receiver domain and their genes are not collocalized in thementioned loci. Transcription of genes in the four cyanobacteriallineage loci are up-regulated only during hormogoniumdifferentiation, except for three genes in different loci (a MCP in locus1, and a cheA and cheW in locus 3) that are not transcribed under anycondition examined. Insertion mutations in genes encoding MCPproteins in the four loci in the cyanobacterial lineage yielded twodistinct phenotypes: a locus 2 MCP mutant fails to differentiatehormogonia and a locus 4 mutant is defective in phototaxis.Hormogonia of a locus 1 mutant have a slight morphologicalalternation, but are phototactic and infect a bryophyte symbiotic plantpartner, while a mutant in the MCP of locus 3 has no detectablephenotypic defect. The chromophore bound to the MCP of locus 3undergoes green-red light photoconversion. The genes in locus 4(phototaxis) include duplicates encoding CheW and partial MCP andCheA proteins. Insertion mutations in the duplicate MCP and cheAgenes yield phototaxis defective phenotypes. Only one MCP of locus5 (NpR0247) is significantly up-regulated late in hormogoniumdifferentiation, but the gene is difficult to manipulate in E. coli.

Conclusions: The transcription and mutant phenotypes indicate avariable organization of the multiprotein sensory transduction

complexes encoded in the four cyanobacterial lineage loci. Wepropose the proteins encoded by locus 4 assemble into a solublecomplex as heterodimeric MCP, CheW and CheA proteins, with one orboth CheY proteins communicating with the motility motor(s).Conversely, the proteins encoded in the membrane-associated locus 2complex (development) assemble as homodimers. The homodimericCheA and CheW of locus 1 may associate with the MCP encoded in adifferent locus. The MCP of locus 3 may assemble as a homodimerwith homodimeric CheW and CheA proteins also encoded in adifferent locus, perhaps locus 1. The MCP of locus five may be achemotactic sensor and interact with proteins encoded in other loci.

OC-14.1

COMPARISON OF NONCODING FEATURES OFCYANOBACTERIAL GENOMES.

Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, JoséCosta5.1Virginia Commonwealth University, VA, USA; 2University of Californiaat Davis, CA, USA; 3University of Pennsylvania, PA, USA; 4UppsalaUniversity, Uppsala, Sweden; 5University of Porto, Porto, Portugal.

Introduction: Insights from genomic sequences have been gainedprimarily analysis of the genes and protein functions inferred fromthem, but much can be learned beyond this analysis. Short repeatedsequences that are not explained by inclusion within multicopy genespose interesting biological questions: (1) By what mechanism are therepetitive units formed? (2) What selective forces act to preservethem? and (3) What effect do they have on cellular function?

Methods: The sequences of 42 cyanobacterial genomes (36complete) were analyzed using BioBIKE, an integrated knowledgebase and programming interface. DNA methylation was inferred bythe presence of orthologs of DNA methyltransferases with knownmethylation activity.

Results: Sequences of at least 24 nucleotides that appear multipletimes in the cyanobacterial genomes were dominated by three types:(1) long dispersed repeats often associated with transposes (henceprobably insertion sequences), (2) repeated sequences interspersedwith non-repeated elements, characteristic of CRISPRs (clusteredregularly interspersed short palindromic repeats), and (3) shortdispersed repeats (termed SDRs) not previously described. All of theseare potentially mobile sequences. We have shown by intergenomiccomparisons that SDR sequences, ranging in size from 21 to 28nucleotides, have moved in recent evolutionary times. They may bethe smallest mobile sequences known. Three families of SDRsequences (SDR4, SDR5, and SDR6). can assume the same conservedsecondary structure, even though they do not share significantsequence similarity. This suggests that they act through an RNAintermediate. One family, SDR5, specifically targets HIP1 sites(GCGATCGC).

For ease in discussion, we define Group F of cyanobacteria to consistof the filamentous cyanobacteria plus those unicellular cyanobacteriawithin the genera Synechocystis, Cyanothece, Crocosphaera,Microcystis, and Acaryochloris, as well as Synechococcus PCC 7002.Group P consists of all small marine Prochlorococcus andSynechococcus. The remaining cyanobacteria, Synechococcus PCC7942/6301, Thermosynechococcus, hot spring Synechococcus, andGloeobacter are ungrouped.

All Group F cyanobacteria were found to possess a high frequency ofHIP1 (GCGATCGC) sequences, as do Synechococcus PCC 7942/6301and Thermosynechococcus but not other genomes. Insertion



sequences were found to be prominant in all Group F cyanobacteriabut were seldom found in Group P cyanobacteria. CRISPR sequenceswere confined to Group F (though absent in many). Orthologs ofdmtA (encoding a GATC-specific DNA methyltransferase) are found inall cyanobacterial genomes outside of Group P. GGCC- and CGATCG-methylation (inferred from the presence of orthologs of dmtB anddmtC, respectively is found exclusively within Group F cyanobacteria.SDR sequences were frequent in heterocystous (filamentous)cyanobacteria and very rare in other genomes.

Conclusions: DNA sequence characteristics – the frequency ofrepeated sequences and methylation patterns – can be used to definecyanobacterial groups that make sense by comparison with otherphylogenetic measures. The groupings may relate to underlyingmolecular properties, e.g. recombination (HIP1) or possibly DNAreplication (GATC methylation). It is interesting that three conservedfeatures have overlapping specificity: GATC-specific methylation,CGATCG- specific methylation, and HIP1 (GCGATCGC), suggestingoverlapping function.

OC-14.2

HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) ASELECTRON DONOR? SEARCHING FOR THE ANSWER IN THEMEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM263.

Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, UlrichFischer1.1Zentrum für Umweltforschung und nachhaltige Technologien (UFT)and Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie,Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie;3Fachbereich Biologie/Chemie, Laboratorium für AllgemeineMikrobiologie, Universität Bremen; Bremen, Germany.

Intoduction: Green sulfur bacteria are anaerobic anoxygenicphotolithoautotrophs provided with a photosystem of type I and withpeculiar organelles, the chlorosomes, in which the light-harvestingpigments are organized. These bacteria couple the fixation of CO2 tothe oxidation of inorganic sulfur compounds or, in only one knowncase, ferrous iron. When sulfide or thiosulfate are the electron donors,elemental sulfur (S0) is an intermediate product, which can be furtheroxidized to sulfate.

S0 is thus a key substrate, whose insolubility in water constitutes aspecial task for the living cells. At present, it is unknown how greensulfur bacteria mobilize S0 and if the protein components involved aresubjected to transcriptional control.

Methods: The mesophilic strain Chlorobaculum parvum DSM 263,whose genomic sequence is now available, was chosen to investigatethe mechanism of S0 mobilization in green sulfur bacteria.

To preliminarily assess whether the cells need a direct contact withsulfur for its oxidation, previously prepared biogenic sulfur wasembedded into agar and brought in contact with a living culture. Theappearance of cleared zones at the border between cell culture andagar was then monitored.

For the proteomics experiment, Clb. parvum DSM 263 was grown onlyon sulfide or on biogenic sulfur previously extracted from a culture ofthe same species. French press was used to disrupt the cells andultracentrifugation to separate the soluble fraction from themembranes. 2-dimension gel electrophoresis with non-equilibrium-pH-gradient-electrophoresis (NEPHGE) as first dimension andmatrix-assisted-laser-desorption/time-of-flight (MALDI/TOF) massspectrometry were used to identify which proteins are differentially

expressed in the membranes of the 2 populations.

Results: Neither a clear zone could be observed in the agar withbiogenic sulfur, nor a culture could grow if the embedded sulfur wasthe only electron donor present. These results allowed a focus on themembrane fraction, in which differences in the analysed proteinpatterns were found, a major one regarding a conserved hypotheticalprotein.

Conclusions: We could show that cells of Clb. parvum DSM 263 needa direct contact with S0 for its mobilization and that at least some ofthe proteins involved in the utilization of this electron donor appear tobe subjected to transcriptional control.

OC-14.3

PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS:INSIGHTS INTO THE SULFUR METABOLISM OF A PHOTOTROPHICGREEN BACTERIUM.

Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, AndersMellerup2, Kirsten S. Habicht3, Raymond P. Cox1, Jens S. Andersen1 &Niels-Ulrik Frigaard2.1Department of Biochemistry and Molecular Biology, University ofSouthern Denmark, DK5230 Odense; 2Department of Biology,University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center forEarth Evolution and Institute of Biology, University of SouthernDenmark, Odense; Denmark.

Introduction: Green sulfur bacteria (GSB) use reduced sulfurcompounds as electron donors for their photosynthetic carbon dioxidefixation. They are often found in stratified lakes at, or below, the levelin the chemocline where sulfide is exhausted. The sulfur metabolism inGSB is complex; almost all green sulfur bacteria oxidize sulfide (HS-)and elemental sulfur (S0) but in addition some are able to utilizethiosulfate (S2O3

2-). GSB have a high affinity for sulfide and thissubstrate is taken up in preference for other sulfur substrates such asthiosulfate. Predictions from the genome sequence of Chlorobaculum(Cba.) tepidum TLS suggest that at least 60 gene products participatein sulfur oxidation pathways (1).

Methods: To obtain information about gene expression during variousstages of the sulfur metabolism of Cba. tepidum, we have grown thecells in batch culture in a medium containing both sulfide andthiosulfate. Proteome analyses were performed at two stages: once inthe early exponential growth phase where the cells are consumingsulfide, and once at the end of the exponential growth phase wherethe cells have utilized almost all thiosulfate. Proteins were extracted,digested with trypsin and the peptides released were analyzed usingan Agilent HPLC coupled to either a LTQ FT Ultra (Thermo) or a LTQOrbitrap XL (Thermo) mass spectrometer. Proteins where identifiedusing Mascot and quantified using MaxQuant. Samples were run induplicate on each system and the relative expression of proteins wascalculated on basis of the measured intensities.

Results: In total 945 proteins of the 2,288 coding sequences on theCba. tepidum genome were identified on the basis of at least twounique peptides. We detected 34 of the 60 proteins proposed to beinvolved in the sulfur metabolism of Cba. tepidum and most of theseproteins were equally expressed in the two samples. However, in cellsin the early exponential growth phase with active sulfide uptake wefound a two-fold up-regulation of the dissimilatory sulfite reductaseenzymes DsrE, DsrF, DsrH and the Rubisco-like enzyme RLPcompared to cells growing on thiosulfate where the sulfur oxidizingenzymes SoxX, SoxY & SoxZ and quinone modifying oxidoreductaseQmoA & QmoB and the soluble cytochrome c555, CycA were up-



regulated. Results will also be presented on proteome analysis of amutant of Cba. tepidum that lacks the DSR enzyme system and isdeficient in elemental sulfur utilization.

(1) Frigaard & Bryant, 2008. p 60-76. In C Dahl and CC Friedrich (eds.),Microbial sulfur metabolism. Springer, New York

OC-15.1

A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALEPHOTOBIOLOGICAL HYDROGEN PRODUCTION UTILIZINGCYANOBACTERIA.

Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue.

Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka,Kanagawa, Japan.

Introduction: In order to meet for the demand for large-scaleproduction of renewable energy, we have proposed nitrogenase-based photobiological production of H2 using mariculturedcyanobacteria. Although most of the technologies are still in thebeginning stages of development, a preliminary trial cost analysis wasmade in order to assess the future prospects for the economicalviability of large-scale renewable energy production.

Methods:

Assumptions:

1) Total solar radiation on the surface of Earth: 2.7 x 106 EJ per year (or5.3 x 109 J m-2 year-1, 1,680 kWh m-2 year-1 or 14.5 MJ m-2 day-1) onaverage.

2) Cyanobacteria photobiologically produce H2 at 1% efficiency vs.total solar radiation (5.3 x 107 J m-2 year-1).

3) Finally, available H2 energy: one and two thirds of biologicallyproduced H2 energy, consuming two and one third needed forharvesting, purification, storage, and transportation to land (1.8 and3.5 x 107 J m-2 year-1, respectively, in the final product).

4) Area required: 38.8 x 1018 divided by 1.8 and 3.5 x 107 = 2.2 and1.1 x 1012 (m2) (=2.2 and 1.1 x 106 km2), respectively.

Results:

1. A plausible general scheme1) Cyanobacteria produce H2 with sunlight as the sole source ofenergy. The substrate is water and products are H2 and O2. Theculture medium is simple in composition and contains mineralnutrients, and the gas phase in the bioreactor is Ar plus lowconcentrations of CO2 and N2.

2) The bioreactor consists of three layers plastic bag (inner: retainsculture medium, middle: hydrogen gas barrier, outer: mechanicallyprotects the inner twos) and is floated on a calm sea surface.

3) Repeated harvesting of gas. When H2 gas attains a certainconcentration level (e.g. 25 - 30%, v/v), a factory ship harvests thegas mixture (main components: Ar, H2, O2) from bioreactors, andpurifies H2 on board.

2. Cost analysis1a) Nutrients. Potentially growth-limiting nutritional elements are

added to the natural water (20 cm in depth) as ‘fertilizers’ akin toagricultural practices. [1.5 - 12 cents m-2 of the bioreactor surface]

1b) Water. Reference price of water for industrial use in Japan at about7.5 - 60 cents per ton. [1.5 - 12 cents m-2]

1c) Bioreactor. The average plastic price of $2 - $4 per kg and theplastics are recycled at the half prices of the feed stocks [50 - 100cents per m2 of bioreactor,]

1d) Gas (mostly Ar). FOB, bulk rate of Ar: $100 - 500 per ton. [9 cents -45 cents per m2]

1e) Other costs: the cost of growing cyanobacteria, the labor cost, thecost of ships, the interest on capital goods, and the cost of marinetransportation of production materials. [Cost E].

1f) Cost of gas harvesting, H2 separation, storage, and marinetransportation to final destination. [Cost F]

2) Depending on the Cost E and F, the final cost of H2 at end-users:1.8 - 13.4 (in cents MJ-1). Comparison; wind electricity: 2.0 - 5.2,solar photovoltanics: 10 - 50, crude oil: 0.8 - 2.4 (50 - 150 dollarsbarrel-1) or 2.4 - 7.2 (conversion efficiency of 1/3 into electricity).

Conclusions: Our trial calculation indicates that it will be difficult forbio-solar H2 production to be competitive with fossil fuels at currentprices, but as specific policies come into play such as carbon taxes,there will likely be an acceleration of its development. In spite of thetechnical as well as economical difficulties, we propose that large-scale H2 production by mariculture is the most promising strategy tobe pursued for “mitigating dangerous anthropogenic interference withthe climate system” (the purpose of UNFCCC).

OC-15.2

SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTIONAMONG DIVERSE HETEROCYSTOUS CYANOBACTERIA.

Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples,Polly Berseth, Tommy Sessions.

Savannah River National Laboratory, Aiken, SC, USA.

Introduction: H2 generated by cyanobacteria is a highly attractiveform of alternative energy. Much progress has been made to this end,yet an important element of the research continuum has been largelyoverlooked in recent years - the physiology and diversity of naturallyoccurring, H2-producing cyanobacteria. The overall goal of theresearch described here was to develop and apply a systematicapproach for evaluating the H2 production capacity among diverseheterocystous cyanobacteria. Specifically we wished to: 1) compareseveral environmental parameters (O2 concentration, N2 concentration,light intensity, and carbon source) that control H2 production amongdiverse heterocystous cyanobacteria, and 2) establish benchmarkactivities/phenotypes against which to routinely evaluate newlydiscovered H2 producing cyanobacteria.

Methods: Cyanobacteria (10 diverse heterocystous strains weretested) were cultured in Roux flasks containing BG11- media bubbledrapidly with air under a light (70-100 µmol m-2 s-1)/dark photocycle(16:8 h). To assay biological hydrogen production, cells wereconcentrated via centrifugation, washed, and placed in sealed vialswhere an argon atmosphere (≥ 98.5% argon) was generated.Headspace gas compositions of the vials were adjusted with N2, O2, orCO2 and gas samples were analyzed with MTI Model M200D microgas chromatograph equipped with a TCD detector.

Results: All strains exhibited an immediate inhibition of H2 productionrates upon the addition of N2. Compared to the activity of cellsincubated in vials containing <1% N2 headspace volume, H2

production rates measured for the individual strains decreased 38-61%in the presence of 5% N2, 71-92% in the presence of 20% N2, and 89-97% in the presence of 80% N2. It was determined that althoughnitrogenase is sensitive to O2, low levels of O2 (typically 2.5-5% atm)are required for respiratory metabolism to generate sufficientreductant and/or ATP to drive H2 production. The addition of glucose(10 mM) accelerated H2 production in 7/10 strains examined, with rateincreases ranging from 1.1-14.5 fold and yields increasing 1.4-88.1fold. When cells were incubated in the absence of an exogenouscarbon source, all of the strains exhibited dramatic increases in H2

yields (13.2-18.0 fold) when the light intensity was increased from 30



to 200 µmol m-2 s-1. The stimulatory effect of increased light intensityon cyanobacterial H2 yield was generally not as pronounced whenstrains were incubated in the presence of glucose (1.4-10.5 foldincreases). Of particular interest, cells of 4 strains exhibited nearmaximal H2 yields at light intensities <100 µmol m

-2 s-1 when incubatedwith glucose

Conclusions: The immediate, inhibitory effect of N2 on H2 productionwas quite similar across a phylogenetically diverse collection ofheterocystous cyanobacteria, enabling us to establish a baseline fromwhich to evaluate the “N2 sensitivity” of H2 production among theseorganisms. Our results also highlight that it is critical to optimizereductant availability for H2 production. Addition of sugars and lowlevels of O2 (2.5-5% atm) may be useful for many strains. Thecommonly held belief that the economic feasibility of cyanobacterialH2 production is dependent primarily on the light to H2 conversionefficiency may be fundamentally flawed. Mixotrophic conditionsshould be explored as a means to produce biohydrogen withcyanobacteria.

OC-15.3

THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLEBACTERIA WITH DARK FERMENTATIVE HYDROGENPRODUCTION AND HYDROGEN ELECTRODE.

A. Tsygankov.

Institute of Basic Biological Problems, Russian Academy of Sciences,Pushchino, Moscow Region, Russia.

Introduction: Green and cost-effective H2 production and H2-dependent electricity generation are of the key problems restrictingthe development H2-based economy. Purple bacteria produce H2

using light energy and simple organic compounds with high rates. Thesource of organic compounds is one of factors preventingdevelopment of biotechnological H2 producing systems based onpurple bacteria. Last years in different laboratories the integration ofpurple bacteria with dark fermentation of organic wastes are underinvestigation. This presentation describes the integrated systemconsisting of dark fermentation of wastes with H2 and concomitantorganic acids production, of purple bacterial H2 production under thelight using organic acids, and of hydrogen enzyme electrode for theelectricity generation using H2.

Methods: Experiments used bioreactors for dark fermentation,photobioreactors for suspension and immobilized purple bacteria, andhydrogen enzyme electrodes with various hydrogenases separatelyand in combination.

Results: The stability of dark fermentation and light-dependent H2

production by purple bacteria in continuous mode of operation wasstudied and it was shown that these processes are stable for months.Operational stability of hydrogen enzyme electrode in buffer system,as well as in medium from photobioreactor was studied also. Differentcombinations of reactors with photobioreactors, hydrogen electrodeswith bioreactors were tested and results will be presented in detail.

Conclusion: Results indicate that there are no principal factorspreventing an integration of bioreactors and hydrogen enzymeelectrode exist. However, the system needs in optimization forelectricity output.

The research was supported by Russian RAS Program for BasicResearch “Chemical aspects of energetics” and by Russian Foundationof Basic Research.

OC-15.4

HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUMRUBRUM UNDER RESPIRATORY DARK CONDITIONS.

Lisa Zeiger, Christiane Rudolf, Hartmut Grammel.

Max-Planck-Institute for Dynamics of Complex Technical Systems,Magdeburg, Germany.

Introduction: The aim of this study was to determine the maximalachievable cell concentration of the facultative photosyntheticbacterium Rhodospirillum rubrum in a bioreactor, thus allowing theestimation of potential volumetric yields in bioprocesses. Masscultivation of purple non-sulfur bacteria, incl. R. rubrum underphotosynthetic conditions is generally inefficient due to the inevitablelimitation of light-supply when cell densities become very high. For R.rubrum however, a cultivation process has been described that allowsthe high-level expression of photosynthetic membranes (PM),equivalent to light-grown cultures, under completely dark conditionswhen grown semi-aerobically in a bioreactor. On the basis of thiscultivation system, we developed a process for obtaining very highcell densities (HCD) in aerobic and semi-aerobic fed-batch cultivations.We also present a biochemical analysis of HCD cells and show that thephysiology of R. rubrum is affected significantly by the appliedconditions.

Methods: For HCD fed-batch cultivations, an exponential feedingalgorithm implemented in a computer process control system wasapplied. An unstructured computational model, based on mixed-substrate kinetics was developed for optimization of the process insimulation studies. For aerobic fed-batch cultivation, the exponentialfeed algorithm was applied in combination with a pH-stat element toprevent substrate accumulation. Semi-aerobic fed-batch cultures wereconducted with a different control strategy, using the culture redoxpotential as the manipulated variable. The obtained high density cellswere characterized by biochemical analyses, absorption andfluorescence spectroscopy, and HPLC-MS.

Results: Key growth parameters (saturation constants, inhibitionconstants, etc. ) were determined prior to the fed-batch experimentand used to parameterize the computational model and the controlleralgorithm. Two carbon substrates, fructose and succinate, werepresent in the feed because of the previously described elevated PMlevels in cells grown semi-aerobically in this medium composition. Theapplied exponential fed-batch strategy resulted in a maximum celldensity of 59 g L-1 cell dry weight in aerobically grown cultures. To ourknowledge, comparable values have never been reached before withR. rubrum or related species. An interesting finding was that highdensity cells accumulated and excreted large amounts of tetrapyrroleintermediates which were identified as protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester. Furthermore, attempts to inducePM expression by applying oxygen-limiting conditions surprisinglyfailed.

Conclusion: In this study we demonstrate that it is possible tocultivate R. rubrum under chemoheterotrophic conditions to, so farinaccessible high cell densities. Biochemical analyses revealedregulatory imbalances in HCD cultures at the branchpoint of aerobicand anaerobic tetrapyrrole biosynthetic pathways. An additionalfinding was that the induction of PM expression is dependent on thecell density and PM formation is severely impaired in HCD cells. Theresults are important for the developments of bioprocesses using thisorganism, and provide an interesting experimental system for theinvestigation of regulatory mechanisms at the heme-bacteriochlorophyll metabolic branch.



OC-15.5

DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA INMISSISQUOI BAY, QUEBEC, USING Q-PCR.

Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, FrancesPick3, David Bird4, Charles W. Greer1.1National Research Council, Biotechnology Research Institute, QC;2Environmental Health Center, Health Canada, ON; 3BiologyDepartment, University of Ottawa, ON; 4Biological Sciences, Universityof Quebec at Montreal, QC, Canada.

Toxic cyanobacterial blooms, and their increasing global occurrence,pose a serious threat to human health, domestic animals and livestock.The frequency and severity of bloom events continue to rise, mostprobably as a direct result of increased nutrient loading of watersystems worldwide. The number of lakes in Quebec, Canada, affectedby blooms has been increasing steadily from 34 (2004), to 45 (2005),84 (2006) and 197 (2007). Government agencies are undertremendous pressure to cope with escalating demands for wateranalysis, specifically for cyanotoxins. In Missisquoi Bay, LakeChamplain, public health advisories were issued from 2000 to 2008,and local microcystin concentrations found in lake water oftenexceeded the Canadian drinking water guideline of 1.5µg L-1.

A quantitative-PCR (Q-PCR) approach was developed for the earlydetection of blooms formed by microcystin-producing cyanobacteria.Primers were designed for the polyketide synthase (mcyD(KS)) and thefirst dehydratase domain (mcyD(DH)) of the mcyD gene, involved inmicrocystin synthesis. Q-PCR was used to monitor the blooms in thelittoral and pelagic zones of Philipsburg, Missisquoi Bay, during thesummers of 2006 and 2007.

Two toxic bloom events were detected by Q-PCR during the summerof 2006: more than 60,000 copies of the mcyD(KS) gene mL-1 weredetected in August and an average of 40,000 copies mL-1 weredetected in September, when microcystin concentations were morethan 4 µg L-1 and approximately 2 µg L-1, respectively. There was agood correlation between gene copy number and microcystinconcentrations. The Microcystis aeruginosa population followed thesame trend as the number of mcyD(KS) copies and microcystinconcentration in the littoral zone. The HPLC profile revealed a changein the toxin composition of the two bloom events. Four microcystinanalogues were observed during the bloom in August (MCYST-LA, LR,RR and YR). The MCYST-LA analogue was predominant in August anddominated the bloom in September. The other analogues identified inthe second bloom were MCYST-LR and YR.

Favorable conditions for a cyanobacterial bloom occurred only late inthe summer of 2007. Approximately 300 copies of the mcyD(KS) genemL-1 were identified in the pelagic zone at the end of August despitethe low concentration of toxin. The Q-PCR method was more sensitivethan standard chemical assays and allowed the detection ofmicrocystin-producing cyanobacteria as early as June. This techniquecould be used for the efficient monitoring of the most at-risk waterbodies.

OC-15.6

EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENESAND THE GENETIC POPULATION STRUCTURE OF TOXICCYANOBACTERIA.

Rainer Kurmayer, Guntram Christiansen.

Austrian Academy of Sciences, Institute for Limnology, Mondsee,Austria.

Introduction: Green- or red-pigmented cyanobacteria of the genusPlanktothrix spp. frequently contain the toxic heptapeptidemicrocystin. We could previously show that non-toxic strains isolatedfrom 28 water bodies in nine European countries have lost more thanninty percent of the large gene cluster (mcy) responsible formicrocystin synthesis (Christiansen, G, Molitor, C, Philmus, B,Kurmayer, R. 2008, Mol. Biol. Evol. 25:1695-1704). This gene lossevent probably happened million years ago. Due to its rather longhistory we hypothesized that the non-toxic strains meanwhile couldhave adapted to a variety of ecological factors not causally related tomicrocystin anymore. This would obscure a clear correlation ofmicrocystin production and ecological fitness.

Methods: In this study 139 red-/green-pigmented strains isolatedfrom lakes of three continents (Europe: 101 strains, North America:29,Sub-Saharan East Africa:9), were analysed (i) for their pigmentationand the production of microcystin, (ii) for the genetic variation withinfive housekeeping gene loci (16S rDNA, 16S rDNA-ITS, PC-IGS, PSA-IGS, RNase P), and (iii) for remnants of the mcy gene cluster (innon-toxic strains). Using established multi locus sequence typingtechniques the strains were phylogenetically assigned to severallineages that may be considered as cryptic ecotypes (Cohan, FM2002, Ann. Rev. Microbiol. 56:457-487). According to common theoryclonal dependence can only be observed if ecological differentiationprevents genetic exchange and therefore favours geneticdifferentiation.

Results: In total 58 non-toxic strains were found that were isolatedfrom all three continents. These non-toxic strains were found to lackthe mcy gene cluster but still contained at least one of the formerflanking regions. The majority of them (42 strains) shared a commonremnant of the mcy gene cluster, mcyT, a type II thioesterase.

The sequencing of the five housekeeping gene loci revealed six majorphylogenetic lineages. Those lineages consisted either of toxic strains(two lineages) or non-toxic strains (three lineages). Only one lineagecontained both toxic and non-toxic strains. Taking all strains togetherthe presence/absence of the mcy gene cluster was not related topigmentation: All toxic lineages and one non-toxic lineage containedboth green- and red-pigmented strains.

Conclusions: The fact that the mcy gene cluster seems to be patchilydistributed is due to a so far unrecognized clonal dependence. Weconclude that the clonal lineages have been adapted to significantlydifferent ecological niches forcing their genetic differentiation.

The identification of mcy gene cluster remnants supports the geneloss hypothesis and provides evidence against the role of a recenthorizontal gene transfer.

Interestingly the reddish type of pigmentation evolved several timesindependently in one toxic and two non-toxic lineages (after the lossof the mcy gene cluster).



OC-16.1

THE RESPIRATORY TERMINAL OXIDASES OF THECYANOBACTERIUM SYNECHOCOCCUS SP. STRAIN PCC7942.

Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder,Dominik Aschenbrenner.

Institute of Physical Chemistry, Vienna, Austria.

Introduction: All cyanobacteria respire with dioxygen as the terminalelectron acceptor. The key enzymes of respiration are the respiratoryterminal oxidases (RTOs) that transfer electrons to oxygen with wateras the reaction product. A search for genes encoding putative RTOs inthose cyanobacteria whose genomic sequence has been determined,has revealed a surprising variability both in the types of RTOs (at least7 different types) and in the number of different RTOs per cell (1 to10). Many cyanobacterial RTOs have not yet been characterizedbeyond their sequence similarity to RTOs from other organisms. Atleast some cyanobacteria have respiratory chains both in theircytoplasmic membranes (CM) and in their intracellular menbranes(thylakoids or ICM). In the ICM the respiratory chain sharescomponents with the photosynthetic electron transport chain. Thebest studied cyanobacterium in this respect is Synechocystis sp. strainPCC6803 that has three RTOs: one genuine aa3-type cytochrome coxidase, one quinol oxidase of the cytochrome bd-type - bothlocalized in the ICM - and one ARTO, (Alternate Respiratory TerminalOxidase, a homolog of cytochrome c oxidase that apparently does notreact directly with soluble cytochrome c) localized in the CM. The aimof this work was to study cyanobacterial genes that show sequencesimilarity to RTO of the cytochrome cbb3 type that is well known fromseveral organisms (including purple bacteria). In cyanobacteria, thecorresponding gene products have so far not been characterized at allto the point that at the beginning of our study it was even unclearwhether they actually encode an RTO. Only a few cyanobacteria(Synechococcus sp. PCC7942, Synechococcus sp. PCC6301 andTrichodesmium erythraeum IMS101) seem to contain these genes andamong these the strain of choice was Synechococcus sp. strainPCC7942, since it is well amenable to genetic manipulation. In otherorganisms cbb3-type RTO accepts electrons from soluble cytochromec and transfers them to dioxygen. In addition to the putative RTO ofthe cbb3 type , the genomic sequence of strain PCC7942 shows thepresence of one set of genes encoding an aa3-type (mitochondrial-type) cytochrome c oxidase and a set of genes encoding a bd-typequnol oxidase.

Methods: Mutants in two putative cbb3 genes (ccoN and ccoO) andalso a mutant with a deletion of the subunit I of cytochrome c oxidase(coxA) were kindly provided by Dr. Susan Golden. We have developeda method to separate and purify cyanobacterial CM and ICM from alarge culture (10 liters) using gradient centrifugation in a zonal rotor.The membranes were tested for in vitro cytochrome c oxidase activityby dual wavelength spectroscopy in a Varian Cary5spectrophotometer using horse heart cytochrome c reduced byascorbic acid as the electron donor.

Results: Wild type strain PCC7942 displayed in vitro cytochrome coxidation both in the CM and in the ICM. Compared to othercyanobacteria, the cytochrome c oxidation activity of PCC7942 CM ishigh on a per mg protein basis. The mutant strain lacking subunit I ofaa3 type cytochrome c oxidase also shows in vitro cytochrome coxidation in both CM and ICM. In contrast, the mutant strain lackingthe ccoN gene displayed no cytochrome c oxidase activity in the ICM ,but some activity in the CM.

Conclusions:We have shown for the first time that the ccoN gene of

Synechoccoccus sp. strain PCC7942 encodes a protein that is part ofan RTO enzyme able to oxidise reduced cytochrome c in vitro. Sinceno activity could be measured in the ICM of the ccoN deletionmutant, our results imply that the cbb3 enzyme of this strain is thesole cytochrome c oxidase in these membranes. The cytochrome coxidase activity activity of the CM of the ccoN mutant of strainPCC7942 must be due to the aa3-type cytochrome c oxidase. This isin striking contrast to Synechocystis sp. strain PCC6803, where theaa3-type cytochrome c oxidase is the only such enzyme in the ICMand is not present in the CM.

OC-16.2

PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA:STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN POST-TRANSLATIONAL MODIFICATION.

Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1,Gaozhong Shen2, Donald A. Bryant2, Wendy M. Schluchter1.1Department of Biological Sciences, University of New Orleans, NewOrleans, LA; 2Department of Biochemistry and Molecular Biology, ThePennsylvania State University, University Park, PA; USA.

Introduction: Cyanobacterial phycobiliproteins can contain one ormore of four different types of isomeric bilins: phycourobilin,phycoerythrobilin, phycoviolobilin (also called phycobiliviolin), andphycocyanobilin (PCB). These linear tetrapyrroles are derived fromheme and are covalently attached via thioether linkages at cysteineresidues within the α- and β-subunits. Phycobiliproteins also contain aunique, post-translational modification of a conserved asparagine(Asn) present at β-72, which occurs only on the β-subunits of allphycobiliproteins. We have identified and characterized several newfamilies of bilin lyases responsible for attaching PCB tophycobiliproteins, and the Asn methyl transferase for β-subunits inSynechococcus sp. PCC 7002.

Methods: Genes encoding putative enzymes involved in post-translational modifications of phycobiliproteins in Synechococcus sp.PCC 7002 were identified and characterized using comparativegenomics, reverse genetics, in vitro enzyme assays of the recombinantproteins, and in vivo heterologous expression in E. coli.

Results: Two new families of bilin lyases that are involved inphycobiliprotein biosynthesis in Synechococcus sp. PCC 7002, firstidentified in Fremyella diplosiphon as cpeS and cpeT, werecharacterized. There are three cpeS-like genes (denoted cpcS, cpcU,cpcV) and one cpeT-like gene (denoted cpcT) within the genome ofSynechococcus sp. PCC 7002. The CpcS and CpcU proteins form aheterodimer (1:1) and catalyze the addition of PCB to Cys-82 on CpcB(β-phycocyanin) and most allophycocyanin subunits (ApcA, ApcB,ApcD, and ApcF). CpcT attaches PCB to Cys-153 on CpcB. ApcE wasshown to have intrinsic bilin lyase activity and has a region withsimilarity to CpcS/CpcU. Many cyanobacteria have one CpcS/CpeSprotein that catalyzes bilin attachment. We hypothesized that theseproteins may form homodimers. Consistent with this, a 3D structureof CpcS-III from Thermosynechococcus elongatus BP1 (tll1699) wasrecently solved, and it crystallized as a homodimer. This is the firststructure solved for any bilin lyase, and we have verified it is the Cys-82 bilin lyase for attaching PCB to CpcB, ApcA and ApcB by in vitroenzyme assays.

The cpcM gene encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases. RecombinantCpcM methylated the β-subunits of phycobiliproteins (CpcB, ApcB,and ApcF) and did not methylate the corresponding α-subunits (CpcA,ApcA, and ApcD). Based upon in vitro studies with various substrates,



we conclude that this methylation probably occurs afterchromophorylation but before trimer assembly in vivo.

Conclusions: All enzymes required for post-translational modificationof all phycobiliproteins in Synechococcus sp. PCC 7002 have nowbeen identified and characterized, and we are beginning tocharacterize the order in which these post-translational modificationsoccur during phycobiliprotein biosynthesis. Now that the first bilinlyase structure has been solved, we hope to gain more mechanisticinsight into how these enzymes catalyze the specific addition of bilinsto their phycobiliprotein substrates.

OC-16.3

SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVELCYANOBACTERIOCHROME-TYPE PHOTORECEPTOR AnPixJ.

Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose,Tomoo Shiba, Shigeru Itoh, Genji Kurisu, Masahiko Ikeuchi.

University of Tokyo, Meguro, Tokyo, Japan.

Introduction: Phytochromes of plants, cyanobacteria and somebacteria are the photoreceptor superfamily that binds a lineartetrapyrrole and exhibits reversible photoconversion between the red-absorbing (Pr) and the far-red-absorbing forms. They exhibitphotochemical isomerization between the C15-Z and C15-E isomersof the linear tetrapyrroles. GAF domain plays a crucial role in thefunction of the chromophore.

Cyanobacteriochromes are the recently emerging photoreceptors incyanobacteria whose GAF domains are related to but distinct fromthose of the phytochromes. Mutational studies in cyanobacteria haverevealed that they are involved in various photoacclimation responsessuch as chromatic acclimation and phototaxis. GAF domains in thephototaxis regulator SyPixJ1 (from Synechocystis sp. PCC 6803) andTePixJ (from Thermosynechococcus elongatus BP-1) bind aphycoviolobilin and undergo the reversible photoconversion betweenblue-absorbing and green-absorbing (Pg) forms. Similar GAF domainsidentified in cyanobacterial genomes may function in novelphotoreceptor proteins.

Methods: The chromophore-binding GAF domains of thecyanobacteriochromes expressed in Synechocystis or phycocyanobilin(PCB) -producing Escherichia coli have been purified. The purifiedproteins were characterized by the spectral and crystallographicanalyses

Results: Putative phototaxis regulator AnPixJ (from Anabaena sp. PCC7120) binds PCB and shows reversible photoconversion between Prand Pg forms. Time-resolved spectral analysis suggest that the Pr formof AnPixJ is almost equivalent to that of the phytochromes and starts aprimary photoreaction with Z-to-E isomerization in a mechanismsimilar to that in the phytochromes, but is finally photoconverted tothe unique Pg form. On the other hand, chromatic acclimationregulator SyCcaS (from Synechocystis) covalently binds PCB andshows similar reversible photoconversion between Pr and Pg forms.However, relationship of chromophore configuration and spectralproperties is opposite between AnPixJ and SyCcaS. To understand thestructural basis, we are now trying the X-ray crystallographic analyses.

We determined the crystal structure of the GAF domain of AnPixJ inthe Pr form. The backbone structure of the protein and thechromophore configuration are independently similar to those of thePr form of bacterial phytochromes, although relative position of thechromophore bound to the protein is significantly deviated.Correspondingly, critical amino acid residues interacting with thechromophore are also diverged in position or orientation, and

resultantly in the function. Together with the role of water molecules, anovel protonation mechanism of the chromophore is proposed.Unique features of a hydrophobic pocket to accommodatephotoisomerization of the chromophore are discussed with regard toits unusual formation of the Pg form. We will discuss about structure-function relationship of the tetrapyrrole-based photoreceptors.

Conclusion: We found various spectral properties of thecyanobacteriochromes. Spectral analyses suggest that theseproperties are yielded by different chromophores, differentconjugated systems or different photoconversion mechanisms.Structure of the Pr form of AnPixJ provides direct insights into thephotoconversion mechanism. This new structural basis shed light onthe universal aspects of the photosensory mechanism of lineartetrapyrrole-based photoreceptors.

OC-16.4

CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OFSYNECHOCOCCUS ELONGATUS REVEALS A NEW ANTIDOTEMOLECULE.

Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz.

Bar-Ilan University, Ramat-Gan, Israel.

Introduction: Toxin-Antitoxin (TA) loci are ubiquitously present onchromosomal DNA of diverse bacteria. These modules were assigneda role in programmed cell death and it has been suggested that thepopulation as a whole benefits from sacrificing individual cells. Inother cases it has been shown that the un-neutralized toxin does notcause cell death, rather, it re-sets cell physiology and adjusts it tostress conditions. In the course of our global transcriptome analyses ofthe cyanobacterium Synechococcus elongatus, we noted that vapC,encoding for a toxin homolog, is highly induced upon nitrogen andsulfur starvation. Chromosomal TA systems of cyanobacteria have notbeen characterized thus far. Additionally, S. elongatus does notpossess a homolog of VapB, the typical cognate antidote of knownVap-systems. Thus, it was highly interesting to examine if VapC acts aspart of a TA system or functions as ‘solitary toxin’ and to address thecellular role of VapC in the context of a cyanobacterial cell.

Methods: We employed genetic and molecular tools including geneinactivation, over-expression and protein tagging to characterize thenature and role of VapC and the new antidote we identified.

Results: To test if VapC has a potential cytotoxic effect on S. elongatuscells, we expressed it using a strong promoter. VapC accumulationupon induction, as verified by Western analysis, was accompanied byrapid cell death. TA modules are typically encoded by operons, inwhich the gene for the antidote molecule precedes the toxin gene.Upstream of vapC, we noted an ORF and termed it VapX; this ORF isnot defined as a potential coding region in the current annotation ofS. elongatus genome. We bring evidence for the function of VapX,which is not homologous to proteins of known function, as anantitoxin. Over-expression of VapX together with VapC counteractsthe killing effect observed when VapC is over-expressed by itself.Furthermore, Northern analysis and RT-PCR indicate that vapXC forma bicistronic operon, a typical feature of TA modules. Experimentsinvolving expression of VapX and VapC in Escherichia coli furthersupport the function of these components as a TA system.Additionally, these experiments indicate complex formation betweenVapC and VapX, in agreement with the suggested antidote function ofthe latter.

Conclusions: VapC of S. elongatus has a cytotoxic effect whenexpressed by a strong promoter. Additionally, we present evidence



based on several observations supporting identification of VapX as anew antitoxin of the Vap-system. To the best of our knowledge, ourstudy provides the first characterization of cyanobacterialchromosomal TA system. The role of the Vap-system of S. elongatuswill be discussed in light of quantitative analyses of natively expressedVapX and VapC and inactivation of vapXC as well as additional TAsystems of this cyanobacterium.

OC-16.5

HOW CYANOBACTERIA BORE (AND WHY LATERALHETEROCYSTS EXIST).

F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao.

School of Life Sciences, Arizona State University, Tempe, AZ, USA.

Introduction: Some cyanobacteria bore into carbonates. They are few,but ancient players in geologic phenomena such as the destruction ofcoastal limestones, the reworking of carbonate sands and thecementation of microbialites. They constitute important tools forpaleoenvironmental reconstruction, and play a significant role as pestsin marine aquaculture. In spite of their importance, the mechanism bywhich cyanobacteria excavate minerals remains unknown. In fact, thisability represents a geochemical paradox, in that autotrophicmetabolism will push the carbonate system towards precipitation, notdissolution. We have advanced mechanistic models that may allowcyanobacterial boring to proceed and be still consistent withgeochemistry. These are based on either temporal or spatialseparation of photosynthesis and boring, or on the active extrusion ofcalcium through a cellular uptake and transport process. Until now,experimental approaches were hampered by the lack of cyanobacteriathat could bore in the laboratory. We report here on investigationsusing a novel isolate of the filamentous heterocystous cyanobacteriumMastigocoleus/Fischerella strain BC008 that maintains boring in thelaboratory.

Methods: Growth and boring of axenic cultures of BC008 wereinvestigated on different crystalline minerals, and under a variety ofconditions. The microenvironments of cyanobacterial filaments activelyboring on calcite chips were monitored quantitatively for free Ca2+

using in vivo fluorophore-enabled confocal fluorescence microscopy.The temporal dynamics of a variety of physical and chemical inhibitorson calcium transport dynamics in the cyanobacterium/mineral systemwere tracked using this system. PCR-based methods were used todetect and track the differential expression of genes putativelyinvolved in boring.

Results:We could easily reject hypotheses involving the action ofaccompanying heterotrophic bacteria or the temporal separation ofphotosynthesis and boring. The range of mineral carbonates bored(Ca-bearing, or those containing its biochemical analog Sr, but notthose with Mg, Mg/Ca, Mn, Fe, K, or Na as metal) implicated Ca asnecessary for boring. Extracellular Ca2+ levels in and around theboreholes of actively boring BC008 were consistent with an activeuptake Ca2+ at the apical end cell (severe under-saturation) andextrusion at the surficial end of the filaments (supersaturation). Thisout-of-equilibrium state could be relaxed by i) ceasing illumination, ii)adding inhibitors of ATP generation, and iii) adding specific inhibitorsof P-type calcium ATPases, indicating that cellular energy is requiredfor this process, as are ATP-ase calcium transport systems. P-typeATPase genes could be detected in BC008’s genome.

Conclusions: Our experiments suggest that cyanobacteria bore byactively taking up Ca2+ ions from the leading end of a filament at thecost of energy, locally lowering extracellular calcium levels to the pointthat mineral equilibrium is displaced towards dissolution there.

Calcium is then subject to trans-cellular transport along the filamentand extruded at the lagging end to the outside aqueous medium.Proton antiport at the apical cell likely maintains charge andneutralizes alkalization by excess carbonate ions. Incidentally, anevolutionary pressure not to interfere with calcium trafficking, may bebehind the unusual property of Mastigocoleus (and BC008) to developlateral heterocysts.

OC-16.6

FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEENIRON HOMEOSTASIS AND OXIDATIVE STRESS.

Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2,Nir Keren1.1The Alexander Silberman Institute of Life Sciences, Department ofPlant and Environmental Sciences, Hebrew University, Givat Ram,Jerusalem, Israel; 2Department of Biological Sciences PurdueUniversity, West Lafayette, IN, USA; 3Department of Botany, Universityof Otago, Dunedin, New Zealand.

Introduction: Iron is an essential nutrient for the survival of allorganisms, including cyanobacteria. However, the same redoxproperties that makes iron a valuable cofactor also lead to oxidativeinteractions resulting in the formation of harmful radicals. Therefore,iron accumulation in the cells should be tightly regulated. Ferritinfamily proteins play an important role in iron homeostasis.Synechocystis sp. PCC 6803 contains two ferritin type storagecomplexes, bacterioferritin (BFR) and MrgA. Previous studiesdemonstrated the role of BFR and MrgA in iron storage. In addition,MrgA was found to play a key role in oxidative stress response.

Methods: In the study presented here, we examined the dual role ofthe ferritin family proteins using physiological and transcriptomicapproaches. These include measurements of growth of cyanobacterialcultures in media limited for iron by various chelators; analysis ofinternal transition metal quotas by ICP-MS; measurements ofphotosynthetic performance and transcriptional profiling by microarrayanalysis.

Results: Microarray analysis of iron limited wild type and ∆mrgAcultures revealed a remarkable up-regulation of oxidative stressrelated genes in the mutant cells. The PerR regulator was found toplay an important role in that process. Furthermore, we were able todemonstrate the connection between internal iron quota, thepresence of the two storage proteins and the sensitivity to externallyapplied oxidative stress. These data suggests a pivotal role for the twoferritin type protein-complexes of Synechocystis 6803 cells incoordinating iron homeostasis and the oxidative stress response.

Conclusions: The combined action of the two complexes allows forthe safe accumulation and release of iron from storage by minimizingdamage caused by the interaction reduced iron and oxygen radicalswhich are abundant in cyanobacterial cells due to the function of thephotosynthetic apparatus.



OC-17.1

RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONASPALUSTRIS, HARBORING THE CRTI REPORTER GENE TOMONITOR ENVIRONMENTAL TOXIC METALS.

Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid.

Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.

Introduction: Genetically engineered bacterial biosensors utilize livingcells as sensing elements that harbor a reporter gene linked to atranscriptional switch. The genetic engineering makes it possible forbacterial cells to elicit signal in response to a small amount ofenvironmental chemicals as effectors. Reporter gene systems that donot require instruments and exogenous chromogenic reagents areadvantageous in establishing simple monitoring protocols. By taking itinto account, the usefulness of several genes involved in carotenoidbiosynthesis pathways has been examined, and the crtI gene has beenselected as the reporter gene system of the purple bacterium, Rps.palustris. In the crtI reporter gene system, the crtI gene placeddownstream of a transcriptional switch is expressed, and complementsthe crtI deletion of host bacterium, only in the presence of effectors. Inthis study, Rps. palustris strains responding to arsenic andlead/cadmium were constructed, and their possibilities as biosensorsfor environmental monitoring were examined.

Methods: The green mutant, no. 711, was constructed throughdeletion of crtI from the chromosome. The arsenic crtI biosensor (As-CrtIBS) was made of the no. 711 strain transformed with the crtI geneunder regulation of the operator/promoter region of the ars operonand the arsR gene from Escherichia coli. The hmrR gene of Rps.palustris, encoding the heavy metal resistance transcriptional regulatorHmrR, and its upstream region were chosen as the transcriptionalswitch of the lead/cadmium crtI biosensor (Pb/Cd-CrtIBS).

Results: As-CrtIBS and Pb/Cd-CrtIBS changed color in response to 10μg/L or less As(III) and Pb(II)/Cd(II), respectively. This change could bedistinguishable to the sense of sight after 24-hour culture withoutfurther manipulation. The hue angle of cultures in the CIE-L*a*b* colorspace shifted from greenish yellow toward red when concentration ofthe metals increased (Fig). As(III) supplemented to mineral waters onthe market and environmental arsenic inBangladesh well waters were monitored.Although the basal color in the absence ofAs(III) shifted to red with an increase in thehardness of mineral water, As-CrtIBS heldresponsibility to As(III). As-CrtIBS couldscreen the well waters indicating an arsenicconcentration higher than 1 μg/L.

Conclusion: The crtI biosensors indicate practical detection limits andare applicable to monitoring of toxic metals in groundwater andfreshwater by the naked eye.

OC-17.2

THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTERCAPSULATUS CELLS VIA ACETATE PERMEASE.

Roberto Borghese, Davide Zannoni.

Department of Biology, University of Bologna, Bologna, Italy.

Introduction: Rhodobacter capsulatus is a purple non-sulfurphototrophic bacterium that shows a highly variable response to thetoxic oxyanion tellurite. It is highly susceptible to the oxyanion whilegrowing by respiration with a MIC (minimal inhibitory concentration) of8 μM, and is very resistant when grown phototrophycally with a MIC of

approximately 1 mM. Further, its susceptibility to tellurite underrespiratory conditions is affected by the carbon source. Under theseconditions the MIC for tellurite varies from 8 μM, with malate andother dicarboxylic acids, to 300 μM with acetate. R. capsulatus is alsocharacterized by a high tellurite uptake rate as compared to otherbacterial species. Uptake activity measurements have shown thattellurite enters the cells at a much lower rate when acetate is present.This effect is not seen when dicarboxylic acids are used instead (1).These early data have led to propose that tellurite exploits an acetatetransport system to enter R. capsulatus cells. In this work we show, forthe first time in a bacterial system, that the main entry gate for telluriteis acetate permease (actP), and that an insertional mutation in thisgene is sufficient to drastically reduce the oxyanion uptake.

Methods: Resistant mutants were isolated on aerobic RCV standardmedium in the presence of 100 μM tellurite. The actP insertionalmutant was constructed using the pJP5603 suicide vector (2). Telluriteuptake was measured as described by Turner et al. (3). MICdeterminations, conjugations, subcloning and PCR amplification wereperformed using standard molecular procedures.

Results: The resistant mutant RTe36, used in this work, showed a MIC50 fold higher than the wild type B100 and an uptake rate that waslimited to 25% of the wild type control. Complementation of themutant with a R. capsulatus cosmid library allowed the isolation of twocosmids which restored high level uptake and reduced the MIC wellbelow that measured in the mutant, but still above the w.t. basal level.From the cosmids it was subcloned the acetate permease gene (actP),which was identified by DNA sequencing, and was shown tocomplement the mutant by restoring both the sensitivity to telluriteand the uptake level of the w.t.. Finally, to confirm the role of actP, anacetate permease insertion mutant was constructed. Initialexperiments indicated that the actP – mutant has indeed a decreaseduptake and a MIC for tellurite higher than wild type.

Conclusions: This work demonstrates, for the first time, that acetatepermease is the main gate for tellurite entry into R. capsulatus cells.The residual uptake, still measurable in the mutant, indicates thattellurite can take advantage of other less efficient entry facilities. Thefailure to bring the MIC for tellurite in the complemented mutant,back to level of the wild type, as opposed to the complete restorationof the uptake rate, indicates that other mutations are present,determining the high level of resistance measured in the mutant. Workis already planned to address this question. Finally, it remains to bedetermined if acetate permease is involved in tellurite uptake in otherbacterial species and, if so, how spread is this mechanism.

Borghese R., Marchetti D. and Zannoni D. (2008) Arch. Microbiol.189:93-100

Penfold R.J. and Pemberton J.M. (1992) Gene, 118:145-146

Turner R.J., Weiner J.H. and Taylor D.E. (1992) Anal. Biochem.,204:292-295

OC-17.3

CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CR-PLATING INDUSTRY WITH EXOPOLYSACCHARIDE-PRODUCINGCYANOBACTERIA.

Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis

Deparment of Agricultural Biotechnology, University of Florence,Firenze, Italy.

Introduction: Chromium (VI) compounds are contained in the wastewaters deriving from a large number of industrial processes. In the lasttwenty years, the possibility to use specific microorganisms for theremoval of heavy metals from industrial waste waters has been widely



studied, but the possibility to use exopolysaccharide (EPS)-producingcyanobacteria for the removal of heavy metals in the anionic form wasnever tested. The aim of this study was the assessment of thecapability of some EPS-producing cyanobacteria to remove chromatefrom waste waters of a plating industry in lab and in semi-pilot systemsand to define the role of the different cell fractions (i.e. biomass andpolysaccharidic layers) in the metal removal process.

Methods: Exopolysaccharide-producing Cyanothece ET5, TI4, PE14,VI22, CE4, Cyanospira capsulata and Nostoc PCC7936, were testedfor their ability to remove Cr (VI) from waste waters of a platingindustry containing Cr (VI) 89.71% (w/w), Cr (III) 8.97%, other metals1.32%. Lab experiments were carried out for 48h with 50 ml of acid-pre-treated cyanobacterial cultures confined in dialysis tubing anddipped in 450 ml of the diluted industrial wastewater (pH 3; Cr (VI)concentrations in the range 10-200 mg L-1). Field experiments werecarried out with acid-pre-treated cultures confined in three differentsemi-pilot systems (a filter press, a filter column and a dialysismembrane system) operating with 30 L of the same diluted wastewater.

Results: In lab experiments, Cr (VI) was removed by Cyanothece CE4at a specific metal uptake of 18.0 ± 0.8 mg of Cr(VI) per g of cell dryweight, while three other strains, Cyanothece ET5 and PE14 andNostoc PCC7936 showed a metal uptake in the range 10 to 12 mg(Cr) (g dry wt)-1. All the other strains showed a lower capability toremove the metal.

The three semi-pilot systems operating with Nostoc PCC7936 biomassshowed a very different behavior in the metal removal process: in thedialysis membrane system, a decrease of the concentration of Cr (VI)and of total Cr was observed, both concentrations reaching, after 72hours, values corresponding to about 60% of the initial ones. In thefilter press system, the metal removal was done by flushing 30L of thediluted waste water containing 112 mg L-1 of Cr (VI). In theexperiment, a decrease in the concentration of Cr (VI) down to a valueof 0.40 mg L-1 was observed after 48 h; at the same time, the total Crconcentration only decreased from 123 to 53 mg L-1, owing to a verysignificant increase in the amount of Cr (III) present in solution. In thecolumn system, after 24 h almost all the Cr (VI) was reduced to Cr (III),without any significant decrease of the total Cr concentration. Thus,dried biomass of Cyanothece TI4, which is known to have a very highCr3+ uptake, was added on the top of the column. In the following 120h, the concentration of total Cr and of Cr (III) decreased to a value of3.6 mg L-1, while the concentration of Cr (VI) reached the value of zero.

Conclusions: The results obtained pointed out the complex roleplayed by the cyanobacterial biomass in the removal of Cr (VI): thecells, when subjected to treatments removing the externalpolysaccharidic structures, carried out the reduction of Cr (VI) to Cr(III), while the polysaccharidic fraction, previously released by the cellsand solubilized in the culture medium, was capable to remove the Cr(III) cations formed in the reduction process. The results obtained inthe semi-pilot systems showed that EPS-producing cyanobacteria canbe profitably used for the removal Cr (VI) from industrial waste waters.

OC-18.1

TRANSCRIPTIONAL REGULATION AND MATURATION OFCYANOBACTERIAL HYDROGENASES.

Peter Lindblad.

Department of Photochemistry & Molecular Science, UppsalaUniversity, Uppsala, Sweden.

Introduction: In cyanobacteria three enzymes are directly involved inthe hydrogen metabolism: a nitrogenase that produces H2 as a by-product of nitrogen fixation, an uptake hydrogenase (HupSL, encodedby hupSL) that recaptures H2 and oxidize it, and a bidirectionalhydrogenase (encoded by hoxEFUYH, forming an enzyme with ahydrogenase part, HoxYH, and an electron transfer partner protein,HoxEFU) that can both oxidize and produce H2, see Tamagnini et al2007. The transcription of cyanobacterial hup and hox operons areknown to be regulated. The maturation of hydrogenases into activeenzymes is a complex process and eg a correctly assembled active siterequires the involvement of at least seven proteins, encoded byhypABCDEF and a hydrogenase specific protease, encoded by eitherhupW or hoxW. We have addressed trancriptional regulation andhydrogenase maturation in a few selected strains.

Methods: The unicellular cyanobacterium Synechocystis PCC 6803contains a single bidirectional hydrogenase. The filamentouscyanobacteria Nostoc punctiforme and Nostoc PCC 7120 may containa single uptake hydrogenase or both an uptake and a bidirectionalhydrogenase, respectively. Molecular experiments were performed asdetailed elsewhere.

Results: Generally, little is known about the transcriptional regulationof cyanobacterial uptake hydrogenases. We showed that NtcA has aspecific affinity to a region of the hupSL promoter of Nostocpunctiforme. Truncated versions of the promoter region of the hupSLoperon were fused to gfp. All constructs showed heterocyst specificexpression (Holmqvist et al 2009). Unexpectedly the shortest promoterfragment, covering 57 bp upstream and 258 bp downstream the tsp,exhibited the highest promoter activity. The cyanobacterial hox geneshave been characterised in some strains. LexA interacts with thepromoter region of the hox operon in Synechocystis PCC 6803. Thehox genes of Nostoc PCC 7120 are transcribed as two differentclusters with LexA interacting with both promoter regions. DNAaffinity assays also identified an AbrB-type DNA-binding protein asinteracting directly with the promoter region of the hox operon inSynechocystis PCC 6803. This protein binds to the its own promoterregion and the promoter region of the hox operon, suggested tofunction as a positive regulator of hox gene expression. The presenceand expression of hyp-genes were examined in the N2-fixingcyanobacterium Nostoc PCC 7120. RT-PCRs demonstrated that the sixhyp-genes may be transcribed as a single operon. TSPs wereidentified 280 bp upstream from hypF and 445 bp upstream of hypC,respectively (Agervald et al 2008). Five upstream ORFs located inbetween hupSL and the hyp-operon, and two downstream ORFs fromthe hyp-genes were shown to be part of the same transcript unit.Transcriptional analyses were performed of hupW in Nostocpunctiforme, and hupW and hoxW in Nostoc PCC 7120. We identifiednumerous transcriptional start points together with putative bindingsites for NtcA (hupW) and LexA (hoxW) (Devine et al 2009). Aphylogenetic tree of hupW & hoxW showed a striking resemblance tothe subgroups previously described for [NiFe]-hydrogenases.Bioinformatic studies revealed a so called “HOXBOX”; an amino acidsequence specific for protease of Hox-type which might be involved indocking with the large subunit of the hydrogenase.



Conclusions: Significant advances have been made in theunderstanding of both the transcriptional regulations and thematuration of cyanobacterial hydrogenases.

References:

Agervald Å, Stensjö K, Holmqvist M, Lindblad P. 2008. BMC Microbiol8: 69

Devine E, Holmqvist M, Stensjö K, Lindblad P. 2009. BMC Microbiol 9:53

Holmqvist M, Stensjö K, Oliveira P, Lindberg P, Lindblad P. 2009. BMCMicrobiol 9: 54

Oliveira P & Lindblad P. 2008. J Bacteriol 190: 1011-1019

Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris D, HeidornT & Lindblad P. 2007. FEMS Microbiol Rev 31: 692-720

OC-18.2

PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THECYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803.

Carrie Eckert, Jianping Yu, and Pin-Ching Maness.

National Renewable Energy Laboratory, Golden, CO, USA.

Introduction: The photosynthetic cyanobacterium Synechocystis sp.PCC6803 has the capacity to integrate light harvesting with chargeseparation and transfer the electrons to a bidirectional NiFe-hydrogenase for the photoproduction of H2 from water. However, theH2-production reaction is short-lived in the presence of O2, the latteran inherent byproduct of oxygenic photosynthesis. We propose twoapproaches to surmount this challenge: (1) to gain more in-depthunderstanding of the Synechocystis Hox hydrogenase for itsimprovement; and (2) to genetically transfer and express a foreign O2-tolerant hydrogenase into Synechocystis. Both approaches will guidethe design of an O2-tolerant system in Synechocystis for sustained H2

production.

Methods: The native Synechocystis Hox hydrogenase is a pentamericcomplex consisting of HoxEFUYH subunits. HoxYH are the catalyticsubunits for H2 production, accepting electrons from NAD(P)Hmediated by the HoxEFU diaphorase moiety. We are geneticallydissecting the 5-subunit Hox complex by generating mutants lackingone or more of the subunits in order to probe their roles both inmaintaining complex stability and in hydrogenase activity. Our secondapproach entails the genetic transfer and expression in Synechocystisa hexameric O2-tolerant NiFe-hydrogenase (CooMKLXUH) from thepurple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus.The Coo hydrogenase is ferredoxin linked; its expression inSynechocystis may improve the overall efficiency of H2 productionusing a photo-reduced ferredoxin as the electron mediator.

Results: We have either acquired or generated Synechocystis mutantlines lacking one or more of the Hox subunits. Western blotsconfirmed the deletion of the subunits using their respectiveantibodies. We determined that mutant containing only the HoxY andHoxH subunits retained hydrogenase activity in vitro, using methylviologen as the redox mediator reduced by sodium dithionite.However, this mutant cannot produce H2 in vivo likely due to the lackof HoxEFU subunits, required for electron mediation from NAD(P)H.Moreover, mutants with deletions in HoxE, F, or U individually or invarious combinations still retained 20 to 50% of its in vitrohydrogenase activity, with most loss observed with deletions of theHoxFU subunits. In the second approach, we have successfullytransformed and expressed four Rubrivivax hydrogenase and itsrelated genes into Synechocystis hosts lacking native hoxH, using

either an integration or expression plasmid. The Rubrivivax transgenesare cooL, U, and H encoding the small and large subunits of a typicalNiFe-hydrogenase as well as hypA, putatively involved in nickelinsertion for hydrogenase maturation. However, no hydrogenaseactivity was detected in the recombinant Synechocystis strain,suggesting the need to transfer additional Rubrivivax hydrogenasegenes to yield activity.

Conclusions: Our approach to generate knockout mutants lacking thevarious Hox subunits led to the conclusion that (1) HoxYH aloneconstitutes in vitro hydrogenase activity and (2) HoxEFU are requiredfor complex stability and for NAD(P)H-supported H2 production, invivo. Work is underway to express additional Rubrivivax coohydrogenase and its hyp maturation genes into Synechocystis,presumably needed to confer activity for sustained photobiological H2

production.

OC-18.3

ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATIONOF THE BIDIRECTIONAL [NIFE]-HYDROGENASE FROMSYNECHOCYSTIS SP. PCC 6803.

Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2,Rüdiger Schulz3 and Frauke Germer3.1School of Life Sciences, Arizona State University, Tempe, AZ, USA;2Max-Volmer-Laboratorium, Technische Universität, Berlin, Germany;3Botanisches Institut, Universität Kiel, Kiel, Germany.

Introduction: In cyanobacteria two different types of hydrogenasesare known. They both belong to the NiFe-hydrogenases and arecalled uptake and bidirectional due to their physiologicalcharacteristics. The cyanobacterial bidirectional hydrogenase is ofparticular interest, since it is rapidly reactivated in the absence ofoxygen and its traits seem to be intermediate between the standardoxygen-sensitive and the oxygen-tolerant NiFe-hydrogenases. Inaddition, this hydrogenase is well suited for solar-driven biologicalhydrogen production in cyanobacteria.

In this study we developed a rapid and gentle method to purify thebidirectional hydrogenase of Synechocystis sp. PCC 6803 forbiochemical as well spectroscopical investigations to characterize itsactive site.

Methods: To overcome the low expression level of the bidirectionalhydrogenase and to simplify its isolation a strain containing the psbAIIpromoter upstream of the hox-operon was constructed. In the samestrain the Strep tag II was fused to the C-terminus of hoxF encoding asubunit of the diaphorase part of the enzyme. In the same strain ahyp-gene cluster containing the genes necessary for the post-translational processing of the hydrogenase was also overexpressed.

After isolation, the hydrogenase was subjected to EPR (electron pulseresonance) and FT-IR (Fourier transform infrared) spectroscopy indifferent redox states.

Results: The insertion of the psbAII promoter in conjunction with theStrep tag led to an increase of total hydrogenase activity of two fold.Overexpressing the hyp-genes led to an additional two fold increase.From these strains the hydrogenase could be isolated in a highly pureand active state after a single run of the affinity column.

EPR measurements reveal the presence of at least two redox activeFeS clusters, but no nickel specific signals. A paramagnetic Ni-specieswas absent under any conditions. This either indicates that the nickelstays in the diamagnetic Ni(II) state and the redox changes take placeat another ligand or that its spin is coupled to a nearby paramagneticcenter in the protein.



In the FT-IR spectra three different signals could be discerned, that areattributed to two cyanides and one carbon monoxide coordinating theiron ion.

The FT-IR spectra under different redox conditions are indicative of aNiFe-hydrogenase attaining a Ni-B like state not the Ni-A state whenoxidatively inactivated even in the presence of oxygen. For otherhydrogenases it is known that they can be rapidly activated when inthe Ni-B state but need much more time when in the Ni-A state.

Conclusions: The spectroscopic investigations of cyanobacterialbidirectional hydrogenase confirm its intermediary behavior betweenthe anaerobic standard enzymes and the aerobic oxygen tolerantenzymes. With these characteristics it is very well adapted to rapidlychanging redox conditions in oxygenic phototrophs. It will be quicklyactivated under anoxic conditions and able to produce hydrogen byfermentation. It will produce hydrogen as an overflow valve whenphotosynthesis resumes from oxygen free conditions, but it will alsobe very rapidly inactivated when photosynthetic oxygen productiontakes over so as not to waste energy.



Notes



P.001

FUNCTIONAL ANALYSIS OF THE PUH PROTEINS INRHODOSPIRILLUM RUBRUM.

Caroline Autenrieth, Robin Ghosh.

Dept. of Bioenergetics, University of Stuttgart, Stuttgart, Germany.

Introduction: Our present view of the photosynthetic unit (PSU) ofRhodospirillum rubrum is that it contains a reaction centre (RC; H, Land M subunits) surrounded by a light-harvesting (LH) 1 complex,composed of alpha and beta subunits which aggregate to form a 16-fold circular assembly. The polypeptides of the LH1 complex as well asthe L and M subunits of the RC are encoded by the puf operon,whereas the H subunit is encoded by the puhA gene of the puhoperon. The puh operon encodes a number other putative geneproducts (puhB, puhC, puhD and puhE) which have been implicated inPSU assembly though their precise role has been unclear so far. In thisstudy we provide new biochemical, functional insight into the role ofsome of the puh proteins in PSU assembly.

Methods and Results: Selected puh proteins were deleted from theR. rubrum chromosome and the phenotype examined. Thephenotype, which is unique so far, was quantified functionally usingspectroscopic and biochemical methods. The results point to anunsuspected role of the puh proteins in PSU assembly.

Conclusions: We will present data as well as a structural hypothesis,that for R. rubrum, the structure of the PSU is more complex thanpreviously thought. In particular, we present a new biochemical insightinto the role of the proteins encoded by the puh operon. If ourhypothesis is correct, our view of the PSU will have wide-reachingconsequences for the interpretations of spectroscopic, cryoEM andAFM data in general.

P.002

THE ROLE OF CYTOCHROME C-554 IN THE ELECTRONTRANSFER PATHWAYS IN THE GREEN SULFUR BACTERIUMCHLOROBACULUM TEPIDUM.

Chihiro Azai1, Yusuke Tsukatani2, Jiro Harada3, Ryo Miyamoto4, ToruKondo4, Hiroumi Murakami4, Shigeru Itoh4, Hirozo Oh-oka1.1Department of Biological Sciences, Graduate School of Science,Osaka University, Toyonaka, Osaka 560-0043; 2Institute for BiologicalResources and Functions, National Institute of Advanced IndustrialScience and Technology (AIST), Tsukuba, Ibaraki; 3Department ofBioscience and Biotechnology, Faculty of Science and Engineering,Ritsumeikan University, Kusatsu, Shiga; 4Division of Material Science,Graduate School of Science, Nagoya University, Nagoya, Aichi; Japan.

Introduction: In the green sulfur bacterium Chlorobaculum (Cba.)tepidum (syn. Chlorobium tepidum), the photo-oxidized primaryelectron donor, P840+, is rapidly rereduced by cytochrome (cyt) cz, oneof subunits of the reaction center (RC) complex. Previous studies haverevealed two different electron donors to the oxidized cyt cz. In thepurified membranes, menaquinol:cyt c oxidoreductase function as thedirect electron donor to cyt cz, while in the in vitro reconstitutionexperiment using the purified RC complex, water-soluble and lowmolecular-weight (approx. 10,000) cyt c-554 (CT0075) does. However,it has still remained unknown whether the electron transfer schemefrom menaquinol:cyt c oxidoreductase to cyt cz via cyt c-554 isoperative when all of them co-exist together in vivo.

On the other hands, green sulfur bacteria use reduced sulfurcompounds (sulfide, thiosulfate, polysulfide and elemental sulfur) as

the photosynthetic electron sources. Electrons obtained from theoxidations of these sulfur compounds are transferred to the RCthrough the photosynthetic electron transfer pathways. Althoughseveral in vitro reconstitution experiments carried out in 1970sdemonstrated that cyt c-555 isolated from Cba. thiosulfatiphilum,which corresponds to cyt c-554 from Cba. tepidum, could acceptelectrons from sulfide/thiosulfate oxidation systems, there has been noexperimental evidence to support this in vivo.

In this study, we constructed three mutants of Cba. tepidum, whichwere devoid of cyt c-554 (cycA::aadA strain), SoxB (CT1021)(soxB::aacC1 strain), and cyt c-554 and SoxB (cycA::aadA/soxB::aacC1strain), respectively, to investigate electron transfer pathways coupledwith sulfide/thiosulfate oxidations.

Results: When purified cyt c-554 was added to the membranesprepared from the cycA::aadA strain, the oxidized cyt cz, immediatelyformed after flash excitation, was rereduced by both cyt c-554 andquinol oxidoreductase independently; the contribution of the formerbecame larger by increasing its concentration, indicating that thereaction between cyt c-554 and cyt cz obeyed the second-orderreaction mode. Unlike the case of the electron transfer reaction fromcyt bc1 complex to cyt c2 in purple bacteria, cyt c-554 in the Cba.tepidum never serves as a shuttle-like carrier between menaquinol:cytc oxidoreductase and cyt cz.

The cycA::aadA strain exhibited a decreased growth rate but normalgrowth yield when compared to the wild type. In accordance withthis, the strain was found to oxidize thiosulfate more slowly than thewild type but completely to sulfate as the wild type. This indicatesthat cyt c-554 is not indispensable for thiosulfate oxidation itself.

Furthermore, both the soxB::aacC1 and cycA::aadA/soxB::aacC1strains did not grow at all in a medium containing only thiosulfate asan electron source. They also grew incompletely even in a mediumcontaining only sulfide when compared to the wild type. Thissuggests that SoxB is not only essential for thiosulfate oxidation butalso responsible for sulfide oxidation.

Conclusions: In Cba. tepidum, cyt c-554 serves as an electron carrierbetween Sox system and cyt cz in the RC. But an alternative electroncarrier or electron transfer path should be operating between them,judging from the complete oxidation of thiosulfate in the cycA::aadAstrain. The Sox system also seems to be involved in the sulfideoxidation, whose issue would be a future work to be resolved.

P.003

EXPRESSION OF HYDSL HYDROGENASE FROM THIOCAPSAROSEOPERSICINA IN ESCHERICHIA. COLI.

Horcheska Batyrova, Anna Khusnutdinova, Galina Shirshikova, OlgaPostnikova, Elena Patrusheva, Aleksander Boutanaev, AnatolyTsygankov.

Institute of Basic Biological Problems, Institutskaya, Pushchino,Moscow Region, Russia.

Introduction: The purple sulfur phototrophic bacterium Thiocapsaroseopersicina BBS contains several Ni-Fe hydrogenases. One of twomembrane-bound hydrogenases appears to be very stable againsttemperature, CO, H2S and proteases. The Ni-Fe hydrogenase HydSLassembly includes the concordant expression of structural genes (hydSand hydL with two additional genes of unknown functional) as well asa number of accessory genes, participating in the maturation processof this enzyme. Structural genes have been cloned by the K.L. Kovacsteam (Szeged, Hungary) earlier and their sequences are available inGenbank database.



The hydSL genes are expressed on the low level in T. roseopersicinawhat makes difficult both basic and applied investigations of theprotein. One possibility to increase HydSL yield might beheterologous expression in a well established system, for instance,such as Escherichia. coli. However, little is known about this possibility.From the other hand, unlike many other hydrogenases, accessorygenes of HydSL are dispersed along the T. roseopersicinachromosome what makes difficult their complementation study in E.coli cells. The aim of this research is the expression of hydS and hydLin heterologous system.

Methods: In this investigation we started from an attempt to expressin E. coli two structural genes hydS and hydL, keeping in mind thatsome of E. coli accessory genes might complement the lack of theircounterparts in T. roseopersicina, as well as the fact that high level ofNi in growth medium might compensate, at least partially, some of T.roseopersicina accessory genes.

Two genes have been cloned by means of PCR and artificial operonhas been constructed in E. coli pET22b expression vector. After that,all two genes have been expressed in E. coli.

Results: An artificial operon from two genes (hydS, hydL) has beencreated to investigate expression of HydSL hydrogenase fromThiocapsa roseopersicina in E.coli. Proteins were overexpressed.However no hydrogenase activity was found. Detailed description ofexperiments will be presented.

Conclusion: The construction with hydS and hydL was created,introduced in E.coli cells and HydS and HydL proteins wereoverexpressed.

P.004

THE RHODOBACTER SPHAEROIDES REACTION CENTERASSEMBLED WITH ZINC BACTERIOCHLOROPHYLL YIELDSEVOLUTIONARY INSIGHTS.

Su Lin*†, Paul R. Jaschke§, Haiyu Wang*, Mark Paddock¶, Aaron Tufts†,James P. Allen†, Federico I. Rosell||, A. Grant Mauk||, Neal W.Woodbury*†, J. Thomas Beatty§

*The Biodesign Institute at Arizona State University, Arizona StateUniversity; †Department of Chemistry and Biochemistry, Arizona StateUniversity; §Department of Microbiology and Immunology, TheUniversity of British Columbia; ¶Department of Physics, University ofCalifornia, San Diego; ||Department of Biochemistry and MolecularBiology, The University of British Columbia.

Introduction: The purple bacterial reaction center RC binds 6 chlorincofactors: the ‘special pair’ (P), a dimer of bacteriochlorophyll (BChl) amolecules on the periplasmic side of the RC; two monomeric BChls(BA and BB) present on either side of P; and two bacteriopheophytin(BPhe) molecules (HA and HB). When P is excited, an electron istransferred through BA to HA, and then to a quinone. Studies on avariety of species and mutants in which cofactors were changedindicated the importance of the energetics of the cofactors ininfluencing ET rates.

The RC from a magnesium chelatase (bchD) mutant of Rhodobactersphaeroides was studied. In this RC, which we call the Zn-RC, thespecial pair (P) and accessory (B) bacteriochlorophyll (BChl) bindingsites contain Zn-BChl rather than BChl. We used spectroscopicmeasurements to fully elucidate the chlorin composition at the P, Band H sites, and to measure the efficiency of ET.

Methods: Mutant and wild type RCs were isolated and the absorptionspectra at room temperature and 10 K were measured, as well as thekinetics of ET in timescales ranging from fs to ms. The

electrochemical midpoint of P was determined by potentiometrictitration and measurement of changes at the P absorption maximum.

Results: Analysis of the room temperature and 10 K absorptionspectra of the Zn-RC revealed that the P, B and H sites all contain Zn-BChl. Furthermore, the absorption peaks in the visible regionindicated that the metals of the Zn-BChls in the P and B sites arepentacoordinated, as is the Mg of BChl in these sites in the wild typeRC, whereas the Zn-BChls in the H sites appear to betetracoordinated.

The midpoint potential of P in the Zn-RC was determined to be 515 ±5 mV, similar to the value of 505 ± 5 mV for the wild type RC.Photobleaching experiments indicated that ET proceeds from P to thequinones, producing a long-lived charge-separated state, as in thewild type RC. The ultrafast kinetics of ET from P to HA in the Zn-RCwere found to be very similar to those in the wild type RC, in contrastto an RC in which HA is BChl.

Conclusions: The Zn-RC H sites contain Zn-BChl instead of BPhe, andso contains 6 identical chlorins. The Zn-BChl H-cofactor spectralproperties in the visible region are proposed to be due to the absenceof a 5th ligand coordinating the Zn. We suggest that this coordinationis a key feature of protein-cofactor interactions that significantlycontribute to the redox midpoint potential of H, and the formation ofthe charge-separated state.

In both the purple bacterial and the cyanobacterial PS2 RC, P is a(B)Chl dimer, B sites contain (B)Chl, and H sites contain (B)Phe. Incontrast, the cyanobacterial RC in PS1 has six Mg-containing Chls in asimilar spatial arrangement, analogous to the Zn-RC, with the A0 Chl inthe PS1 RC equivalent to the HA BPhe in the WT RC. The Mg of theChl electron acceptor A0 in the PS1 RC protein is weakly ligated by thesulfur of a Met side chain, in contrast to the usual His. We speculatethat evolution has resulted in two strategies for a high rate of ET inRCs: 1) in the PS2-type of RCs, the primary electron acceptor is a(B)Phe surrounded by protein that excludes (B)Chl and water from theH sites; 2) in the PS1-type of RCs, the primary acceptor is a Chl (A0),but Chl functions well as the primary acceptor because the Mg in theA0 Chl is ligated weakly by a sulphur atom coming from the protein,endowing this Chl with tetracoordination-like electrochemicalproperties.

P.005

A NEW PROTEIN INVOLVED IN THE OCP-RELATEDPHOTOPROTECTIVE MECHANISM IN CYANOBACTERIA.

Clémence Boulay, Adjélé Wilson, Diana Kirilovsky.

Commissariat à L’Energie Atomique (CEA), Institut de Biologie etTechnologies de Saclay (iBiTec-S) et Centre National de la RechercheScientifique (CNRS) URA 2096, 91 191 Gif-sur-Yvette, France.

Introduction: The most characterised photoprotective mechanism incyanobacteria is named qEcya. Under high light conditions, itdiminishes the formation of dangerous oxygen species by increasingenergy dissipation into heat and thus, decreases the energy arriving atthe reaction centers from the phycobilisomes (PBS, the cyanobacterialexternal antennae). The mechanism is induced by the absorption ofblue-green light by the carotenoid of the soluble photoactive OrangeCarotenoid Protein, OCP [1, 2]. The process is accompanied by areversible decrease (quenching) of fluorescence. When the“quenched” cells are then transferred to darkness or low lightintensities, the initial level of fluorescence is recovered within 15-20minutes. The aim of this study was to discover other proteins involvedin this photoprotective mechanism.



Methods: A mutant unable to recover the lost fluorescence along withseveral mutants which complemented it were constructed. Theirphenotype, with respect to the photoprotective mechanism, wasstudied by measuring fluorescence kinetics in a pulse amplifiedmodulated fluorimeter (PAM-fluorimeter). Co-purification and co-immunoprecipitation experiments were used to investigate theinteraction of a new protein with the OCP.

Results: We have demonstrated that a 13kDa membrane protein,which we have named Fluorescence Recovery Protein (FRP), isinvolved in the PBS fluorescence emission recovery after a strongblue-green light illumination. In the absence of this protein, there is nofluorescence recovery, while overexpression of the same proteinaccelerates the recovery. When the His-tagged FRP protein wasisolated from cells overexpressing this protein, traces of the OCP wereco-isolated. Moreover, OCP and FRP co-immunoprecipitated using anantibody against the His-tag present only in FRP. These results stronglysuggested that OCP and FRP interact.

Conclusion: We have discovered a new protein involved in the OCP-related-photoprotective mechanism. This protein, named FRP, isessential for fluorescence recovery.

[1] A. Wilson, G. Ajlani, J.M. Verbavatz, I. Vass, C.A. Kerfeld, D.Kirilovsky, A soluble carotenoid protein involved in phycobilisome-related energy dissipation in cyanobacteria, Plant Cell 18 (2006)992-1007.

[2] A. Wilson, C. Punginelli, A. Gall, C. Bonetti, M. Alexandre, J.M.Routaboul, C.A. Kerfeld, R. van Grondelle, B. Robert, J.T. Kennis,D. Kirilovsky, A photoactive carotenoid protein acting as lightintensity sensor, Proc Natl Acad Sci U S A 105 (2008) 12075-12080.

P.006

PROBING THE STRUCTURAL CHANGES INVOLVED IN D1PROTEIN TURNOVER OF PHOTOSYSTEM II IN SYNECHOCYSIS SP.PCC6803.

Aparna Nagarajan, Hong-Jin Hwang, Robert Burnap.

Dept. Microbiology & Molecular Genetics, Oklahoma State University,Stillwater, OK, USA.

D1 protein subunit is deeply buried within the PSII complex and has ahigh turnover rate (approximately 30min., depending upon lightintensity). The process of turnover involves an efficient process ofdamage recognition and repair, which is largely unknown.Rearrangements in the structure of the D1 protein and PSII complexare inevitable during this turnover process.

We are adopting two technical approaches to detect the structuralchanges during this process. The first approach involves the use ofreagents causing a more general modification of various residues thatbecome exposed to the aqueous phase during the turnover process.Different chemical modification reagents (e.g. carbodiimides) arebeing explored to probe changes in conformation. These modifiedPSII subunits can be analyzed by mass spectrometry (MALDI-TOF). Wehave obtained good coverage for the core PSII proteins with 64% ofD1, 57% of CP43 and 47% of MSP full-length sequence. The regionsof coverage include the domains that are hypothesized undergochanges. A comparison of peptides before and after treatment coulddemonstrate the changes in the solvent exposure post modification.The alternative and more specific approach is to detect thesestructural changes using cysteine-scanning mutagenesis. Analysis ofthe structure of D1 has allowed us to select a few domains that mightbe involved in solvent exposure. Based on this structure, a set ofmutants have been generated which have all the four native cysteines

removed and one cysteine substituted at a specific location. Thesecysteine residues are probed using thiol reactive reagents like 5-iodoacetamido-fluorescein. A cysteine residue that is solvent exposedcan react with the probe and can thus be hypothesized as the domainthat undergoes a conformational change. These chemically modifieddomains of D1 protein can be detected by using gel electrophoresis.This system involves the use of PSII particles and will allow us to probethe solvent exposed residues both under native and damageconditions in vitro.

Supported by National Science Foundation NSF MCB-0818371

P.007

KINETIC ANALYSIS OF CCMR AND CMPR (LYSR-TYPETRANSCRIPTIONAL REGULATORS) WITHIN SYNECHOCYSTIS SP.PCC6803.

Shawn M. E. Daley1, Marla Carrick1, Robert L. Burnap2.1Biochemistry & Molecular Biology; 2Dept. Microbiology & MolecularGenetics; Oklahoma State University, Stillwater, OK, USA.

Aquatic photosynthetic organisms are generally dependent uponactive transport of inorganic carbon to supply the Calvin-Basham-Benson carbon fixation process. This is an energetically expensiveactive transport uptake system termed the carbon concentrationmechanism (CCM). The control of the high-affinity/low-flux inorganicCCM in cyanobacteria has been of great interest for many years. TwoDNA-binding proteins, CcmR and CmpR, are responsible for thetranscriptional control of the expression of transport genes for theCCM. CmpR has been shown to be an activator of cmpABCD(Takahashi et al 2004). CcmR has been shown to be a repressor invivo as established by microarray analysis appearing to control severaldifferent genes/operons that are spread throughout the genome ofSynechocystis sp. PCC6803 (Wang et al 2004). Therefore thegenes/operons under the control of CcmR are referred to as the‘CcmR Regulon’. This putative regulon contains 5 distinctgenes/operons; ccmR, ndhF3 [operon], ndhD5 [operon], sbtA [operon]and ubiX. However, direct physical evidence for the interactionbetween CcmR and the proposed genes comprising this regulon islimited. This work aims at testing the hypothesis that CcmR indeedinteracts with the DNA regulatory region of the genes previouslyidentified by microarray analysis and to provide a more completemolecular characterization of the regulatory features of this putativeregulon. For the first time in vitro data supporting direct binding ofCcmR to all of the regulon components will be presented. These havebeen determined by electrophoretic mobility shift assay. The controlof this energy intensive system of carbon sequestration has also beenof great interest for many years. Recently Nishimura et al (2008) hasdetermined the ligand molecule for CmpR within Synechococcuselongatus PCC7942. Efforts to determine the ligand molecule, usingsurface plasmon resonance, and its associated kinetic constants (ka, kd,kD) will be reported, for CcmR and CmpR within Synechocystis sp.PCC6803.

Supported by US Department of Energy, Energy Biosciences DivisionDOE DE-FG02-08ER15968



P.008

SUPRAMOLECULAR ORGANIZATION OF PHYCOBILIPROTEINS INTHE CHLOROPHYLL D-CONTAINING CYANOBACTRIUMACARYOCHLORIS MARINA.

Min Chen1, Tom Bibby2, Matthias Floetenmeyer3.1School of Biological Sciences, University of Sydney, Australia,2National Oceanography Centre, Southampton University, UnitedKingdom; 3Centre for Microscopy and Microanalysis, University ofQueensland, Australia.

Acaryochloris marina MBIC11017 is an ecologically important,biochemically interesting and evolutionarily fascinating marinecyanobacterium using the unique chlorophyll, Chl d. In addition to theaccessory Chl d-binding light-harvesting protein complexes as itsmajor light harvesting system, which feeds energy to photosystems Iand II, it possesses a primitive phycobiliprotein complex as anaccessory peripheral antenna system. Using advanced cryo-electronicmicroscope technologies we have revealed the high resolution detailsof the organisation of phycobiliprotein structures of AcaryochlorisMBIC11017. Interestingly, the new strain isolated from the Salton Sea (Acaryochloris CCMEE5410) lacks phycobiliproteins and no suchsupramolecular structure of phycobiliproteins was observed.

Cryo-electron transmission-microscopy on native cell-sections ofAcaryochloris MBIC11017 shows extensive patches of near-crystallinephycobiliprotein rods that are associated the thylakoid membranes.They are organized as an array of rods along the stromal (orcytoplasmic) side of the thylakoid membranes and divide the thylakoidmembranes into an uneven “ribbon” with about 25 nm spaces (forphycobiliprotein region) or a closely stacked region leaving no spaces(less than 10 nm, without phycobiliproteins between the membranes).The dimensions of the individual rods are 10 nm × 25 nm. Aligning thePSII supercomplexes to rod-arrays of phycobliprotein complexes, wesuggest that one array of phycobiliprotein rod-complexes lines up withone array of PSII dimer-antenna supercomplexes.

Additionally, Acaryochloris possesses a huge genome with size up to8.3 Mb including nine plasmids from 2.1 kb to 370 kb. The genesencoding phycocyanin related peptides but allophycocyanin α/βsubunits are located in the plasmid pREB3. The genes encodingallophycocyanin α/β subunits are located in the main chromosome,and are not clustered together. With the aid of SDS-PAGE analysis wehave identified a number of phycobiliprotein polypeptides and theproposed model structure of phycobiliproteins in Acaryochloris agreeswith the structure observed by using electronmicroscope. Thissupramoleuclar photosynthetic structure represents a novelmechanism of organizing the photosynthetic light harvestingmachinery. The relationship among photosystems and antennasystems in Acaryochloris will be discussed.

P.009

CRYSTALLOGRAPHIC STRUCTURE OF A LIGHT AND REDOXSENSING PROTEIN MUTANT, AppA17-133 Q63E, FROMRHODOBACTER SPHAEROIDES LOCKED IN PSEUDO-SIGNALINGSTATE.

Vladimira Dragnea, George Feldman, Arun Ignatius, David Giedroc,Carl Bauer.


AppA is a light and redox sensing protein and an anti-repressor ofphotosynthetic gene transcription in Rb. sphaeroides. Structure of thelight sensing domain (called Blue-Light- Using-FAD) from several

different species has been solved. AppA BLUF domain binds oxidizedFAD and is able to undergo a slow photocycle, represented by a 10nm red-shift in the absorption spectrum of the flavin. Despite theefforts of many groups, the exact mechanism of the photocycle andthe structure of its signaling state is still a mystery. Several mutants ofAppA were constructed and their influence on AppA photocycle isdiscussed. Particularly interesting is a Q63E mutant, which exhibits nophotocycle, permanent 3 nm red-shift of the flavin absorptionspectrum and permanent fluorescence quenching of flavin peak.Acrylamide quenching of tryptophan fluorescence indicated that thismutant is “locked” in the light (signaling) conformation. We haveconfirmed this conclusion further by recording 2D-NMR spectra of theQ63E mutant, which exhibits almost identical magnetic shifts as AppA-light state. We have solved the crystal structure of AppA17-133 Q63Emutant that represents “light” conformation of this clone.

P.010

HETEROLOGOUS EXPRESSION OF [FeFe]-HYDROGENASES INANABAENA SP. STRAIN PCC 7120 FOR IMPROVED HYDROGENPRODUCTION.

Katrin Gaertner1, Ivan Khudyakov2, Sigal Lechno-Yossef3, HajimeMasukawa4, C. Peter Wolk3, and Eric L. Hegg1.1Department of Biochemistry and Molecular Biology, Michigan StateUniversity, East Lansing, MI, USA; 2All-Russia Research Institute forAgricultural Microbiology, Saint Petersburg - Pushkin, Russia 3MSU-DOE Plant Research Laboratory, Michigan State University, EastLansing, MI, USA; 4Department of Microbiology and MolecularGenetics, Michigan State University, E. Lansing, MI; USA.

Introduction: Problems related to the massive use of fossil fuels arewell known. H2 is a promising, clean fuel, but currently the vastmajority of industrially produced H2 is generated from fossil fuels andis therefore neither environmentally friendly nor renewable. Somephotosynthetic organisms produce H2 from sunlight and water,utilizing the reductant released during water oxidation to reduceprotons. Although sunlight and water are abundant and inexpensive,the application of photosynthetic organisms to H2 production on anindustrial scale is challenging. The major classes of enzymes capableof synthesizing H2 are nitrogenases (N2ases); [FeFe]-hydrogenases(H2ases); and [NiFe]-H2ases, such as uptake H2ase (Hup) and reversibleH2ase (Hox). O2, produced by water-splitting photosynthesis, inhibitsor inactivates all known N2ases and H2ases. Unlike N2ases, H2asesrequire no ATP. [NiFe]-H2ases are less O2-sensitive than [FeFe]-H2ases,but have lower specific activities and turnover rates. Anabaena spp.are filamentous N2-fixing cyanobacteria that contain differentiated,micro-oxic cells called heterocysts that protect N2ases and H2ases fromO2. Our goal is to enhance H2 production by Anabaena byheterologously expressing [FeFe]-H2ases in the heterocysts.

Methods: We are using standard genetic and molecular genetictechniques to target [FeFe]-H2ases, and the proteins needed for theirmaturation, to the heterocysts of Anabaena. To obtain optimalexpression, these genes from various organisms are being driven byheterocyst-specific promoters (P) starting with Anabaena Pnif with andwithout amplification (Wolk et al., Mol Microbiol 7: 441, 1993), usingintegrating and replicating vectors, in Hup- and Hup-Hox- strains.Different combinations of [FeFe]-H2ases and maturation proteins willbe tested, beginning with Chlamydomonas reinhardtii hydA andClostridium acetobutylicum maturation genes hydE, hydF, and hydG(King et al., J Bacteriol 188: 2163, 2006).

Results: We have integrated the gene encoding T7 RNA polymeraseinto the genome of Anabaena in the nif operon, eliminating N2ase



activity. The hyd genes will be expressed from PT7 on a replicatingplasmid that has been transferred to Anabaena. All H2 production byour Hup-Hox- recipient strains will be due to heterologous expressionof an [FeFe]-H2ase.

Conclusion: Micro-oxic heterocysts provide a promising environmentfor the expression of O2-sensitive [FeFe]-H2ases under oxic externalconditions. If strains expressing [FeFe]-H2ases in heterocysts producemore H2 than strains containing only the native N2ases, the formerstrains may provide a route to economically practicable production ofH2 from renewable resources.

P.011

CHARACTERIZATION OF AN ACTIVE CYTOCHROME B6FCOMPLEX FROM THERMOSYNECHOCOCCUS ELONGATUSINCLUDING THE NEW SUBUNIT PETP.

Dorothea Gomolla1, Regina Oworah-Nkruma1, Corinna Lüer1, MeikeGendrullis1, Gábor Bernát1, Sascha Rexroth1, Frauke Baymann2,Matthias Rögner1.1Plant Biochemistry, Ruhr-University Bochum, 44801Bochum,Germany; 2BIP/CNRS, 31 Chemin Joseph-Aiguier, 13402Marseille Cedex 20, France.

Introduction: The cytochrome b6f (cyt b6f) complex of oxygenicphotosynthesis from the thermophilic cyanobacteriumThermosynechococcus elongatus connects the electron transportbetween Photosystem 2 and Photosystem 1 (PS1). Each monomer ofthe dimeric integral membrane complex consists of four majorsubunits: cyt f (PetA, 33 kDa), cyt b6 (PetB, 24 kDa), the Rieske iron-sulfur protein (PetC, 19 kDa) and subunit IV (PetD, 17 kDa).Additionally, there are four small subunits - i.e. PetG, PetL, PetM andPetN with molecular masses ranging from three to seven kDa.Recently, we identified an additional 7.2 kDa subunit of the cyt b6fcomplex of Synechocystis sp. PCC 6803 (S. 6803), which we namedPetP [1].

Methods: In order to investigate structure-function relationships in astable and fully active complex we developed a new strategy for thepurification of the cyt b6f complex from T. elongatus, involving His-tagged cyt f and two chromatographic steps. The purified dimericcomplex was analyzed by MALDI-MS and ESI-MS, as well as EPRmeasurements.

Results: The resulting dimeric complex contains all cyt b6f subunitsand a 7.2 kDa protein. ESI-MS and MALDI-MS analysis revealed thatthis protein is encoded by the open reading frame tlr0524 – with highhomology to PetP (ORF ssr2998) of S. 6803. Disruption of tlr0524showed impaired growth especially under high light conditions and aslower electron donation to cyt f and PS1 according to cyt f and P700absorption kinetics.

Crystal structure analysis of cyt b6f from the thermophiliccyanobacterium Mastigocladus laminosus [2] and the green algaChlamydomonas reinhardtii [3] showed an additional redox cofactorheme ci. This cofactor is located in the Qi pocket of the complex (closeto heme bH) and covalently linked to a cystein residue of the cyt b6

protein via a single thioether. Preliminary EPR analysis of heme ci fromT. elongatus in presence and absence of the inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide showed similar spectra as obtained with thecyt b6f complex from C. reinhardtii [4].

Conclusions: In summary, we suggest that PetP, which is missing in thecrystal structures of M. laminosus and C. reinhardtii, is a new – possiblyregulatory – subunit of the cyt b6f complex. Similar to S. 6803 ourresults showed a direct association of PetP with the electron transportof the cyt b6f complex, although characterization of the PetP-less

mutant indicated a non-essential function. Spectral analysis by EPRindicates the existence of the redox cofactor heme ci in T. elongatus(as reported before for M. laminosus and C. reinhardtii). Due to itstransformability, the purification of fully active dimeric cyt b6f complexfrom T. elongatus now enables an in-depth characterization includingsite-directed mutants.

References:

[1] Volkmer, T., Schneider, D., Bernát, G., Kirchhoff, H., Wenk, S-O.,Rögner, M., J Biol Chem, 2007, 282, 3730-7

[2] Kurisu, G., Zhang, H., Smith, J.L., Cramer, W.A., Science, 2003, 302,1009-14

[3] Stroebel, D., Choquet, Y., Popot, J-L., Picot D., Nature, 2003, 426,413-18

[4] Baymann, F., Giusti, F., Picot, D., Nitschke, W., PNAS, 2007, 104,519-24

P.012

PROTEOMIC ANALYSIS OF KNOCKOUT MUTANTS FORPLASTOCYANIN AND CYTOCHROME C6 IN THECYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803

Manuel Hervás, Ornella Castielli, José A. Navarro, Berta De la Cerdaand Miguel A. De la Rosa.

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevillaand CSIC. Centro Isla de la Cartuja, Américo Vespucio 49, E-41092Sevilla, Spain.

Introduction: Iron and copper participate as essential proteincofactors in photosynthesis and respiration. However, both metals athigh concentration promote the generation of reactive oxygenspecies. Thus, metal homeostasis is crucial in maintaining theadequate concentration of metal ions inside the cell [1].

Plastocyanin (Pc) and cytochrome c6 (Cyt) are two alternative electrontransport proteins that can replace each other depending on copperavailability [2]. In this work, we describe a comparative proteomicanalysis of Synechocystis wild-type (WT) and two knockout mutants foreither Pc or Cyt growing with or without copper, thus yielding relevantinformation on metal homeostasis in cyanobacteria.

Methods: Changes in the proteome of Synechocystis have beenstudied by comparing 2-DE gels of cytoplasmic soluble fractions whencultured with or without copper. Soluble proteins were first resolvedby isoelectric focusing on a pH range from 4 to 7, followed by SDS-PAGE. Qualitative differences, as well as quantitative comparison ofspots, were analyzed using the PDQuest software package (Biorad).The selected spots were further identified by MALDI-TOF.

Results: Due to the relevance of copper and iron homeostasis incyanobacteria, we have studied the effect of copper deprivation onthe proteome of Synechocystis [3,4]. Particular emphasis has beenmade on the combined effect of copper deficiency and deletion of thepetE and petJ genes coding for Pc and Cyt, respectively, whoseexpression is regulated by this metal [2].

The differences in the proteome of WT cells reveal that copperinduces not only the over-expression of 19 enzymes involved in themain metabolic pathways, but also the synthesis of the GroEL1chaperone and of the ATP-dependent ClpP protease induced understress. On the contrary, the lack of copper enhances the expression ofan ABC transporter, which is the major contributor to ferric irontransport across the plasma membrane.

When comparing the proteomes of WT and DpetE cells grown in thepresence of copper, substantial expression changes are observed in



43 proteins involved in the main metabolic pathways. In particular, thechanges observed in the mutant concern proteins involved inphotosynthesis and enzymes related to regulation of phycobilisomescoupling to PSII.

Similarly, the proteomes of WT and DpetJ cells grown in the absenceof copper show different expression levels in 29 proteins involved inthe main metabolic pathways. Actually, the expression level ofproteins involved in carbohydrate metabolism, along with the antennaproteins and enzymes participating in the synthesis of pigments aresignificantly altered in the mutant cells.

Moreover, Synechocystis cells grown with neither Pc nor Cyt sufferfrom redox stress due to the blocking of the electron transport chains.Accordingly, the over-expression of thioredoxins, peroxiredoxin,superoxide dismutase and the DnaK chaperone, among otherproteins, can be observed.

Conclusions: The proteomic analyses of Pc and Cyt deletion mutantsof Synechocystis grown in the absence or presence of copper, ascompared to WT cells, can be used as a powerful tool to understandmetal homeostasis in cyanobacteria.

References

[1] Messerschmidt A et al (eds) (2001) Handbook of Metalloproteins.John Wiley & Sons, Chichester[2] Hervás M, Navarro JA, De la Rosa MA (2003) Accounts Chem Res36, 798–805[3] De la Cerda B et al (2008) Brief Funct Genomic Proteomic 6, 322–329[4] Castielli O et al (2009) FEBS Lett (in press)

P.013

TRANSCRIPTIONAL REGULATION OF THE NITROGEN FIXATIONRELATED GENES (NIF-GENES) BY NTCA IN A UNICELLULARCYANOBACTERIUM GLOEOTHECE SP. 68DGA.

Tadashi Kawabata1, Yukiko Taniuchi2, Shinya Yoshikawa1, MitsunobuKamiya1, Kaori Ohki1.1Faculty of Marine Bioscience, Fukui Prefectural University, Obama,Fukui, Japan; 2Department of Marine Biotechnology and Resources,National Sun Yat-sen University, Kaohsiung, Taiwan.

Introduction: The gene set encoding proteins required for thediazotrophic cyanobacteria synthesis of functional nitrogenase (nif-genes) are clustered and organized as several transcriptional units.Expression of the nif-genes (including the structural genes fornitrogenase, nif H, D and K) occurs under nitrogen-limiting conditions.However, it is unclear whether expression of the nif-genes is controlledby a single transcriptional regulator as found in enteric bacteria. Apossible candidate for a transcriptional regulator is NtcA that is knownas a global nitrogen regulator in cyanobacteria. NtcA is thought to beactivated by 2-oxoglutarate (2-OG) whose level increases duringnitrogen depletion. A typical binding site for NtcA on the DNA wasfound to contain the palindromic sequence, GTAN8TAC. In thefilamentous cyanobacteria, Anabaena sp. PCC7120, NtcA is requiredfor heterocyst formation where the expression of nif-genes occurs inthe late stage of heterocyst differentiation. However, directinteractions between NtcA and nif-genes have not been clear yet. Todetermine the role of NtcA in transcriptional regulation of nif-genes,we cloned the nif-genes from the unicellular diazotrophiccyanobacteria, Gloeothece sp. 68DGA (Gloeothece sp.); andcharacterized the binding of NtcA in the upstream region of the nif-genes.

Methods: A genomic library of Gloeothece sp. constructed in EMBL3was used for screening and sequencing of nif-genes. The geneencoding NtcA (ntcA) from Gloeothece sp. was cloned into theexpression vector, pET-28a (+). The vector was transformed into E. coliBL21 (DE3) and recombinant NtcA was purified using a His trapcolumn. An electrophoretic mobility shift assay (EMSA) was performedto determine the NtcA binding site in the upstream region of the nif-genes.

Results: The nif-genes were arranged as a continuous cluster spanningapproximately 19 kb and organized into eight operons (in order:orf4orf5, nifVZT, nifP, nifBfdxnifSU, nifHDK, nifENX, orf2 and nifW).

A typical NtcA-binding sequence was not found at an upstream regionof each operon. However, sequences similar to the typical NtcA-binding site were found between nifP and nifBfdxnifSU (TGTN10ACA)that corresponds to the upstream regions of these operons and anupstream region of nifVZT (GTGN10ACA). EMSA showed NtcA boundonly to the region between nifP and nifBfdxnifSU. The binding affinityof NtcA to the region between nifP and nifBfdxnifSU was similar to thetypical NtcA-binding sequence present in an upstream region of theglnA in Gloeothece sp. The binding of NtcA to the region betweennifP and nifBfdxnifSU was enhanced by the addition of 2-OG. BothnifP and nifBfdxnifSU were expressed under nitrogen-limitingconditions in Gloeothece sp.

Conclusion: Transcriptional regulation of nifP and/or nifBfdxnifSU isthought to be mediated by NtcA. Enhancement of NtcA binding byadding 2-OG suggests regulation occurs in response to the N-statuswithin the cells according to the level of 2-OG. The data suggeststhere may be other transcription factor(s) that regulate thetranscription of nif-genes other than nifP and/or nifBfdxnifSU.

P.014

THE CBB3 OXIDASE FROM RUBRIVIVAX GELATINOSUS.

Bahia Khalfaoui Hassani 2, Ileana Agalidis2, Chantal Astier2, SoufianOuchane 2, Robert van Lis1, Wolfgang Nitschke1 and BarbaraSchoepp-Cothenet1.1Laboratoire de Bioénergétique et Ingénierie des Protéines (UPR9036), Institut de Microbiologie de la Méditerrannée, CNRS, Marseille;2CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette;2Université Paris-Sud, Orsay; 2Université Pierre et Marie Curie, Paris;France.

Introduction: Among the three groups of heme/copper O2

reductases, cbb3–type oxidases are the least well-understood. Weobserved that the β-proteobacterium Rubrivivax (R.) gelatinosusexpresses high quantities of a cbb3–oxygen reductase when grownunder oxygen-limited conditions. As a result of the high level ofenzyme, this species thus lends itself both for the study of the enzymein membranes and as starting material for biochemical purifications.

Methods: The operon coding for the enzyme together with flankingregions containing genes involved in its regulation and maturationwere sequenced and analysed. The enzyme was studied in membranefragments and purified samples by UV/Vis and EPR (including studieson oriented material) spectroscopy and electrochemistry. The twoextrinsic c-type cytochrome subunits CcoO and CcoP and site-directed mutants thereof were heterologously expressed in E. coli.Attribution of EPR spectral features to heme cofactors in the wholeenzyme was achieved via a CcoP null mutant.

Results: EPR spectral characteristics, redox midpoint potentials andorientations with respect to the membrane of all heme components



present in the R. gelatinosus cbb3 oxidase were determined. Theheme contained in CcoO was found to be spectrally andelectrochemically heterogeneous. The spectroscopic results obtainedon the product of the ccoO gene and site-directed mutants thereofsuggest that during the enzyme’s catalytic cycle, CcoO switchesbetween Met143 and His130 as axial 6th ligand rationalizing theobserved redox heterogeneity. This ligand switch is furthermore in linewith the absence of a well-defined orientation of the CcoO heme withrespect to the membrane as opposed to the situation which weobserve for all other hemes of the complex and which are in specific,well-determined orientations.

Conclusions: The obtained results together with the observation thatboth Met143 and His130 are conserved in cbb3 oxidases indicate thatthe ligand switch is an essential part of the catalytic turnover of thisgroup of O2 reductases.

P.015

OPTICAL PROPERTIES OF LIGHT-HARVESTING COMPLEX 2 FROMTHERMOPHILIC PURPLE SULFUR BACTERIUM,THERMOCHROMATIUM TEPIDUM.

Masayuki Kobayashi, Hiroaki Suzuki, Zheng-Yu Wang, TsunenoriNozawa.

Ariake National College of Technology, Omuta, Fukuoka, Japan.

Introduction: In photosynthetic purple bacteria, light energy isabsorbed efficiently by antenna complexes and the captured photonsare transferred to photosynthetic reaction center. Purple sulfur bacteriahave two types of antennas which are core light-harvesting complex 1(LH1) and peripheral light-harvesting complex 2 (LH2).Thermochromatium (Tch.) tepidum belongs thermophilic purple sulfurbacterium and its optimum growth temperature is the highest of allpurple bacteria [1]. In this study, we tried to isolate the LH2 complexfrom Tch. tepidum and found interested optical properties of that.

Methods: Intracytoplasmic membranes (ICM’s) from Tch. tepidum cellwere prepared by a sonication method [2]. LH2 complexes wereisolated from the ICM’s by treatment with 0.35 % (w/v) lauryl-N,N-dimethyl amine-N-oxide (LDAO) at room temperature for 1 hour.Purification of the LH2 was carried out DEAE-anion exchanged columnchromatography. Absorption and circular dichroism (CD) spectra weremeasured with Shimadzu U-3100 and Jasco J720-W spectrometers,respectively.

Results: In near IR region, absorption spectrum of ICM’s from Tch.tepidum has three peaks at 917, 855 and 800 nm. These peaks hasbeen assigned to B915 for LH1-RC complex and B850 and B800 forLH2 complex, respectively [3]. The profiles and peak position ofabsorption and CD spectra of the solubilized LH2 complex in 0.05%LDAO were very similar to those of native LH2 complex in ICM [3].These suggest that the solubilized LH2 complex maintains nativepigment-protein structure.

We observed the changes of absorption and CD spectra at higherconcentration of LDAO. The peak position of Qy transition of theB850 band was blue-shifted to 852 nm in 0.1% LDAO, 847 nm in 0.2%LDAO and 841 nm in 0.4% LDAO and the magnitude of that wasdecreasing according to increasing of the concentration of LDAO. Thepeak position of Qy transition of the B800 band was not shifted butthe intensity of that was increasing by addition of LDAO. The B800and B850 of LH2 from Tch. tepidum shows positive and negative CDsignal from shorter wavelength, respectively [3]. Although the couplettype CD signal of the B850 was changed to one negative CD signal,the couplet type CD of the B800 was unchanged by increasing of the

concentration of LDAO. Since LH2 complex has the ring structure, theCD signals of B800 and B850 indicate two couplet type CD signals [4].These results reflected the differences of interaction of BChls a in theB850 and the B800 and suggested that the interaction between BChlsa in the B850 became weaker in high concentration LDAO. We alsofound the blue-shifted LH2 complex was returned to 855 nm bydilution of the concentration of LDAO to 0.05%.

Sucrose density gradient of the blue-shifted LH2 complex did notshow clearly band. The absorption spectra of the solution of upperand lower area showed blue-shifted LH2 complex and native like LH2complex, respectively. This result suggested that the size of the blue-shifted LH2 complex smaller than that of the native LH2 complex

Conclusions: We found that the reversible spectral changes of theLH2 complex from Tch. tepidum were induced by the increasing of theconcentrations of detergents. The blue-shifted LH2 complex wassmaller than native LH2 complex. The interaction among BChls a ofthe B850 in blue-shifted LH2 complex were weaker than that in nativeLH2 complex.

Reference

[1] M. T. Madigan, Science, 225, 313-315(1985)

[2] H.Suzuki, Biochim. Biophys. Acta 1767, 1057-1063(2007)

[3] T. Nozawa et. al., Biochim. Biophys. Acta 852, 191-197(1986)

[4] S. Georgakopoulou et. al., Biophys. J. 82: 2184-2197(2002)

P.016

PURIFICATION AND CHARACTERIZATION OF A PHYCOBILISOMESUBCOMPLEX CONTAINING THE LARGE ISOFORM OFFERREDOXIN:NADP OXIDOREDUCTASE.

A. Korn, G. Ajlani, B. Lagoutte, A. Gall, P. Sétif.

CEA, Institut de Biologie et de Technologies de Saclay, Gif sur Yvette,France

The main role of the Ferredoxin:NADP oxidoreductase (FNR) inphotoautotrophs is to provide the NADPH for CO2 reduction. In plant-root plastids, a distinct FNR isoform functions in the oppositedirection, providing electrons for nitrogen assimilation at the expenseof NADPH generated by carbohydrate oxidation. A multiple genefamily encodes FNR isoenzymes in plants, whereas there is only oneFNR gene in most cyanobacteria. Nevertheless, we detected two FNRisoforms in the cyanobacterium Synechocystis sp. strain PCC6803.One of them (FNRS specifically induced under heterotrophicconditions) is similar in size to the plant FNR (34 kDa) while the otherone (FNRL 47 kDa) contains an extra N-terminal domain that allows itsassociation with the phycobilisome (PBS). Mutants have beenconstructed that contain only one of the two isoforms present in thewild type. The mutants that express exclusively the small isoform andthe long isoform attached to the PBS are called FS and MI6,respectively. Following an NADP+/NADPH quantification, significantdifferences have been found for the NADP+/NADPH ratio in thesemutants compared to the wild type.

Using a genetically engineered strain of Synechocystis, we successfullypurified a native complex composed of a phycocyanin hexamer, therod-core linker and FNRL (FNRL-PC), in a 1:1:1 stoichiometry. Wecompared the NADPH-oxidase and NADP+-reductase activities of thetwo isoforms. Both FNRS and FNRL-PC exhibit similar oxidase activitieswhen using potassium ferricyanide as an artificial electron acceptor,but differences in catalytic constants have been observed whenanalyzing the Fd mediated reduction of cyt c. This corresponds to a 40% decrease in the affinity of FNRL-PC to Fd compared to FNRS.



Furthermore, we found a significant decrease of the reaction rates ofthe first reduction of FNR by reduced Fd for the complex FNRL-PCcompared to FNRS in the absence of NADP

+. We also performedmultiple catalytic turnover experiments and found that FNRS andFNRL-PC reductions are only limited by electron transfer from reducedFd. Our results suggest a modified affinity for Fd in the purified FNRL-PC complex compared to FNRS, which may be due to steric hindrance,by PC. These differences obtained in the catalytic activities do notexplain the different phenotypes of the respective mutants thatexpress only one FNR isoform. Therefore we propose that the activitymust be determined by the differential localization of the FNRisoforms, which also probably determine their substrates availability,and their involvement in different pathways of electron flow. Further invivo studies are underway to elucidate the function of each FNRisoform.

P.017

STRUCTURAL AND FUNCTIONAL ANALYSIS OF A CONSERVEDTYROSINE TRIAD IN CYTOCHROME C1.

J.A. Kyndt, J.C. Fitch, T.E. Meyer, M.A. Cusanovich.


Introduction: Cytochrome bc1 (bc1), is a ubiquitous membraneprotein found in all chloroplasts and mitochondria of eukaryotes andthe cytoplasmic membrane of many respiratory and photosyntheticbacteria. Bc1 serves two functions—to transfer an electron to thesoluble electron carrier cytochrome c, and to generate a protongradient used in photosynthesis and oxidative phosphorylation. Thec1 subunit forms the pivotal connection between the bc1 complexand its soluble partners. A triad of tyrosine residues in the c1 subunitof R. capsulatus bc1 (Y152-154) are ideally positioned to be involvedin electron shuttling or correlate conformational communicationbetween the Rieske protein, c1 and c2. The role of these residues wasinvestigated by mutagenesis followed by spectral and kinetic analysis.

Methods: R. capsulatus bc1 mutants and WT protein were producedin R. sphaeroides ∆fbc (gift from Prof. A. Crofts) with a c1 His tag. Allcultures were grown photosynthetically, except for the Y154Acomplement which could only be grown aerobically in the dark.Proteins were purified on Ni-NTA and eluted in MOPS (pH 7.8) with20% glycerol, 200mM His, 100mM NaCl, 1mM MgCl2, 0.01 % DM and15 μg/ml PC. Decylubiquinol was used in the catalytic assays assubstrate and the reduction of cytochrome c was measured bymonitoring the absorbance change at 550 nm.

Results: Mutation of Y153 to Q or A resulted in a spectrally altered cytbc1. Both absolute and reduced minus oxidized spectra have theSoret peaks slightly blue shifted (by ~ 4 nm). Moreover, the intensitiesof the cyt c1 α and β peaks are significantly lower than in wild-type cytc1 upon reduction. Measuring the redox midpoint potential (Em) forY153Q showed that the Em for the c1 heme in this mutant was loweredby about 115 mV. Since this alone cannot explain the lower intensity ofthe c1 α and β peaks upon reduction with dithionite, and the fact thatthere is a slight Soret blue shift, we believe that the mutation of Y153creates both redox and spin heterogeneity in cyt c1. In addition, thecatalytic activity, measured in amount of cyt c reduction per second, isabout 10 fold lower for the Y153Q mutant compared to WT. SinceY153 is positioned on the opposite side of the c1 hinge region as thec1 heme, it is likely that mutagenesis at that position creates stericclashes with the hinge region, which will affect the position of the sixthligand. This was confirmed by structural homology modeling.Mutagenesis of Y to Q at position 152 did not change the spectral and

electrochemical properties of c1, and showed WT enzymatic c2reduction rates. However, changing Y154 resulted in very low yieldand non-functional protein.

Conclusion: Structural homology modeling and directed mutationalanalysis of these three tyrosines showed that mutations to Q and A atposition 153 clearly affect the electrostatic properties of c1 and theelectron transfer rate from quinol to cyt c, presumably throughalteration the sixth heme ligand position. Therefore, a model whereY153 has a structural function is more likely than a role as an electronpath. The fact that Y152Q has properties very similar to WT indicatesthat this mutation did not introduce major structural changes in c1 anddoes not create rate limiting changes in the activity of bc1.Mutagenesis of Y154 displayed a significant destabilization of c1,underscoring its structural importance in bc1.

P.018

EFFORTS TO ENHANCE SOLAR HYDROGEN PRODUCTION BYHETEROCYST-FORMING CYANOBACTERIA.

Sigal Lechno-Yossef, C. Peter Wolk.

Michigan State University, East Lansing, MI, USA.

Introduction: The negative effects of fossil fuel use on our economyand environment make the utilization of sustainable energy sourcesdesirable. A principal such source is sunlight. Its energy may be usedby diverse microorganisms to generate H2 from water viaphotosynthesis by use of nitrogenases (N2ases) or hydrogenases(H2ases). Because these enzymes are oxygen(O2)-sensitive, processesof oxygenic photosynthesis and H2 production are normally separatedtemporally or spatially. Anabaena spp. and closely relatedcyanobacteria form specialized cells, heterocysts, in which N2ases andH2ases are protected from O2 by inactivation of O2-producing PSII,accelerated respiration, and a thick envelope of glycolipids andpolysaccharide that impedes penetration of O2. Reductant required forN2 fixation and H2 production is generated by photosynthesis invegetative cells and moves to heterocysts as sugar. We are trying, bymodifying Anabaena metabolism, to increase H2 production to acommercially practicable level. In preliminary experiments, in which ashort version of the nifH promoter was inserted before hoxYH in anuptake H2ase(Hup)-minus nifHD mutant of Anabaena variabilis strainATCC 29413, no H2 was produced under oxic conditions.

Methods: Our revised approach is to replace N2ase by a native,bidirectional H2ase (Hox) in a Hup-minus derivative of Anabaena sp.strain PCC 7120, in which Hox is encoded by the genes hoxE, F, U, Y,and H that (with other ORFs) are located in two independenttranscriptional units separated by ca. 9 kb (1). We are generating aconstruct in which those two operons are contiguous, togetherreplacing much of nifD, so as to over-express hox, rather than nifD, inheterocysts from the strong nif promoter. We retain nifH because itmay provide part of the mechanism to reduce the concentration of O2

in heterocysts (2). The products of genes hydF, C, D, B, and A arerequired for synthesis of the NiFe cluster of Hox, and of gene hoxW isrequired for proteolytic processing of HoxH (1). These genes will beexpressed on a replicating plasmid from the heterocyst-specificpromoters of coxBII and patB (3,4). Abstracts by H. Masukawa, K.Gaertner, J.J. Park and co-authors describe complementaryapproaches to enhancement of H2 production: modifying N2ase suchthat it reduces more protons to H2 and fewer N2 to NH3, utilizing notHox but exogenous Fe-only H2ases, and studying electron donors andpathways in heterocysts.



Results: To facilitate further genetic modification of the host strain, wehave successfully modified a flp-FRT-based technique for removal ofantibiotic markers from the genome (5) for use in Anabaena sp. strainPCC 7120. The constructs described above are initiated, and some arequite advanced.

Conclusions: It remains to be determined whether the native hoxgenes, expressed in heterocysts in an oxic milieu, can servepracticably as a source of H2 at the level of a household or atransportation system. Although available strains of Anabaena areconvenient experimental objects, it is likely that hitherto unstudiedstrains may produce H2 more abundantly.

References: 1. Tamagnini et al., FEMS Microbiol Lett 31: 692, 2007; 2.Thorneley and Ashby, Biochem J 261: 181, 1989; 3. Valladares et al.,Mol Microbiol 4&: 1239, 2003; 4. Wang and Xu, J Bacteriol 187: 8489,2005; 5. Hoang et al., Gene 212: 77, 1998.

P.019

TRANSCRIPTIONAL ANALYSES IN SYNECHOCOCCUS SPECIESPCC 7002.

Marcus Ludwig, Zhenfeng Liu, Craig A. Praul, Lynn P. Tomsho, StephanC. Schuster, Donald A. Bryant.

Dept. of Biochemistry and Molecular Biology, University Park, PA, USA

Introduction: The cyanobacterium Synechococcus sp. PCC 7002grows in brackish and marine water, tolerates very high lightintensities, and has an extremely short doubling time. BecauseSynechococcus sp. PCC 7002 is easily manipulated genetically, theorganism is a good candidate for biotechnological applications. In thisstudy, we performed transcriptional analyses in Synechococcus sp.PCC 7002 by mRNA/cDNA sequencing. We compared thetranscriptomes of cells cultivated under four different conditions inorder to find differentially transcribed genes.

Methods: Cells used for transcriptional analyses were cultivated inmineral medium at 38oC, 250µmol photons*m-2*s-1 and continuousbubbling with air containing 1% CO2 (standard conditions). Growthunder CO2 limitation was the same except that the air was notenriched with CO2. The cultures were grown to an OD730 of 0.7. In thecase of cultures incubated in the dark, the cultures (at OD 0.7) wereincubated in dark for 1h with either air or N2 containing 1% CO2 toproduce anoxic conditions. cDNA libraries were synthesized after RNAisolation and subjected to 454 pyrosequencing or SOLiD sequencing.The obtained sequences were mapped against the Synechococcus sp.PCC 7002 genome using BLAST (maximum expect value 0.01,minimum identity 78%). The genes with the best hits were counted,and the counts were used for subsequent comparisons.

Results: 454 sequencing resulted in about 100000 sequences percondition. Mapping against the genome of Synechococcus sp. PCC7002 resulted in about 370000 hits in open reading frames and about2000 in intergenic regions in total. The major fraction (87-96%) of thehits was identified as 16S and 23S rRNA, although procedures hadbeen undertaken to deplete rRNA. Further analyses were only basedon the mRNA sequences. The most abundant transcripts resulted fromgenes coding for the photosystems and light-harvesting complexes.After dark incubation (both aerobic and anaerobic) the transcriptionlevel of these genes is significantly lowered. Genes of the CO2

concentration and fixation mechanism are also quite abundant. Thetranscription level of these genes decreased upon dark incubation,whereas under CO2 limitation these genes showed an increase oftranscription. Interestingly, transcription of pyruvate:ferredoxinoxidoreductase and phosphoenolpyruvate synthase is strongly

increased after dark incubation, and is even higher after darkanaerobic incubation. Although the numbers of hits were quite low, asignificant increase of transcription of hydrogenase and hydrogenase-related genes was also observed upon dark incubation.

Conclusions: The data obtained in this study demonstrate that both454 pyrosequencing and SOLiD sequencing of cDNA are appropriatetechniques to monitor the transcriptome in Synechococcus sp. PCC7002. A comparison of four different conditions revealed that underCO2 limitation the transcription of genes involved in CO2 fixation arestrongly up-regulated. Transcription of genes coding for componentsof the photosystems and light harvesting complexes are down-regulated upon dark incubation. An interesting finding is theup-regulation of transcription of two genes of pyruvate metabolismupon dark incubation. These transcriptome data provide valuable datafor finding strongly regulated promoters for expression systems andgive information about metabolic pathways, which is important forfuture metabolic engineering for biotechnological applications.

P.020

OCCURRENCE OF THE OXYGEN-BINDING SPHAEROIDES HEMEPROTEIN AND DIHEME CYTOCHROME C INRHODOBACTERACEAE.

T.E. Meyer, J.A. Kyndt, M.A. Cusanovich.


Introduction: The oxygen-binding Sphaeroides Heme Protein (SHP)and soluble Diheme Cytochrome c (sDHC) were originallycharacterized from Rhodobacter sphaeroides, but have since beenidentified in more than 36 proteobacterial genomes. They usuallyoccur in a three-gene operon along with a membrane-spanningcytochrome b (CytB), which in Rb. sphaeroides is fused with aduplicated mDHC domain (CytB/mDHC). In most species, the SHPoperon includes regulatory genes. However, not all strains of Rb.sphaeroides have the SHP operon, it is not associated with regulatorygenes, and has not been reported in other species ofRhodobacteraceae. We therefore set out to analyze a variety ofRhodobacter strains for the presence of the SHP operon anddetermine whether the SHP genes could have been acquired throughrandom gene transfer or are functionally related.

Methods: Rhodobacter species were obtained from our owncollection, from the DSMZ, Braunschweig Germany, CV Ramana,Hyderabad India, and DK Newmann, Pasadena California and grownphotosynthetically on the recommended media. PCR primers weredesigned based upon the conserved heme binding sites in SHP andDHC. Universal primers were also used to sequence 16S rRNA as aquality control.

Results:We found that SHP and DHC are present in 9 out of 10 strainsof Rb. sphaeroides, the only negative strain was 2.4.3. We wereunable to detect any differences between our own isolates, strains TJ4and JB15, but they differ from strains 2.4.1 and 2.4.9 by about 30bases in the 2kb operon. Strain FY differs by about 41 bases andstrain KD131 by 56 bases. Strain SCJ differs by about 87 bases.Strains 2.4.18 and IL106 differ from one another by only 5 bases, butare 365 bases distant from strain 2.4.1. As might be expected, thereare also significant differences in the translated sequences of the 3proteins from these two strains, which are 76-82% identical to those ofstrain 2.4.1, which is about the same extent as observed for distinctspecies. The SHP operon was also found in Rhodobacter sp. SW2, Rb.changlensis, Rhodovulum adriaticum, Rv. marinum, and Rv.robiginosum. PCR was negative for Rb. capsulatus strains SB1003,2.3.1, and SP108, Rhodobacter sp. TJ12, Rb. blasticus, Rb. veldkampii,

Rv. imhoffii, and Rv. sulfidophilum W4 and BSW8, however this doesnot prove that the genes are absent.

Conclusions: The SHP operon is present in 9 out of 10 R. sphaeroidesstrains studied and was found in five related species ofRhodobacteraceae. Its occurrence in this family is therefore not dueto random gene transfer but must provide a selective advantage.Furthermore, duplication of DHC and formation of the chimeraoccurred before speciation. SHP is postulated to function as anoxygenase and to share the same role and substrate specificitythroughout the Rhodobacteraceae. The function is likely to bedifferent in the related Rhodospirillaceae and in bacteria that haveoperons with associated sensor kinases and response regulators.

P.021

DRAFT GENOME SEQUENCE OF THE PHOTOSYNTHETIC PURPLESULFUR BACTERIUM, ECTOTHIORHODOSPIRA VACUOLATA.

T.E. Meyer, M.A. Cusanovich.


Introduction: There are now more than 700 complete bacterialgenome sequences, but only five species of non-sulfur purple bacteriaand one purple sulfur species completed. By way of contrast, thereare more than a dozen green sulfur bacterial genomes and more than20 distinct cyanobacterial genome sequences. There are also morethan 20 species of aerobic anoxygenic phototrophic species that havebeen sequenced. Although there is tremendous diversity within thepurple bacteria, they have been neglected to date with only a handfulof species in the pipeline. We have thus initiated sequencedetermination of the purple sulfur bacterium, Ectothiorhodospiravacuolata (aka Ect. shaposhnikovii). The Ectothiorhodospiraceae arenormally found in desert soda lakes that are characterized by alkalinepH, high carbonate coupled with low calcium and magnesiumconcentrations, and high productivity. When oxidizing sulfurcompounds such as sulfide and thiosulfate, elemental sulfur isdeposited outside the cells. Ect. vacuolata is unique as the onlyspecies in the family to produce gas vesicles.

Methods: Ect. vacuolata strain DSM2111 was obtained from DSMZ,Braunschweig Germany and grown photosynthetically on therecommended medium. DNA was extracted with the Qiagen DNeasykit, sonically sheared (250 base average size), and fragmentssequenced in a single run on the Roche 454 sequencer. Sequenceswere assembled into contigs using Newbler and annotated with Rast.

Results: 51 Mb high quality DNA sequence data were obtained andassembled into 121 contigs greater than 500 bases resulting in 3.3 Mbunique sequence (15-fold coverage). The CG content was found to be63%. Automatic annotation resulted in 2900 coding sequences. The16S rRNA sequence was in agreement with the published results andall 24 genes previously identified in Ect. vacuolata and Ect.shaposhnikovii were found to be in complete agreement except forthe fccB gene for sulfide dehydrogenase which differed by 50%. It islikely that there are two genes for FCSD, only one of which wasrecovered. The alternative sulfide dehydrogenases, SoxEF and SQRare apparently absent. In the sulfur oxidation pathway, the sulfurcarrier SoxYZ and the hydrolase SoxB were clustered, but the SoxAXcytochrome was located on a separate contig. The only possiblesulfite dehydrogenase we found was a homolog of YedYZ. A closerelative of Ect. vacuolata from Mono Lake was reported tophotosynthetically oxidize arsenite to arsenate, but we could only findgenes for arsenic resistance, ArrAB and ArsDABC. The GvpANFGSKJgenes for gas vesicle synthesis were recovered. There were 4 clustersof chemotaxis genes and 22 MCPs. The PilABCD and PilGHIJL genes

for twitching motility were also present. There are 26 histidinekinases, 43 response regulators, and 40 diguanylate cyclases.

Conclusions: It is now possible to obtain high quality draft genomesequence data at an affordable cost in less than a month and it doesnot require any special expertise. Most of the expense of genomesequencing comes at the finishing stage, although there is nocompelling reason to determine complete sequences. Draftsequences provide most information required and any additionalsequence can be readily obtained by walking PCR.

P.022

OPERATION OF THE CYTOCHROME BC1 COMPLEX OFRHODOBACTER CAPSULATUS AT HIGH PH VALUES.

Katrin Jahns1, Natalia E. Voskoboynikova1, Maria A. Kozlova2,Armen Y. Mulkidjanian1,2.1School of Physics, University of Osnabrück, Osnabrück, Germany;2Moscow State University, Moscow, Russia.

Introduction: In a dimeric cytochrome bc1 complex an ubiquinolmolecule is oxidized in one of the two catalytic centers P and the tworeleased electrons go to different acceptors. One is taken by themobile domain of the [2Fe–2S] iron–sulphur Rieske protein to bepassed further to the c-type cytochromes. The other electron crossesthe membrane, via the low- and high-potential hemes of cytochromeb, to reduce a stable semiquinone molecule in one of the two centersN from the opposite membrane side (see refs. [1-3] for reviews). Atneutral pH values, a single Zn2+ ion, which can bind close to the centerP [4], not only retarded the proton release from this center and themovement of the FeS domain towards cytochrome c1, but also sloweddown the oxidation of heme bh and the formation of ubiquinol incenter N [5]. This correlation was attributed to the mechanisticcoupling between the two quinone-binding centers [1-3,5,6].

Methods: The flash-induced generation of membrane voltage wastraced, via spectral shifts of native carotenoid pigments, in vesicularpreparations of the inner cellular membranes (chromatophores) ofphototrophic α-proteobacteria Rhodobacter capsulatus and correlatedwith the optically monitored redox changes of the cytochrome hemesas described in ref. [5].

Results: The aforementioned transmembrane coupling between thequinone-binding centers implies that the retardation of events incenter N should, reciprocally, affect the events in center P. To checkthis prediction, we have measured the pH-dependence of flash-induced reactions in the cytochrome bc1 complex, both in the absenceand in presence of Zn2+ ions. The difference between kinetic traces, asobtained in the absence and presence of Zn2+ ions, respectively,diminished both under acidic and at alkaline conditions. At acidic pHvalues, the impact from the addition of Zn2+ ions diminished; mostlikely, the binding site became protonated with apparent pK of ca. 7.0and could not bind Zn2+ ions anymore. Under alkaline conditions, incontrast, the oxidation of heme bh slowed down even in the absenceof Zn2+ ions. At pH of ca. 9.0, the kinetics of reactions in thecytochrome bc1 complex resembled those measured in the presenceof Zn2+ ions at neutral pH.

Conclusions: The same kinetic behaviour of the cytochrome bc1complex could be achieved either by blocking the proton release fromcenter P by Zn2+ ions or via preventing the protonation of ubiquinonein center N by high pH. These observations support our suggestion ofa cross-membrane mechanistic/thermodynamic coupling between thequinone-binding sites of this enzyme [1-3, 5, 6].



References:

[1] Mulkidjanian A.Y. (2005) Biochim Biophys Acta 1709, 5-34.

[2] Mulkidjanian A.Y. (2006) Biochim Biophys Acta 1757, 415-427.

[3] Mulkidjanian A.Y. (2007) Photochem Photobiol Sci 6, 19-34.

[4] Giachini L. et al. (2007) Biophys J. 93, 2934-2951.

[5] Klishin, S.S., W. Junge, and A.Y. Mulkidjanian (2002) BiochimBiophys Acta 1553, 177-182.

[6] Mulkidjanian, A.Y. and W. Junge (1995) in: Photosynthesis: fromLight to Biosphere: (P. Mathis, Ed.), Kluwer Academic Publishers,Dordrecht, Vol.II, pp. 547-550.

P.023

CYANOBACTERIAL RNA HELICASES: A NOVEL ROLE INREGULATING CARBON ASSIMILATION.

Laura Patterson-Fortin1, Lige Wu1, Meghana Ventakesh2,George S. Espie3, George W. Owttrim1.1Department of Biological Sciences, University of Alberta, Edmonton,AB; 2Department of Biology, University of Toronto, Mississauga, ON,;3Department of Cell and Systems Biology, University of Toronto,Mississauga, ON; Canada.

Introduction: RNA participates actively in the regulation of geneexpression in all organisms. To be functional, RNA molecules must foldinto the appropriate secondary structure. RNA helicases are enzymesthat alter RNA or RNP structure, thereby participating in the post-transcriptional regulation of gene expression. We have utilized amultidisciplinary approach to study the mechanisms by whichalteration of RNA secondary structure by RNA helicases influencesgene expression in cyanobacteria. Expression of the helicase, crhR(cyanobacterial RNA helicase Redox), is regulated by the redoxpotential of the electron transport chain and is therefore enhanced bylow temperature (20ºC). Biochemically, CrhR rearranges RNAsecondary structure by both unwinding dsRNA and annealingcomplementary ssRNA. Here we explore the broad ranging effects ofa crhR null mutation in the cyanobacterium Synechocystis sp. PCC6803.

Methods: CO2 / HCO3- transport and accumulation was measured

using an aqueous inlet mass spectrometer. Photosynthetic O2

evolution was determined with a Clarke electrode. For microarrayanalysis of RNAs interacting with CrhR, CrhR was immunoprecipatedfrom Synechocystis cells grown at 20ºC for 3 hours. After phenol-chloroform extraction the RNA was labeled and hybridized to aSynechocystis microarry consisting of three copies of all identifiedORFs. Transcripts detected in all 9 replicates from 3 biological sampleswere considered positive as potential RNAs interacting with CrhR invivo. Proteomic analysis was performed using 2-D gels followed bymass spectrophotometric identification of proteins whose levelsaltered between wild type and crhR mutant cells.

Results: Microarray analysis indicates CrhR interacts with a limitednumber of transcripts encoding genes involved with photosyntheticelectron transport, energy metabolism, protein synthesis/degradationand unexpectedly transponsons. Initial proteomic analysis indicatesthat crhR mutants lack CcmK, a major component of the carboxysomeshell. Phenotypically, the crhR mutant grows at near-normal wild-typerates at 30ºC, but not at 20ºC. The reduced rate of growth at 20ºCwas correlated with a 4-fold reduction in the rate of photosyntheticoxygen evolution. Interestingly, after brief exposures to 20ºC,photosynthetic capability was restored upon return to 30ºC. In short-term experiments, mass spectrometric analysis revealed that transport

and intracellular accumulation of CO2 / HCO3- was comparable to

wild-type levels, indicating a capacity for photosynthetic electrontransport.

Conclusions: Microarray and proteomic analysis indicates CrhR RNAhelicase interacts with transcripts involved with maintenance ofphotosynthetic capacity and protein synthesis. The inability of the crhRmutant to utilize the intracellular CO2 / HCO3

- pool implies a defect inphotosynthetic carbon assimilation. This is the first time that a RNAhelicase has been associated with carbon assimilation inphotosynthetic organisms.

P.025

IDENTIFICATION AND CHARACTERIZATION OF HIGHLYCONSERVED PROTEINS IN THE PHOTOSYNTHETIC BACTERIUMRHODOBACTER SPHAEROIDES.

Aaron Setterdahl, Allan Huth, Olga Simmons, Wes Halfacre.

Indiana University Southeast, New Albany, IN, USA.

Introduction: Genome sequencing of many organisms in recent yearshas revealed an enormous database of gene sequences that code formany previously unknown proteins (Zhou, Kvikstad et al. 2003). Anumber of these proteins are highly conserved between many diverseorganisms. Sequence analysis of the purple photosynthetic bacteriumRhodobacter sphaeroides reveals several protein sequences that areuncharacterized and are highly conserved among bacteria such asEscherichia coli, Staphylococcus aureus, Xanthomonas axonopodis,Bradyrhizobium japonicum, Rhodopirellula baltica, Gluconobacteroxydans, Pseudomonas syringae, Shigella sonnei, Tenacibaculum,Flavobacterium, Blastopirellula marina, Rickettsiella grylli,Mesorhizobium, Streptomyces ambofaciens, Enterobacter spp.,Planctomyces maris, and Klebsiella pneumoniae. Even with detailedannotation of genomes in which sequences of DNA from oneorganism are compared to other DNA sequences of differentorganisms, not all genes and their respective gene products can becorrectly identified. Numerous instances exist where annotationreveals many genes to be “unknown function” or “hypotheticalprotein.” Recent studies used microarray analysis to determine theexpression of hundreds genes in response to anaerobic-light toaerobic-dark conditions (Arai, Roh et al. 2008). Of the genes analyzedin that study at least 89 genes code for hypothetical proteins. Manyof the genes that show sequence similarity and have been assigned afunction by sequence comparison have been previouslyuncharacterized in R. sphaeroides or any other photosyntheticbacteria.

Methods: The program Artemis was used to scan the annotatedgenomic DNA database of R. sphaeroides. Gene sequences that werelabeled as unknown function or uncharacterized protein, were BLASTusing the non-redundant NCBI database. Conserved proteinsequences were targeted for PCR. Genomic DNA of R. sphaeroides ispurified and used as a template for PCR. Primers were designed witha specific sequence that will amplify each several genes.

Results: PCR has confirmed the presence of the gene sequences ofthe Iron-containing alcohol dehydrogenase, Putative FAD-dependentglycerol-3-phosphate dehydrogenase, a conserved hypotheticalprotein, and a Probable FAD oxidoreductase. Gene sequences thatwere targeted but were unconfirmed by PCR were a putative copperbinding protein, a conserved hypothetical protein, probablebacterioferritin, putative iron-regulated protein, and a probable c-typecytochrome.



Conclusions: PCR has confirmed the presence of the gene sequences,but more work needs to be done to confirm or discover the functionof the gene products. This is a first step in the elucidation of the genefunctions. The next step is to systematically clone, express and purifythese and many other gene products. The confirmation of these geneidentities is paramount to our understanding of these organisms.Many genes have proposed functions, yet still need to besystematically characterized in order to confirm or discover theirproper function within R. sphaeroides.

References:

Arai, H., J. H. Roh, et al. (2008). “Transcriptome dynamics during thetransition from anaerobic photosynthesis to aerobic respiration inRhodobacter sphaeroides 2.4.1.” J Bacteriol 190(1): 286-99.

Zhou, S., E. Kvikstad, et al. (2003). “Whole-genome shotgun opticalmapping of Rhodobacter sphaeroides strain 2.4.1 and its use forwhole-genome shotgun sequence assembly.” Genome Res 13(9):2142-51.

P.026

THE INVESTIGATION OF THE ACSF PROTEIN AND PIGMENTBIOSYNTHESIS IN CHLOROFLEXUS AURANTIACUS.

Kuo-Hsiang Tang, Jianzhong Wen, Xianglu Li, Robert E. Blankenship.

Departments of Biology and Chemistry, Campus Box 1137,Washington University, St. Louis, MO, USA.

Introduction: The green phototrophic bacteria contain a uniquecomplement of chlorophyll pigments, which self-assemble efficientlyinto antenna structures known as chlorosomes with little involvementof protein. The few proteins found in chlorosomes have previouslybeen thought to have primarily a structural function. The biosyntheticpathway of the chlorosome pigments, bacteriochlorophyll (BChl) c, dand e is not well understood.

Methods: In this report, we used spectroscopic, proteomic and geneexpression approaches to investigate the chlorosome proteins of thegreen filamentous anoxygenic phototrophic bacterium Chloroflexusaurantiacus (C. aurantiacus).

Results:We characterized the chlorosome proteins, CsmM, CsmN,and unexpectedly, Mg-protoporphyrin IX monomethyl ester (oxidative)cyclase, AcsF, under anaerobic growth conditions for C. aurantiacus.The AcsF protein was found in the isolated chlorosome fractions, andthe proteomics analysis suggested that significant portions of the AcsFproteins are not accessible to protease digestions. Additionally, real-time (RT)-qualitative PCR studies showed that the gene expression ofcsmM, csmN, and bchE is up-regulated under anaerobic conditions,but that the transcript level of the acsF gene is not lower in anaerobicgrowth than in semi-aerobic growth (Tang, K.-H. et al. J. Bacteriol. inpress). Finally, we have investigated on the location of AcsF in C.aurantiacus and other photosynthetic bacteria, as well as probed onthe biosynthesis of the isocyclic ring formation in BChl a and BChl c.

Conclusion: Together, it is clear that even for the most investigatedFAP bacterium, C. aurantiacus, the role(s) of AcsF is far away frombeing understood. Additionally, since no studies have been reportedpreviously for AcsF and BchE in any green bacteria, which have aspecialized photosynthetic compartment— the chlorosome, caution isneeded to compare the properties and mechanism of action of AcsFand BchE in GSB and FAP with the studies of higher plant, algae, andother bacteria. We are currently investigating these two enzymes withboth in vitro and in vivo studies.

P.027

PURIFICATION AND CHARACTERIZATION OF THE FMO PROTEINFROM THE NEWLY DISCOVERED AEROBIC PHOTOTROPH,CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUM.

Yusuke Tsukatani, Donald A. Bryant.

The Pennsylvania State University, PA, USA.

Introduction: We have discovered a new thermophilic, aerobicphototroph from the phylum Acidobacteria which had not includedphotosynthetic members. The discovery of the new acidobacteriumCandidatus Chloracidobacterium (Cab.) thermophilum enables us toexplore new mechanisms of photosynthesis and may provide newinsights into evolution of photosynthesis. Genomic analysis of Cab.thermophilum revealed that this organism possesses genes encodinga reaction center core subunit (pscA), a baseplate protein ofchlorosomes (csmA), and a light-harvesting Fenna-Matthews-Olson,so-called FMO protein (fmoA). Thus, Cab. thermophilum appears todo type-1 reaction center-based photosynthesis in the presence ofoxygen, whereas green sulfur bacteria and heliobacteria that also havetype-1 reaction centers are all strictly anaerobic phototrophs. Here wereport the first isolation of the FMO protein from Cab. thermophilum.The FMO protein shows different absorption and fluorescenceproperties compared to that from green sulfur bacteria.

Methods: Membranes prepared from photosynthetically grown cellsof Cab. thermophilum were incubated with sodium carbonate andthen a supernatant was collected by ultracentrifugation. After dialysis,the supernatant was subjected to anion-exchange DEAE columnchromatography. The blue-colored FMO protein was eluted by 500mM NaCl.

Results: The purified FMO protein appeared as a single band uponCoomassie blue-stained SDS-PAGE. The molecular mass predictedfrom SDS-PAGE was about 44 kDa, which corresponds to the masspredicted by the amino acid sequence of the FMO protein (40.5 kDa).The 44-kDa protein band was confirmed as the FMO protein bypeptide mass fingerprinting analysis. Absorption spectrum ofpigments extracted from the FMO protein showed that the proteinbinds bacteriochlorophyll a. The FMO protein showed a largeabsorption peak at 797 nm and shoulder peaks at around 810 and 824nm at room temperature. The fluorescence emission spectrum of theFMO protein showed two peaks at around 814 and 827 nm at roomtemperature. On the other hand, the FMO protein of green sulfurbacteria is known to have a single fluorescence peak at 818 nm atroom temperature and the peak is sharpened and shifted to 831 nm at77K. We will report the low-temperature spectroscopic properties ofthe FMO protein. We are also characterizing components inmembrane preparations and attempting to isolate an active reactioncenter.

Conclusions: We successfully purified the FMO protein from the newaerobic phototroph Cab. thermophilum. The FMO protein was abacteriochlorophyll a-containing protein like that of green sulfurbacteria. However the FMO protein of Cab. thermophilum haddistinctive spectroscopic properties compared to that of green sulfurbacteria. The results suggest that the energy transfer scheme insidethe FMO protein of Cab. thermophilum may be significantly differentfrom that of green sulfur bacteria.



P.028

GLOBAL PROTEOMICS REVEAL NOVEL NITROGEN UTILIZATIONMECHANISM UNDER NUTRITIONAL STRESS BY SYNECHOCYSTIS6803, A MODEL PHOTOTROPH.

Kimberly M. Wegener1, Jon M. Jacobs2, Abhay K. Singh1, ThanuraElvitigala3, Eric A.Welsh1, 4, Nir Keren1, 5, Marina A. Gritsenko2, Bijoy K.Ghosh1, 6, Richard D. Smith2, and Himadri B. Pakrasi1.1Department of Biology, Washington University, St. Louis, MO, USA;2Pacific Northwest National Laboratory, Richland, WA, USA;3Department of Electrical and Systems Engineering, WashingtonUniversity, St. Louis, MO, USA; 4Pfizer, Inc., St. Louis, MO, USA; 5Dept.of Plant and Environmental Sciences, Silberman Institute of LifeScience, The Hebrew University of Jerusalem, erusalem, Israel;6Department of Mathematics and Statistics, Texas Tech University,Lubbock, TX, USA.

Introduction: Cyanobacteria, oxygenic phototrophic prokaryotes, arethe progenitors of the modern chloroplast. They are crucial to globaloxygen production and worldwide carbon and nitrogen cycles.Synechocystis sp. PCC 6803 has long been a model cyanobacteriumfor photosynthesis research. Here we present the first global proteomeanalysis of an autotrophic photosynthetic bacteria and the mostcomplete coverage of a photosynthetic prokaryotic proteome to date.

Methods: Synechocystis 6803 cells were subjected to iron, phosphate,nitrogen, or sulfate depletion and subsequent repletion. Cells werealso subjected to environmental stresses including heat shock, coldshock, salt stress, high CO2 (3%) or growth under photoheterotrophicconditions.. Samples were collected at a total of 33 time points acrossconditions, fractionated, and subjected to proteomic analysis usingthe Accurate Mass and Time (AMT) tag approach. These results wereanalyzed statistically to combine technical replicates. Protein foldchanges were calculated by comparing levels of an individual proteinin a stress condition to those in cells grown in complete BG11.

Results: The resulting proteome dataset consists of 22,318 uniquepeptides, corresponding to 2,369 unique proteins, covering 65% ofthe predicted proteins. These proteins uniformly represent allfunctional categories and include proteins encoded by theSynechocystis 6803 chromosome as well as the plasmids pSYSM,pSYSX, pSYSA, and pSYSG (Cyanobase). In contrast, previousproteome studies of Synechocystis 6803 have identified just 1,099proteins (30% of the predicted proteome). 1,359 of the proteinsidentified in our study were novel proteomic identifications while1,010 proteins were common proteins in our study and previouslypublished proteomes. Additionally, we have identified 758 proteinsthat are currently annotated as hypothetical. These proteins can nowbe reclassified as unknown proteins, reducing the number ofhypothetical proteins in Synechocystis 6803 by over one half.Importantly, quantitative analysis of protein abundance ratio incultures grown under nutritional stresses revealed that Synechocystis6803 resorts to a universal mechanism for nitrogen utilization underphosphate, sulfate, iron, and nitrogen depletion. Of the 2,369 proteinsidentified, 1,233 proteins were differentially regulated underenvironmental stress conditions.

Conclusions: Global proteomics offers a detailed picture into the stateof an organism that reveals insight that cannot be garnered fromtranscriptomic studies alone. Here we present the first globalproteome analysis of an autotrophic photosynthetic prokaryote todate. This study offers the most complete coverage of aphotosynthetic prokaryotic proteome with quantitative data as toprotein response under various critical nutritional stresses.

Acknowledgements: This work was supported by National ScienceFoundation Grants FIBR 0425749 and MCB 0745611 to H. B. P. Thiswork was additionally supported as part of the Membrane BiologyScientific Grand Challenge project at the W. R. Wiley EnvironmentalMolecular Science Laboratory, a national scientific user facilitysponsored by the U.S. Department of Energy’s Office of Biological andEnvironmental Research program (Pacific Northwest NationalLaboratory).

P.029

INFLUENCE OF THE CAROTENOID NATURE AND OF THE AMINO-ACIDS AROUND THE CAROTENOID IN THE ORANGECAROTENOID PROTEIN ACTIVITY IN PHOTOPROTECTION.

Adjélé Wilson, Claire Punginelli, Jean-Marc Routaboul, DianaKirilovsky.

School of Biological Sciences (A08), University of Sydney, New SouthWales, Australia.

Introduction: In most cyanobacteria high irradiance induces aphotoprotective mechanism that downregulates photosynthesis byincreasing thermal dissipation of the energy absorbed by thephycobilisome, the water-soluble antenna. The light activation of asoluble carotenoid protein, the Orange-Carotenoid-Protein (OCP),binding hydroxyechinenone, a keto carotenoid, is the key inducer ofthis mechanism. Light causes structural changes within the carotenoidand the protein, leading to the conversion of a dark orange form intoa red active form. We studied the effects on the activity of the OCP ofchanges in the nature of the bound carotenoid and of modifications ofthe 100% conserved amino-acids surrounding it.

Methods: Synechocystis PCC6803 mutants overexpressing the OCPand containing a mutated OCPs, or lacking the crtO gene wereconstructed. The existence of the photoprotective process was testedby fluorescence measurements using a PAM fluorometer. The OCPprotein was isolated and its photoactivity tested.

Results and Conclusions: In the ∆crtO mutant, lackinghydroxyechinenone and echinenone, the OCP was found to bindzeaxanthin but the stability of the binding appeared to be lower andlight was unable to photoconvert the dark form into a red active form.Moreover, in the strains containing zeaxanthin-OCP, blue-green lightdid not induce the photoprotective mechanism. In contrast, in mutantsin which echinenone is bound to the OCP, the protein isphotoactivated and photoprotection is induced. Our results confirmedthat the red OCP is the active form and strongly suggest that thepresence of the carotenoid carbonyl group that distinguishesechinenone and hydroxyechinenone from zeaxanthin is essential forthe OCP activity.

Trp288 and Tyr201 forming a hydrogen bond with the carbonyl of thecarotenoid are essential for OCP photoactivity and induction of thephotoprotective mechanism. They cannot be replaced by otheramino-acid without loose of activity. While Trp110 and Tyr 44 can bechanged by a Phe without loose activity, they cannot be replaced bySer suggesting that the interaction of the carotenoid hydroxyl ringwith aromatic groups is important for the formation and/or stability ofthe red form.

Trp 277 also is involved in the conversion and /or stabilization of thered form. Finally, while Arg155 is essential for the induction of thephotoprotective mechanism is not involved in the light-inducedorange to red form conversion.



P.030

CHARACTERIZATION OF A NITROGENASE-LIKEPROTOCHLOROPHYLLIDE REDUCTASE IN THECYANOBACTERIUM LEPTOLYNGBYA BORYANA.

Haruki Yamamoto, Shohei Kurumiya, Rie Ohashi, Yuichi Fujita.

Graduate School of Bioagricultural Sciences, Nagoya University,Nagoya, Japan.

Introduction: ark-operative protochlorophyllide (Pchlide)oxidoreductase (DPOR), the determinant enzyme for the greening inthe dark, catalyzes the stereo-specific D-ring reduction of Pchlide toconvert porphyrin ring to chlorin ring in thebacteriochlorophyll/chlorophyll biosynthesis. DPOR is a three-subunitenzyme of BchL/ChlL, BchN/ChlN and BchB/ChlB, and their aminoacid sequences show significant similarity to the nitrogenase subunitsNifH, NifD, and NifK, respectively. A series of initial biochemicalanalysis of DPOR from the anoxygenic photosynthetic bacteriumRhodobacter capsulatus confirmed the nitrogenase-like features.DPOR is distributed widely not only among anoxygenicphotosynthetic bacteria but also among oxygenic phototrophs such ascyanobacteria, green algae and gymnosperms, which raises a questionhow the nitrogenase-like DPOR operates in oxygenic photosyntheticcells. Here we report in-vivo complementation of cyanobacterialDPOR mutants by the expression vectors, reconstitution of DPOR withtwo components purified from cyanobacterial cells, and the oxygensensitivity of the DPOR components.

Methods: Two mutants YFC2 (∆chlL) and YFB14 (∆chlB) of thecyanobacterium Leptolyngbya boryana strain dg5 were used as thehost cells to overexpress the DPOR subunits, ChlL and ChlN-ChlB. Weconstructed two shuttle vectors for overexpression of Strep-taggedChlL (pHBL2) and Strep-tagged ChlN-ChlB (pHBNB2) in L. boryana.YFC2 and YFB14 were transformed with pHBL2 and pHBNB2,respectively, by electroporation. These transformants were cultivatedin BG-11 supplemented with 20 mM HEPES-KOH; pH7.4, 30 mMglucose and 10 µg ml-1 chloramphenicol. Pigments of cells wereextracted in 90 % methanol and the Chl concentration wasspectroscopically determined. Crude extracts of these transformantswere prepared by sonication in an anaerobic chamber. Strep-taggedChlL and ChlN protein were purified by Strep-Tactin Sepharosecolumns. DPOR assays were carried out as described for DPOR fromR. capsulatus. Purified components were exposed to air followed bythe standard DPOR assay.

Results: The ability of chlorophyll biosynthesis in the dark wasrestored in both transformants, suggesting that both DPORcomponents are expressed as active forms. Strep-tagged ChlL andChlN proteins were purified from the crude extracts of thetransformants. ChlL protein was purified as a single band. ChlN wasco-purified with a protein that was cross-reacted with an anti-ChlBantibody, indicating that ChlN forms a stable complex with ChlB asshown in NB-protein from R. capsulatus. DPOR assay was carried outwith the purified proteins. When both proteins, L-protein and NB-protein, were added to the reaction mixture, a marked chlorophyllide(Chlide) formation was observed, while there was no detectableconversion of Pchlide to Chlide in the reaction mixtures containingonly one of the purified components. Then we evaluated the oxygensensitivity of L-protein and NB-protein. While the activity of NB-protein maintained more than 60% of the original level upon 30-minexposure to air, and the L-protein activity rapidly disappeared with ahalf-life of about less than 3 min upon the exposure to air.

Conclusion: The results suggested that L-protein is the primary target

of inactivation by oxygen and that the oxygen-sensitive DPOR,especially L-protein, is protected by some molecular mechanisms inoxygenic cyanobacterial cells.

P.031

PPSR AS A HEMIN SENSOR.

Liang Yin, Vladimira Dragnea, Carl Bauer.


PpsR and AppA coordinate light and redox control of photosynthesisgene expression in R. sphaeroides. PpsR is a DNA-bindingtranscription factor that contains two PAS domains followed by acarboxyl terminal DNA binding domain. Under aerobic conditions,PpsR blocks the transcription of tetrapyrrole biosynthesis andphotosynthetic genes that are responsible for biosynthesis of thepigments and the light harvesting II complex (1). AppA is flavincontaining photoreceptor that functions as an antirepressor of PpsR byforming an inactive PpsR2-AppA complex (1). In the past year, ourresults indicate that hemin provides another level of control in thissystem, showing the first direct link between the biosynthesis of hemewith that of the photosystem.

In this study we demonstrate that PpsR is a hemin-binding proteincontaining a 1:1 stoichoimetry of [PpsR]/[hemin]. The presence ofhemin significantly inhibited the DNA-binding ability of PpsR, whileother tetrapyrrole products did not show any inhibition. Experimentswith truncated PpsR suggest that hemin binds to the C-terminalregion. Our results suggest that PpsR is likely a hemin sensor withheme controlling the DNA binding activity or PpsR. Most of wellcharacterized hemin-binding proteins have tightly-coordinated heminto perform many different tasks, such as electron transfer, oxygenstorage and gas molecule sensing (2). However in recent years severalheme sensors, have been identified from bacteria (3) to mammals (4)that are able to sense the free hemin in vivo. Our study of hemebinding by PpsR will provide new insights about hemin sensors.

1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623

2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L.,Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073

3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A96, 13056-13061

4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc NatlAcad Sci U S A 105, 76-81

P.032

EXCITATION ENERGY TRANSFER IN FILAMENTS ANDHETEROCYSTS FROM NOSTOC PUNCTIFORME.

Tanai Cardona, Ann Magnuson.

Department of Photochemistry and Molecular Science, The ÅngströmLaboratories, Uppsala University, Uppsala, Sweden.

The filamentous cyanobacterium Nostoc punctiforme sp. ATCC 29133is able to differentiate a type of cell specialized in N2 fixation, aheterocyst. Heterocysts undergo dramatic changes associated withnitrogen starvation and the protection of the oxygen sensitivenitrogenase enzyme, like the partial degradation of phycobilisomesand the inactivation of Photosystem II. It is poorly understood whetherheterocysts are capable or not, to control the distribution of excitationenergy to Photosystem I and maximize N2 fixation under different lightconditions. Using fluorescence spectroscopy, we compared theresponsiveness of filaments and isolated heterocysts to green and red



light illumination. It was found in both cell types that 5 minutes greenillumination triggered the quenching of a 680 nm emission peakcorresponding to fluorescence from the terminal emitter of thephycobilisome and the appearance of a fluorescence peak at 750 nmcorresponding to the trimerization of Photosystem I. We concludedthat the quenching of the terminal emitter might be due to directenergy transfer from the phycobilisome core to the Photosystem Itrimer. It is suggested that heterocysts possess a functional, possiblymodified phycobilisome capable of distributing excitation energy toand away from Photosystem I to optimize cyclic photophosphorilation.

P.033

TRANSCRIPTION OF A “SILENT” CYANOBACTERIAL PSBA GENEIS INDUCED BY MICROAEROBIC CONDITIONS.

Cosmin Ionel Sicora1,2, Felix M. Ho3, Tiina Salminen4, StenbjörnStyring3, Eva-Mari Aro1.1Department of Biology, University of Turku, FI-20014, Finland;2Biological Research Center Jibou, 455200, Romania; 3MolecularBiomimetics, Department of Photochemistry and Molecular Science,Uppsala University, SE-75120 Uppsala, Sweden; 4Department ofBiochemistry and Pharmacy, Åbo Academy University, Finland.

Introduction: Cyanobacteria, contrary to higher plants, have a smallpsbA gene family encoding the reaction centre D1 protein subunit ofphotosystem II, the first macromolecular pigment-protein complex ofthe photosynthetic electron transport chain. Modulation of expressionof multiple psbA genes in the family allows cyanobacteria to adapt tochanging environmental conditions. To date, two different strategiesfor regulation of the psbA genes have emerged. One, characterized inSynechocystis PCC6803 and Gloeobacter violaceus PCC7421 involvesthe increased expression of one type of D1 protein to cope with theincreased rate of damage. The other strategy, in SynechococcusPCC7942 and Anabaena PCC7120, is to replace the existing D1 with anew D1 form for the duration of the stress. However, most of the psbAgene families characterized to date contain also a divergent,apparently silent psbA gene of unknown function. This gene, presentin Synechocystis, Anabaena and

Thermosynechococcus elongatus BP-1 was not induced by any stresscondition applied so far.

Methods: The main investigative methode used was Real-time RT-PCRfor the estimation of expression of each individual gene in the familyusing specific primers. We also used molecular modeling to comparethe structural differences between different forms of D1 and flashfluorescence to estimate the functional changes between isoforms.

Results: Our data shows a reversible induction of the divergent psbAgene during the onset of argon-induced microaerobic conditions inSynechocystis, Anabaena and Thermosynechococcus elongatus. Theunitary functional response of three unrelated cyanobacterial species,namely the induction of the expression of the divergent psbA gene asa reaction to the same environmental cue, indicates that these genesand the protein they encode are part of a specific cellular response tomicroaerobic conditions. There are no specific primary structuresimilarities

between the different microaerobic inducible D1 forms, designated asD1′. Only three amino acid residues are consistently conserved in D1′.These modifications are: G80 to A, F158 to L and T286 to L. In silicomutation of the published D1 structure from Thermosynechococcusdid not reveal major modifications. The point by point effects of themutations on the local environment of the PSII structure are alsopresented.

Conclusion: The presence of an anaerobically induced gene in threeunrelated cyanobacteria, makes us to conclude that a new form of D1is present and important to cellular adaptation to specific conditions.

P.034

IDENTIFICATION AND CHARACTERIZATION OF BILIN LYASES FORPHYCOERYTHRIN I BIOSYNTHESIS IN MARINESYNECHOCOCCUS.

Yasmin M. Vasquez, Avijit Biswas, and Wendy M. Schluchter.

Department of Biological Sciences, University of New Orleans, NewOrleans LA, USA.

Introduction: Cyanobacteria have evolved photosynthetic light-harvesting complexes called phycobilisomes. The main component ofthe phycobilisome is phycobiliproteins. In marine Synechococcus, oneof the most abundant phycobiliproteins is phycoerythrin (PE). PE iscomposed of α and β subunits that have the linear tetrapyrrolephycoerythrobilin (PEB) and/or phycourobilin (PUB) attached tospecific cysteine residues via thioether linkages. We are interested inidentifying bilin lyases involved in attaching the PEB chromophores toPE I subunits in marine Synechococcus.

Methods: Putative bilin lyases from Synechococcus sp. WH8020(CpeU, CpeY, CpeZ) or from Synechococcus sp. WH8102 (CpeS) wereamplified by PCR and individually cloned in different Duet vectors.Bilin biosynthetic genes for making PEB from heme (pebS and ho1)were cloned together in pACYC (kind gift of Dr. Nicole Frankenberg-Dinkel). The cpeA and cpeB genes were cloned into the pMAL vector(fusing each to the gene encoding the maltose binding protein). Thecultures were initially grown at 37ºC until the OD at 600 nm wasbetween 0.5-0.6, then they were cooled down to 18ºC and inducedwith 0.5 mM IPTG for 15-18 hours for protein expression. Thepurification of MBP-CpeA and MBP-CpeB was accomplished using anamylose resin affinity column.

Results: We achieved successful cloning of all the genes, which wasconfirmed by sequencing. Bilin lyase as well as phycobiliproteinsubunit expression was found to occur optimally at 18 ºC with IPTG fora period of 15 hours. Soluble subunits CpeA and CpeB were purifiedwith amylose resin column. SDS-PAGE confirmed bands at 57.1 kDcorresponding to CpeA with maltose binding protein tag and a bandat 59.1 kD corresponding to CpeB with the maltose binding proteintag. SDS-PAGE also confirmed bands for CpeZ, CpeY, CpeS and CpeUat 20.6 kD, 47.4 kD, 20.8 kD and 22.7 kD respectively. Solubility ofthese bilin lyases is currently being analyzed. Once conditions areoptimized to obtain soluble lyases, these will be tested for bilinaddition activity in in vitro reactions with PEB.

Conclusions: CpeZ, CpeY, CpeS and CpeU bilin lyases arehypothesized to participate in PEB addition to the α and βphycobiliprotein subunits for PE I. Further work will focus on cloningthe suspected lyases with a histidine tag in pCDF vector. The histidinetag will allow for further purification of the lyases using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. Purified proteins will beused to carry out in vitro reactions to confirm the specificity of the bilinlyase for specific cysteine residues on the α and β PEI subunits.



P.035

USE OF BARLEY STRAW FOR THE CONTROL FRESHWATERCYANOBACTERIAL BLOOMS.

Rob Iredale, Japareng Lalung, David Adams.

University of Leeds, Leeds, West Yorkshire, United Kingdom.

Introduction: The potential of barley straw to control nuisancegrowths of cyanobacteria in freshwater has been well documented in anumber of laboratory and field studies, but its mode of action is stillunclear. In light of this, a study was designed to investigate the effectof straw preparation and decomposition conditions on treatmentefficacy, in the hope that the collected data would allow us tocomment on current theory. Additionally, the relative straw-sensitivityof different cyanobacteria, some of which were freshly isolated, wasdetermined, and we attempted to tentatively identify some of thecompounds leached from straw during the early stages ofdecomposition.

Methods: A specially constructed decomposition chamber containinga combination of natural spectrum and UV fluorescent tubes was usedto vary the light conditions under which straw was decomposed.Laboratory bioassays were used as a means of testing organismresponse to the presence of straw and straw liquor, with all assaysconducted in triplicate. Solid phase extraction and reverse-phaseHPLC were used in order to identify compounds in straw leachate, andflow cytometry was employed as a means of estimating chlorophyll aper cell for a strain of Microcystis.

Results:We found varying sensitivity of different cyanobacteria strainsto the effects of straw, consistent with previous studies. Finechopping of fresh straw resulted in instant activity even at lowconcentrations, whereas fresh, whole straw exhibited little activity. Wehave shown that microbial activity is essential for straw to becomeactive, and that straw decomposed under UV light appeared to bemore active than batches decomposed under natural spectrum lightalone. The addition of catalase to UV-decomposed straw bioassaysreduced the inhibitory action of straw, suggesting hydrogen peroxidemay play a role in the activity of straw. Flow cytometry data showedthat Microcystis cells exposed to straw for 48 hours contained around35% of the chlorophyll a content of cells not exposed to straw. HPLCspiking experiments suggested that a mixture of low molecular weightaromatic compounds were leached from the straw in the first week ofdecomposition.

Conclusions: It is currently accepted that the activity of straw is atleast partly attributed to the solubilisation of lignin by microorganisms,possibly yielding a cocktail of various smaller aromatic compounds.Our findings support this theory, both in terms of our HPLC detectionof compounds which could have originated from lignin, and the factthat fresh, finely chopped straw was inhibitory to growth, whereasfresh whole straw was not. This implies that the breakdown of ligninmight be the rate-limiting step determining the onset of activity, whichcan be enhanced by the combination of lignin disruption andincreased surface area resulting from fine chopping of the straw. Thepossibility of hydrogen peroxide playing a role in the activity of strawhas been previously hypothesised, but this study has provided the firstexperimental data that this may be the case. Data from this studyhave also highlighted the potential use of fresh, finely chopped strawas a cyanobacterial growth control agent, and larger scale studies,possibly involving the use of chopped and whole straw in combinationshould be considered in the future. Although many questions remainregarding its exact mode of action, barley straw remains the leadingcandidate for environmentally sound control of cyanobacteria infreshwater.

P.036

DIFFERENTIAL RESPONSES OF EPIPHYTIC AND PLANKTONICTOXIC CYANOBACTERIA TO ALLELOAPTHIC ACTIVITY OF THESUBMERGED MACROPHYTES STRATIOTES ALOIDES.

Abdulrahman M. Al-Shehri, Zakaria Mohamed.

Department of Biological Sciences, College of Science, King KhalidUniversity, Abha, Saudi Arabia.

Introduction: Cyanobacteria are prevalent bloom-forming algae,commonly occurring in most water bodies, such as lakes andreservoirs. The cyanobacterial bloom can lead to the deterioration ofwater quality through production of toxins that pose a risk to animaland human health. Therefore, control and prevention of the growth ofsuch harmful organisms is a significant goal in environmental science.Bioremediation using macrophyte allelochemicals for algal bloomcontrol has been widely accepted because of its higher environmentalsafety and the ability of macrophytes to restore and renew thedamaged aquatic ecosystem. The present study investigates whetherepiphytic and planktonic toxic cyanobacteria respond similarly toallelopathic activity of the submerged macrophyte stratiotes aloides.

Methods: Changes in growth, toxin production, and activities ofalkaline phosphatase (APA) and antioxidative enzymes (Glutathione S-transferase (GST) & Glutathione peroidase (GPX) of toxic epiphytic(Merismopedia tenuissima & Leptolyngbya boryana) and planktonic(Anabaena variabilis) of cyanobacteria were studied in a batchexperiment over a 15-day exposure to different concentrations of the50 % aqueous acetone extract of the submerged macrophyteStratiotes aloides.

Results: The results showed that epiphytic and planktonic species ofcyanobacteria exhibited differential responses to the allelopathicactivity of this macrophyte. Stratiotes aloides extract reduced thegrowth, alkaline phosphatase activity and toxin production ofplanktonic cyanobacterim, with an increase in lipid peroxidation,glutathione and activities of antioxidative enzymes (GST & GPX) ofthis alga. In contrast, this extract increased the growth and toxinproduction in epiphytic cyanobacteria, but it did not significantly affectlipid peroixdation or the activities of APA and antioxidative enzymes.

Conclusions: The macrophyte Stratiotes aloides has an allelopathicinhibition on planktonic cyanobacteria only, but can stimulate thegrowth and toxin production of epiphytic cyanobacteria attached to itssurface. Therefore, this study suggests that macrophytes stimulatingthe growth of toxic epiphytic cyanobacteria should be avoided as abiological control of harmful cyanobacteria in water resources.

P.037

LEAD(II) UPTAKE BY THE CYANOBACTERIUM SYNECHOCYSTIS SP.STRAIN PCC 6803 IMMOBILIZED IN CALCIUM-ALGINATE BEADS.

L.F. Allison, J-H. Yau, N. T. Flynn, M.M. Allen.

Wellesley College, Wellesley, MA, USA.

The presence of unsafe levels of lead(II) in water systems is a problemworldwide as lead is known to cause irreversible health problems.Bioremediation, the use of living organisms to remove pollutants, hasemerged as an efficient and less expensive method for environmentaldecontamination. The cyanobacterium Synechocystis sp. strain PCC6803 was immobilized in calcium-alginate beads in order to enableeasy removal of contaminated bacteria from the environment.Alginate is a polymer derived from brown algae, which has shownpotential to bind cations. Beads were prepared by dropping varyingconcentrations of alginate and cells through a syringe needle into 0.1



M calcium chloride. Lead accumulation experiments were conductedfor 4 hours in 200 mL of 500 ppm lead; the concentration of lead insolution was monitored with an ion-selective lead electrode. Theamount of lead removed from the system was measured relative to thedry weight of the beads and reported as mg Pb2+ per gram dry weightof alginate. In order to examine the effect of immobilization on thecyanobacteria, 5% EDTA (pH 9) was used to degrade the calcium-alginate. Cells were immobilized in beads and grown in BG-11medium with 100 ppm lead; the calcium-alginate was then degradedand optical density at 750 nm was measured. It was demonstrated thatthe cells exposed to lead in beads grew at the same rate as free cellsin BG-11 medium with no lead. The cells were observed to remain inclusters after the alginate was degraded. Rapid aggregation was alsoobserved for free cells inoculated into a 500 ppm lead solution. Theseresults suggest the cyanobacteria may be forming a biofilm whenstressed in the calcium-alginate beads and when exposed to lead. Theability of calcium-alginate to absorb lead from solution was evaluated;beads prepared with 4% (w/v) alginate and without cells were capableof removing an average of 340±30 mg Pb2+ per gram. WhenSynechocystis sp. strain PCC 6803 cells were embedded in the beads,the maximum accumulation of lead for all cell samples measured was479 mg Pb2+ per gram; this sorption was produced by cells with anoptical density at 750 nm of 1.0 in the exponential phase of growth.Stationary phase cells at the same optical density were found toremove 466 mg Pb2+ per gram. However, cells in the exponential andstationary phases of growth, when tested at higher optical densities of2.0 and 4.0, had decreased lead uptake by 110 to 170 mg Pb2+ pergram. Beads embedded with killed cyanobacteria were able to absorblead at levels approximately equal to the 4% beads without cells. Theaverage uptake for the dead cells in 4% alginate was 330±15 mg Pb2+

per gram. Thus, cyanobacteria have been shown to remove lead fromsolution at levels significantly higher than calcium-alginate beadswithout cells. Both the growth phase and density of the cells at thetime of uptake appear to significantly affect the amount of lead thecells are capable of removing from solution.

P.038

USED OF ARBUSCULAR MYCORRHIZAL FUNGI ANDPSEUDOMONAS PUTIDA IN PHYTOTOXICITY TESTS OFSEWAGED SOIL.

M. Attia, Namet M. Awad, Azza Sh. Turky.

Agricultural Microbiology Dep. National Research Centre, Dokki,Cairo, Egypt.

A pot experiment, with tomato plants, was carried out to judge theuse of bio-indicators for phytotoxicity in sewaged soil. A sandy soilheavily fertilized with compost was used for comparison. Results showthat mycorrhizal root colonization of tomato plants and totalmycorrhizal spore numbers were significantly low in sewaged soilcompared to organic fertilizer amended soil. More dense populationof Pseudomonas spp., bacterial counts as well as high dehydrogenaseactivity was retrieved in the rhizosphere of tomato plants grown in soilamended with organic fertilizers compared to those grown in sewagedsoil. Relatively different CO2 release potentials were showed amongdifferent treated soils (from about 15 to about 72 mg CO2 100g

-1

within 7 days). Soils biofertilized with both microorganisms liberatedmore CO2 than non-biofertilized ones. Microbial respiration looks asthough unaffected even at heavy metal concentrations. AM fungi andPs. putida elevated soil bio-activity regime as a result of improving soilquality, as indicated by the dehydrogenase activity, microbial density,CO2 evolution, as well as percentage of mycorrhizal infection andspore numbers.

P.039

USING METABOLIC NETWORKS TO EVALUATE PATHWAYCONSTRAINTS OF BIOENERGY PRODUCTS IN SYNECHOCYSTISSP. PCC 6803.

K.L. Tibbles, S. Bryan, N.J. Burroughs.

Warwick Systems Biology Centre, Coventry, United Kingdom.

A stoichiometric metabolic network of Synechocystis sp. PCC 6803was constructed from genomic data and coupled with models forrespiration, photosystem electron transport and bioenergy productgeneration- hydrogen, ethanol and bio-diesel, building on theprevious models of Shastri & Morgan, 2005 and Fu, 2008. Bydetermining the elementary models of the system we examined theoptimal pathways for growth and bioenergy product generation. Thisallowed determination of essential genes for production of theseproducts and their commonality with requirements for growth, therebyascertaining the biotechnological constraints of synechocystis forbioenergy utilisation. Further, we determined which of these 3products is theoretically the most efficient as regards light energycapture.

P.040

NOVEL PIGMENT PROFILES OF AEROBIC ANOXYGENICPHOTOTROPHS INDUCED BY HEAVY METAL STRESS.

Julius T. Csotonyi, Vladimir Yurkov.

Department of Microbiology, University of Manitoba, Winnipeg, MB,Canada.

Aerobic anoxygenic phototrophs (AAP) are obligately aerobic bacteriathat can nonetheless supplement their energy requirements with light,using the pigment BChl a, which is functionally incorporated into areaction center and light harvesting complexes. Many AAP possessconstitutive high level resistance to several toxic oxyanions of heavymetals or metalloids – collectively, metal(loid)s – such as Se, Te or V.The reason for their high level resistance is incompletely understood,but the involvement of photosynthetic pigments has beenhypothesized. To gain an understanding of the response of theirpigment systems to heavy metal duress, AAP from hypersalinesupralitoral marine microbial mats, hypersaline springs andhydrothermal systems were cultured in the presence of highconcentrations of K2TeO3 (up to 1000 µg/ml), Na2SeO3 (up to 1000µg/ml) and NaVO3 (up to 10000 µg/ml). Spectrophotometry wasperformed on pigments extracted with acetone/methanol (7:2, v/v).From Erythrobacter litoralis strain T4, extracted pigments were furtherpurified by thin layer chromatography for identification. AAPexpressed a diversity of altered pigment profiles under heavy metalstress, including decreased or enhanced BChl and carotenoidsynthesis, and the appearance of novel pigments not expressed inmetal-free cultures. In the presence of tellurite, Erythrobacter litoralisstrain T4 accumulated a putative precursor to BChl a, tentativelyidentified by spectrophotometric analysis as Mg-protoporphyrin IX orits monomethyl ester. Identification of accumulated intermediates ofpigment synthesis pathways helps to reveal the nature of the responseof AAP to heavy metal stress. Elucidation of their molecular defensestrategies will be invaluable to the development of wastemanagement techniques in heavy metal industries.



P.041

HYDROGEN PHOTO-PRODUCTION FROM VARIOUS DARKFERMENTATION EFFLUENTS BY TWO RHODOPSEUDOMONASPALUSTRIS STRAINS.

Alessandra Adessi, Lucia Bianchi, Luca Collina, Michela Palomba,Roberto De Philippis.

Department of Agricultural Biotechnology, University of Florence,Piazzale Cascine 24, I 50144 Firenze, Italy.

Introduction: Among the biological H2 production processes so fartested or still under study, the sequential fermentations carried out bychemo- and photo-heterotrophic bacteria seems to be the mostpromising one, owing to the possibility given by the combination ofthe two types of fermentation to approach the theoretical maximumyield of 12 mol of H2 produced per mol of glucose consumed.However, in spite of the theoretical advantages of the integration ofthe two processes, only few experimental data are available on theuse of the combined H2 production system.

The aim of this work was to test the H2 photo-evolution performancesof two Rhodopseudomonas palustris strains growing on three differentspent media deriving from dark fermentations carried out by chemo-heterotrophs.

Methods: Rp. palustris strains 42OL and AV33 were grown in 250 mlbioreactors under anaerobic conditions, 30°C, light intensity of 200µmol (photons) m-2 sec-1.

The media utilized for photo-fermentation were:

(1) TDF, constituted by the effluent of the dark fermentation of glucosecarried out by the thermophilic bacterium Thermotoga neapolitanaDSM 4359 (2.1 g L-1 acetate and 0.3 g L-1 lactate).

(2) MDF, constituted by the effluent of the dark fermentation ofglucose carried out by the mesophilic bacterium Paenibacilluspolimixa ISSDS-851 (0.8 g L-1 acetate and 2.7 g L-1 lactate).

(3 VDF, constituted by the effluent of the dark fermentation ofvegetable residues carried out by the autochthonous microflorapresent on the residues (2.1 g L-1 acetate and 7.8 g L-1 lactate).

Results: A first set of experiments showed that the addition of ferriccitrate to the effluents is necessary in order to have H2 production.Both R. palustris strains showed good performances with VDF: a meanH2 production rate of 15.6±1.0 ml H2 (L*h)

-1, with a substrateconversion of 43%, and a mean H2 production rate of 10.7±2.6 ml H2

(L*h)-1, with a substrate conversion of 44%, were obtained with 42OLand AV33, respectively. Photo fermentation of the other two effluentsshowed a lower degree of substrate conversion in H2 as aconsequence of the considerable growth observed with bothsubstrates. Indeed, with TDF the highest H2 production rate, obtainedwith strain 42OL, was about 4 ml H2(L*h)

-1, with a substrate conversionof 16.5%, while with MDF the highest H2 production rate, obtainedwith strain AV33, was about 2 ml H2(L*h)

-1, with a substrate conversionof 7.1%. NMR analyses of TDF and MDF substrates showed thepresence of large amounts of N-containing compounds, which causedthe utilization of fatty acids as carbon sources for cell growth insteadof as electron donors for the synthesis of H2.

Conclusions: Photo-fermentation of vegetable residues or of effluentsderived from previous dark fermentations is a promising process,giving the possibility to recover energy from organic wastes bycoupling the production of H2 with waste disposal, but needs to beoptimized, in particular in the presence of N-containing compoundswhich cause the shift of the microbial metabolism from H2 productionto cell growth.

Acknowledgments The Authors would like to thank the ItalianMinistry of University and Research (MIUR) (FISR Project IDROBIO) andEnte Cassa di Risparmio di Firenze (Firenze Hydrolab Project), whichpartially supported this research, and Dr Agata Gambacorta, ICB-CNR,Pozzuoli, Italy, and Dr Giulio Izzo, ENEA - Casaccia, Roma , Italy, whokindly provided the spent media of Thermotoga neapolitana andPaenibacillus polimixa, respectively.

P.042

NEW MOLECULAR MARKERS FOR QUICK IDENTIFICATION OFTHE HEPATOTOXIN-PRODUCING CYANOBACTERIAL STRAINSBELONGING TO GENUS MICROCYSTIS.

Bogdan Drug�1,2, Martin Welker3, Bogdan Fren�iu1, Adriana Bica1,2,Cristian Coman1,2, Nicolae Drago�1,2.1Babe�-Bolyai University, Cluj-Napoca, Romania; 2Institute of BiologicalResearch, Cluj-Napoca, Romania; 3Technische Universität, Berlin.

Introduction The cyanobacteria belonging to genus Microcystis arewell-known for their capacity to produce hepatotoxins, also known as“microcystins”. These are secondary metabolites of peptide nature,and they are nonribosomally synthesized by a multi-enzymaticcomplex, consisting of peptide synthetases and poliketide synthases.

The microcystins characteristics make their identification andcharacterization very difficult, and thus, the cyanobacterial bloomsmay be extremely dangerous for the humans or animals that interactwith the water in which these blooms are developed. The cellsdestruction by chemical means does not represent a proper solution,as long as the toxins are usually found only inside of the cells, thusbeing released if the cells are lysed. That is why the most suitablesolution to this problem is to find ways to identify the toxiccyanobacterial blooms during their initial phases, in order to limit therisk of intoxication. The purpose of this study was to find suitablemolecular markers able to provide the strict and quick detection of thecyanobacterial strains with toxic potential of genus Microcystis.

Methods The hepatotoxic potential of the strains was analyzed byPCR amplification of certain genomic regions considered responsiblefor the synthesis of the enzymes involved in toxin production. Theresults achieved by this molecular method were verified throughcertain toxicity tests that consisted in the intraperitoneal injection ofmice with cyanobacterial suspension concentrated by centrifugation.The strains toxicity was also verified by MALDI-TOF (Matrix-AssistedLaser Desorption/Ionization) using a mass spectrometer. In order toachieve a correlation between PCR results and toxicity tests we triedto discriminate the amplicons by DGGE (Denaturing Gradient GelElectrophoresis). Eventually, we have sequenced the PCR amplicons,and based on the sequences multiple alignments we have designednew primer pairs able to generate amplicons only in those strainswhich produce hepatotoxins.

Results The intraperitoneal injection of mice with cyanobacteria hasemphasized the presence of toxicity in 5 of the 24 analyzed strains.The MALDI-TOF MS spectra have also displayed the presence ofmicrocystins in these 5 strains, while cyanopeptolins and othersecondary metabolites were detected in other strains.

The primers used for the amplification of certain genomic regionscorrelated with the toxic potential were found in several articles, butduring PCR they have generated amplicons in 7 strains, not only in the5 toxic ones. The DGGE profiles could not differentiate these 7amplicons according to the strain toxicity. The sequences multiplealignments allowed us to design new primer pairs, which were specificfor the 5 toxic strains.



Conclusions During this study we have observed that theoligonucleotide primers found in various articles did not generateamplicons during PCR in our toxic strains, as expected, but also inother two non-toxic strains. The DGGE technique has not proved tobe proper to discriminate between amplicons according to strainstoxicity.

Based on the sequences multiple alignments, we have obtained newprimer pairs which are able to amplify certain regions of the genecluster responsible for toxicity, these being highly specific for the toxicstrains. One of these 5 strains belongs to species Microcystiswesenbergii, which was thought to be the only species of genusMicrocystis which does not produce microcystins.

P.043

PARTS AND MODULES FOR H2 PRODUCTION ANDCONSTRUCTION OF A CYANOBACTERIAL CHASSIS.

Daniela Ferreira1,2, Filipe Pinto1,2, Catarina C. Pacheco1,3, Miguel Lopo1,Pedro Moradas-Ferreira1,3 & Paula Tamagnini1,2.1IBMC - Instituto de Biologia Molecular e Celular; 2Faculdade deCiências, Departamento de Botânica; 3Instituto de CiênciasBiomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto,Portugal.

Introduction: The BioModularH2 project (FP6, NEST-2005-Path-SYN,Contract 043340) aims at designing reusable, standardized molecularbuilding blocks that integrated into a chassis will result in aphotosynthetic bacterium containing engineered chemical pathwaysfor competitive, clean and sustainable hydrogen production [1]. Thechassis is being constructed using as basis the unicellularcyanobacterium Synechocystis sp. PCC 6803. Synthetic parts andmodules related to oxygen consumption and hydrogen productionwere designed, and are being constructed/synthesized.

Methods: Design and construction of standardized vectorscompatible with the BioBrick™ format. The vectors are being used toremove redundant parts (e.g. genes) from the bacterial genomeand/or to integrate synthetic parts/devices.

Generation of mutants through double homologous recombination,using the constructed standard vectors.

Mapping of the Synechocystis chromosomal neutral sites usingbioinformatics tools and RT-PCRs.

Design and construction of synthetic parts and assembly of modulesfollowing the BioBrick™ system philosophy.

Characterization of parts and devices in the cyanobacterial chassis,using reporter genes and measuring enzyme activities.

Results: The chassis preparation started with the removal ofredundant parts, like the deletion of genes encoding theSynechocystis native bidirectional hydrogenase. In addition, theSynechocystis genome was screened for putative neutral sites, whichwill facilitate the integration of the synthetic parts/devices (e.g. anefficient heterologous hydrogenase).

The construction of optimized synthetic modules/devices requires thepreliminary characterization of parts, such as promoters, transcriptionfactors, RBSs and terminators, which is currently being performed.Furthermore, several oxygen consuming devices were designed andsynthesized, and are in the process of being tested. One of thesedevices will be chosen to provide a microaerobic/anaerobicenvironment required for the optimal expression of a heterologoushydrogenase.

Conclusions: The characterization of the different parts will allow the

design and construction of functional modules that can be assembledinto circuits with predictable behaviour. The final goal is theconstruction of the cyanobacterial chassis accommodating thecharacterized synthetic modules will allow a sustainablephotobiological H2 production, and the parts and devices producedwill become available for other biotechnological applications.

References:

[1] http://www.biomodularh2.org

P.044

CHANGES IN MICROCYSTINS AND OTHER OLIGOPEPTIDESPROFILES IN THE CYANOBACTERIUM RADIOCYSTIS FERNANDOIEXPOSED AT THREE DIFFERENT LIGHT INTENSITIES.

Alessandra Giani, Daniel de Albuquerque Pereira.

Department of Botany, Institute of Biological Sciences, UniversidadeFedearl de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.

Introduction: Cyanobacteria are known to produce various kinds ofbioactive oligopeptides. The most common peptides are microcystins,cyanopeptolins, anabaenopeptins and microviridins. There are severalhypotheses about the functions of these metabolites, which includesprotection against grazing, allelophatic effects, quorum sensing andinternal metabolism, but some of these functions might be only ofsecondary importance. The comprehension of the factors affecting theproduction of these secondary metabolites may give some cluesabout the ecological and physiological functions of these compounds.

Methods: In this study, the influence of light intensity on the patternof oligopeptide production of a Radiocystis fernandoi strain wastested. The strain used in this work was isolated from a reservoirlocated in the southeastern region of Brazil. This species usually formsblooms in eutrophic aquatic systems. The experiments were done intriplicates and consisted of three treatments, each one at a differentlight intensity: 25 µmol.m-2.s-1, 65 µmol.m-2.s-1 and 95 µmol.m-2.s-1.Experiments lasted 10 days and growth conditions were 12h light: 12hdark photoperiod at 20°C. At the end of the experimental time,cultures were freeze dried and the peptides extracted with 75%methanol and purified with C18 SPE cartridges. The peptides werequantified by HPLC (Waters Alliance) and the main peaks werecollected and analyzed in a MALDI-TOF Bruker Autoflex III massspectrometer.

Results: Seven different peptides were identified, 1 cyanopeptolin(Cy-1071), 1 microviridin (Mv-1709) and 5 microcystins (Mc-RR, dMc-YR, Mc-YR, Mc-FR and Mc-WR). The production of Mc-YR, Mc-FR,Mc-WR and the sum of all five microcystins were highest at 25 µmol.m-

2.s-1, lowest at 65 µmol.m-2.s-1 and intermediate at 95 µmol.m-2.s-1. Theproduction of Mc-RR was intermediate at 25 µmol.m-2.s-1, higher in the65 µmol.m-2.s-1 treatment and lower at 95 µmol.m-2.s-1. The Mv-1709was produced in the greatest amount at 65 µmol.m-2.s-1 and in loweramounts in the other two treatments. The production of the sevenpeptides together was highest in the 25 µmol.m-2.s-1 treatment andlower in the other two treatments. The production of Cy-1071 and thedMc-YR showed no significant differences among the treatments.

Conclusions: It is possible that differences observed between the MC-RR and other microcystins could be explained by genetic regulationand/or aminoacids and enzyme activities modulated by different lightintesities. The fact that the different classes of peptides do not presentsimilar responses to environmental factors, such as light, suggests thatthese compounds may have different functions.



P.045

ENGINEERING BIODEGRADABLE PLASTICS USINGEXTRACELLULAR MATRIX IN THE TERRESTRIALCYANOBACTERIUM NOSTOC COMMUNE.

K. Inoue-Sakamoto1, T. Ogawa2, T. Sakamoto3.1Department of Applied Bioscience, College of Biotechnology andChemistry, Kanazawa Institute of Technology (K.I.T.), Ohgigaoka,Nonoichi; 2Department of Applied Chemistry, College ofBiotechnology and Chemistry, K.I.T., Ohgigaoka, Nonoichi; 3Divisionof Life Sciences, Graduate School of Natural Science and Technology,Kanazawa University, Kakuma, Kanazawa; Japan.

Introduction: The terrestrial cyanobacterium Nostoc commune iswidely distributed on earth and is adapted to various externalconditions by acquiring tolerance to desiccation, ultraviolet light andextreme temperatures. N. commune forms colonies in which thefilamentous cells are embedded in extracellular matrix. The majorcomponent of extracellular matrix in N. commune is extracellularpolysaccharides (EPSs). EPSs absorb large amount of water rapidly andhelp the cells to maintain biological activities including photosynthesisand respiration. The hydroscopic or absorbent ability of EPSs can beapplied to non-hydroscopic materials so the composites are used as apackage to regulate moisture level inside the package. Alternatively,the composites can be used for agricultural soil cover sheet as abiodegradable sheet to enhance degradation of the composite bymicroorganisms in soil. In this study, colonies of N. commune orisolated EPSs were blended with plastics to test the capacity of waterin the composites, and the physical properties of the composites wereinvestigated.

Methods: The colonies of N. commune naturally grown in the fieldwere collected, washed and air-dried. The dried colonies were crushedin a kitchen mixer and then powdered in a frozen pulverizer. Thepowder with diameter less than 100 µm were selected with a sieveand blended directly with pellets of polybuthylene succinate (PBS) orlow density polyethylene (LDPE) in weight ratio of 10:90, 15:85 and20:80 by using a compression molding machine and an extruder incombination. EPSs were prepared from N. commune colonies byextraction with acetone and diluted acetic acid and precipitation byethanol. EPSs were freeze-dried, powdered and blended with PBS orLDPE as described above. The sample sheets of the composite wereused for the analyses. For mechanical properties, tensile stress andtensile strain at break were measured by a tensile tester. For the waterabsorption capacity, the samples were immersed in deionized waterand the increase in weight of the samples was measured. For thebiodegradability assay, the samples were buried in wet soil and theirsurfaces were periodically observed by scanning electron microscopy.

Results: The surface of sheets made from plastic composites with N.commune powders were rougher than that of control, showing thatadhesion between plastics and N. commune powder is poor and thetwo polymers are incompatible. The tensile stress of pure LDPEpolymer was about 10 MPa, whereas the stresses of LDPE compositeswere 6-9 MPa. The composites absorbed 1.2-1.5% (w/w) of water andthe tensile stresses of the water-absorbed composites were 4-5 MPa,which was approximately 50% of control. The surface of the compositesheets kept in soil for several months had more irregular structure thanthat of untreated control, suggesting that the polymer from N.commune absorb water and enhance degrability of the composites.

Conclusions: The composites were successfully made from plasticpellets and powders of N. commune colonies or EPSs. The plasticcomposites still remained over 50% of the tensile stress of non-

blended control, indicating that the composite has the strength forpractical usages as bioplastics. The composites have water-absorbingcapacity and can be used as a container for either fresh vegetables ordry medicament. N. commune EPSs enhance biodegradability of thecomposites in soil and it maybe an advantage for the disposal of theplastics.

P.046

ONE-STEP PLASMID CONSTRUCTION FOR GENERATION OFKNOCK-OUT MUTANTS IN CYANOBACTERIA.

J.H. Jacobsen1, D. Paiva2, N.-U. Frigaard1, Y. Sakuragi2.1Department of Biology, Faculty of Science, University of Copenhagen,2Department of Plant Biology and Biotechnology, Faculty of LifeSciences, University of Copenhagen, Frederiksberg, Denmark.

Introduction: Cyanobacteria include some of the most efficientphototrophic organisms known, featuring doubling times of less than4 hours at exponential growth for certain strains. They are thereforeideal for biomass and bioenergy production through metabolicengineering, including gain-of-function and loss-of-functionmutagenesis. The recently reported uracil-specific excision reagent(USER) cloning method1, 2, previously applied on plants and fungi,allows highly efficient ligase- and restriction-enzyme-independentconstruction of knock-out vectors. In the present work we adapted the“four fragment USER-cloning method”1, for utilization in cyanobacteriain order to streamline the construction of vectors for loss-of-functionmutagenesis.

Methods: Synthetic oligonucleotides encoding the USER-cloningcassette, comprising a BamHI restriction site, flanked by PacI andNt.BbvCI restriction sites, was inserted into pUC19. Antibioticresistance cassettes suitable for selection in cyanobacteria wereinserted at this unique BamHI restriction site within the USER-cloningcassette. Digestion of the resulting USER-cloning vectors with PacI andNt.BbvCI generates two fragments, the vector backbone and afragment containing the antibiotic resistance gene, respectively.Regions flanking the genes encoding glycogen phosphorylase (glgP)and photomixotrophic growth related protein (pmgA) fromSynechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 wereamplified with uracil-containing oligonucleotides and annealed withthe digested vector and directly used for Escherichia colitransformation. Constructs were confirmed by PCR and restriction mapanalysis. The final constructs were used to transform Synechococcussp. PCC 7002 and Synechocystis sp. PCC 6803.

Results: A set of eight different plasmids for “four fragment USER-cloning” has been constructed, each construct containing one of theantibiotic resistance conferring genes aadA (SmR,SpR), aacC1 (Gmr),ermC-cat (Emr,Cmr), nptII (Kmr,Neor), inserted in either parallel orantiparallel orientation. Knock-out vectors for insertional deletion ofglgP or pmgA in Synechococcus sp. PCC 7002 and Synechocystis sp.PCC 6803 have been constructed in the plasmid vectors containingaacC1 and aadA for glgP and pmgA, respectively. Restrictiondigestion patterns of selected clones confirmed the presence of thecorrect inserts in all the clones, demonstrating the high efficiency ofthe cloning strategy. Transformants from cyanobacterial transformationwith glgP and pmgA deletion constructs have been obtained, andhomozygous mutants are currently being selected. The results fromanalysis of glycogen contents, thus biomass capacity, of the mutantswill be presented.

Conclusions: The USER-cloning method provides a fast and reliablestrategy for generation of knock-out mutants in the cyanobacterialspecies Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803



through single step construction of knock-out vectors.

References:1 Frandsen, R.J., Andersson, J.A., Kristensen, M.B., Efficient fourfragment cloning for the construction of vectors for targeted genereplacement in filamentous fungi. BMC Molecular Biology (2008), 9.70

2 Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H., Advancing uracil-excision based cloning towards an ideal technique for cloning PCRfragments. Nucleic Acids Research (2006), 34. e122

P.047

MAXIMIZING HYDROGEN PRODUCTION IN RUBISCO-COMPROMISED MUTANTS OF NONSULFUR PURPLEPHOTOSYNTHETIC BACTERIA.

Rick A. Laguna, F. Robert Tabita.

Department of Microbiology, The Ohio State University, Columbus,OH, USA.

Introduction: The Calvin-Benson-Bassham (CBB) reductive pentosephosphate pathway allows for the reduction of CO2 into cellularcarbon under aerobic chemoautotrophic and anaerobicphotoautotrophic growth conditions. By contrast to its use as a majorcarbon source, during photoheterotrophic growt CO2 is primarily usedas an electron acceptor. The CBB cycle thus performs the importantfunction of maintaining the redox balance of the cell under thesegrowth conditions. Ribulose 1,5-bisphosphate carboxylase-oxygenaseis the key enzyme of the CBB pathway that catalyzes the reduction ofCO2. Over the years we have shown that nonsulfur purple (NSP)photosynthetic bacteria possess an array of metabolic and regulatorycapabilities that allow for the utilization of alternative redox sinks whenthe primary electron sink, CO2, is nullified via the inactivation ordeletion of the RubisCO genes. For example, in many instances thederepression of nitrogenase synthesis occurs under normal repressiveconditions. Such gain-of-function adaptive mutant strains have beenobtained from Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodospirillum rubrum, and Rhodopseudomonas palustris, wherebysuch strains balance their redox potential via nitrogenase-catalyzedreduction of protons to hydrogen gas. Over the years we have shownthat nitrogenase-derepressed mutant strains produce copiousquantities of hydrogen gas by virtue of using the nitrogenase enzymecomplex exclusively as a hydrogenase. NifA is the transcriptionalactivator of the nif (nitrogen fixation) operons. In general nifAexpression is regulated by the cascade of proteins that encompass thenitrogen fixation regulon (Ntr), while post-translational regulation ofNifA occurs through protein-protein or protein-metabolite mediatedcontact.

Method: To determine if a mutation occurred in NifA of RubisCO-deletion nitrogenase derepressed mutant strains, the nifA genes weresequenced. Next, the mutant NifA protein was inserted into a wild-type background to determine if derepression of the nitrogenasecomplex occurred. Hydrogen production values were recorded fromthe RubisCO-deletion nitrogenase derepressed mutant strains.

Results: A single point mutation in the NifA protein was identified inthe RubisCO-deletion nitrogenase derepressed mutant strains of R.capsulatus, R. sphaeroides, and R. palustris. A mutant NifA proteinappeared to be responsible for derepression of the nitrogenasecomplex in R. palustris. In R. sphaeroides an additional unidentifiedmutation appears to be involved in the derepression of thenitrogenase complex. Interestingly, no such nifA mutation was foundin a nitrogenase-derepressed strain of a RubisCO knockout strain of R.

rubrum. The RubisCO-deletion strain of R. palustris produced themost hydrogen compared to the other mutant strains.

Conclusion: These results suggest that some NSP bacteria utilizedifferent mechanisms to derepress nitrogenase synthesis in CBB-compromised mutant strains. Inactivating competing redox balancingprocesses, such as the CBB pathway, plays a crucial role in maximizinghydrogen production in nitrogenase-derepressed strains. Furtherstudies are directed at understanding the molecular mechanisms thatallows the cbb and nif systems to compete for reducing equivalentsand become subject to reciprocal control.

P.048

SCREENING OF SOIL CYANOBACTERIA FROM VIETNAM FORANTIBACTERIAL ACTIVITY.

Le Thi Anh Tuyet1, Sabine Mundt2.1Department of Natural Sciences, Hong Duc University, Thanh Hoa,Vietnam; 2Institute of Pharmacy, Ernst-Moritz-Arndt-UniversityGreifswald, Germany.

Introduction: A large number of interesting metabolites of widechemical diversity has been isolated from cyanobacteria andcompounds with antibacterial, antiviral, antifungal and cytotoxicactivity have been detected. Currently we are investigatingbiologically active chemical substances from cyanobacteria with theobjective of finding new antibacterial compounds which could be asource of new lead structures for development of antibacterial drugs.As only a few cyanobacterial species from Vietnam have beenscreened for antibacterial activity , in the present study 11cyanobacterial strains isolated from acidic soils of paddy and cottonfields in Vietnam belonging to the genera Nostoc, Anabaena,Calothrix, Scytonema, Oscillatoria and Westiellopsis were cultured andtested.

Methods: All the organisms were grown under controlled laboratoryconditions in Greifswald University, Germany. The biomasses werelyophilised and extracted with n-hexane, methanol, and water,respectively. Culture media were extracted with ethyl acetate. Allcrude extracts were tested for antibacterial activity against Bacillussubtilic ATCC 6051, Staphylococcus aureus ATCC 6538, E.coliATCC11229, and Pseudomonas aeruginosa ATCC 22853 in the agardiffusion test system using 6mm diameter paper disc containing 2 mgextract per disc. An inhibition zone of 8 mm or more was consideredas good antibacterial activity. Ampicilline was used as control.

Results: Antibacterial evaluation demonstrated that 91 % of methanolextracts exhibited activity to Bacillus subtilis and Staphylococcusaureus, whereas the maximum inhibition zones of 19mm werepresented by methanol extracts of Westiellopsis sp. VN and Calothrixelenkinii. 45 % of the methanol extracts inhibited the growth of E.coli,while 18 % of methanol extracts possessed antibacterial activityagainst Pseudomonas aeruginosa. 17 % of n-hexane extracts inhibitedthe growth of Gram-positive bacteria and 6 % of n- hexane extractsexhibited activity against Gram- negative bacteria. No aqueous extractshowed antibacterial activity. 75% of the ethyl acetate extracts ofculture medium exhibited significant activity against Gram-positivebacteria (a maximum inhibition zone of 25mm was exhibited byWestiellopsis sp. VN) and 50 % of ethyl acetate extracts inhibitedgrowth of Gram-negative bacteria. Of the 11 cyanobacteriainvestigated, at least one of the prepared extracts from biomass orcultivation medium showed antibacterial activity. Among the activeextracts, the best antibacterial activity was detected for methanol andethyl acetate extracts of Westiellopsis sp. VN and methanol extract ofCalothrix elenkinii.



Conclusions: This screening proves that Vietnamese cyanobacteria area promising source of new antibacterial substances. Based on theseresults Westiellopsis sp.VN and Calothrix elenkinii were selected forbioassay-guided isolation of the active compounds. Structuralelucidation of the substances is in progress.

P.049

SITE-DIRECTED MUTAGENESIS OF ANABAENA SP. PCC 7120NITROGENASE TO INCREASE PHOTOBIOLOGICAL H2PRODUCTION.

Hajime Masukawa1, Kazuhito Inoue2,3, Hidehiro Sakurai3,4, and RobertP. Hausinger1,5.1Department of Microbiology and Molecular Genetics, Michigan StateUniversity, East Lansing, MI, USA; 2Department of Biological Sciencesand 3Research Institute for Photobiological Hydrogen Production,Kanagawa University, Hiratsuka, Kanagawa, Japan; 4Department ofBiology, School of Education, Waseda University, Shinjuku, Tokyo,Japan; 5Department of Biochemistry and Molecular Biology, MichiganState University, East Lansing, MI, USA.

Introduction: Hydrogen gas (H2) produced photobiologically bycyanobacteria that use sunlight as the sole energy source and water asthe ultimate electron donor has the potential to become a large-scale,environmentally-friendly, renewable energy commodity. H2 isgenerated by hydrogenases and, as an inevitable by-product of N2

fixation, by nitrogenases. The best studied nitrogenase consists of twoseparable metal-containing proteins, the Fe protein (dinitrogenasereductase encoded by nifH) and the MoFe protein (dinitrogenaseencoded by nifDK). The latter contains the FeMo cofactor (FeMoco) atwhich substrate is bound and reduced. Mutagenesis of Azotobactervinelandii MoFe protein has shown that selected amino acidsubstitutions in the vicinity of FeMoco allow for effective protonreduction, while eliminating or greatly diminishing N2 fixation. Wehave examined whether modifications of residues near FeMoco of thecyanobacterial MoFe protein would allow electron flow throughnitrogenase to be directed preferentially toward proton reduction toincrease the rate of photobiological H2 production.

Methods: The nifHDK genes, with the insertion element within nifDexcised, were obtained from Anabaena sp. (sometimes called Nostocsp.) PCC 7120 genome and a non-replicating vector expressingnifHDK was constructed. Site-directed mutations were introduced intonifD. To express the mutated version of nifHDK and eliminate thewild-type version, the nifHDK genes were deleted within a ∆hupL,uptake hydrogenase-negative, variant of Anabaena sp. PCC 7120 asthe parental strain and the vector was integrated into the nifHDKdeletion mutant chromosome by homologous recombination.

Results: We performed site-directed mutation to replace six targetresidues, all predicted to lie within 5 � of FeMoco, with differentresidues for a total of 49 variants. H2 production and nitrogenase(acetylene reduction) activities of the site-directed mutants weredetermined and several mutants were shown to exhibit significantlyhigher H2 production rates in the presence of N2 than did the parental∆hupL strain. Additional targets for site-directed mutagenesis werebased on the proposed hydrophobic substrate channel that connectsFeMoco to the protein surface in the X-ray crystal structure of A.vinelandii MoFe protein. Corresponding residues were predicted inthe cyanobacterial enzyme for this region. Four hydrophobic residuesin the putative channel were replaced with bulky or charged residuesin an attempt to inhibit access of N2 to FeMoco. Thirteen single andsix double site-directed mutants were constructed; however, none of

these mutants exhibited significantly higher H2 production rates thandid the parental ∆hupL strain.

Conclusion: Particular mutations of certain residues close to theFeMoco prosthetic group were shown to lead to increased partitioningof electron flow toward proton reduction relative to acetylenereduction.

P.050

CYANOBACTERIA - A PROLIFIC SOURCE OF NEWPHARMACEUTICALS.

Sabine Mundt1,2, Elmi Zainuddin1, Thi Ngoc Ha Bui1, Susann Kreitlow1,GeroldLukowski2, Rolf Jansen 3, Victor Wray3, Manfred Nimtz3.1Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald;2Institute of Marine Biotechnology e.V., Greifswald; 3Helmholtz Centrefor Infection Research, Braunschweig; Germany.

Introduction: In the last decades screening programs have revealedthat cyanobacteria are potential sources of new active substances, butthe rich resources of German lakes, the Baltic Sea and Asian countriesare hardly screened so far. Due to recent development in bacterial andviral resistance and the increase in incidence of tumour diseasesworldwide we focused on screening of antibiotic andcytostatic/cytotoxic activity of such hardly tested cyanobacterial strainsand isolation and structural elucidation of the active substances. Forpractical use of biomass or isolated substances the form of applicationis very important.

Methods: Laboratory cultures were established and biomass andcultivation medium were extracted with solvents of different polarity.Lipophilic and hydrophilic extracts have been tested in agar-platediffusion test for antibacterial and antifungal activity, for antiviralactivity against Influenza virus A and Herpes virus Typ1 and forcytotoxic/cytostatic activity against MCF7 (breast cancer cell line),5637 (urinary bladder) and FL (amnion) cell lines. Bioassay-guidedisolation of the active compounds was done by columnchromatography including HPLC. Structure was elucidated by analysisof ESI-MS-MS, ESI-TOF-MS, 1D (1H and 13C) and 2D (COSY, TOCSY,ROESY, NOESY, HMQC and HMBC) NMR spectra and amino acidanalyses. The preparation of micro- and nanoparticles was doneaccording to PCT/DE 03/00747. A pre-suspension of the biomass anda surfactant-water mixture was produced by stirring followed by highpressure homogenization.

Results: Separation of the n-hexane extract of Limnothrix redekei HUB051 (Müggelsee, Germany) resulted in the identification of threeunsaturated fatty acids, α-linolenic acid, coriolic acid and α-dimorphecolic acid showing antimicrobial activity in vitro. From themethanol extract of the filamentous Nostoc strain CAVN 10 from a soilsample collected in North Vietnam four new compounds,carbamidocyclophanes, with cytotoxic activity against tumor cell lineshave been isolated. Separation of the methanol extract of aMicrocystis strain (Plön, Germany) led to identification of two newcyclic peptides with activity against Influenza virus A. Application ofmicro- or nanoparticles prepared from the biomass of an Anabaenastrain Bio 33, isolated from the Baltic Sea, prevents the dermalcolonisation of MRSA.

Conclusions: Our first results show that so far hardly investigatedcyanobacteria from the Baltic Sea, German lakes and from Asiancountries are sources of structural new active compounds withpotential therapeutically value. Especially for external applicationmicro- and nanoparticles are a promising application form.



P.051

GENES PUTATIVELY INVOLVED IN THE SYNTHESIS OFCYANOBACTERIAL EXOPOLYSACCHARIDES.

Sara Pereira1,2, Ângela Brito1,2, Andrea Zille1, Pedro Moradas-Ferreira1,3

& Paula Tamagnini1,2.1IBMC - Instituto de Biologia Molecular e Celular; 2Faculdade deCiências, Departamento de Botânica; 3Instituto de CiênciasBiomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto,Portugal

Introduction: Many cyanobacterial strains possess outside their outermembrane, additional surface structures, mainly of polysaccharidicnature, that are usually referred to as exopolysaccharides (EPS). Eventhough EPS are common in cyanobacteria, their biosynthetic pathwaysand the factors that regulate these processes are far from being fullyunderstood. However, the mechanisms involved in the synthesis ofEPS are relatively conserved throughout bacteria, involving threeclasses of proteins: (i) enzymes involved in the biosynthetic pathwaysof nucleotide sugars (ii) glycosyltransferases, and (iii) those required forthe oligosaccharide or polysaccharide extension and processing. Thesugar activation/modification enzymes and the glycosyltransferasesare strain dependent, whereas the proteins involved in thepolymerization, chain length control and export are generallyconserved among bacteria [1].

Methods: Genome sequences were retrieved from GenBank andcomputer-assisted sequence comparisons were performed usingBLASTp, cDART (NCBI), ClustalW (EMBL-EBI), and SMART.

Transcription profiles of the genes were evaluated by RT-PCR usingCyanothece sp. CCY 0110 cells grown under different physiologicalconditions that are known to affect EPS production: presence/absenceof combined nitrogen and different light regiments. Carbohydratecontent of the cultures was measured using the Dubois method [5].

Results: An in silico analysis of genome sequences performed in threemorphological distinct types of cyanobacteria (unicellular, filamentousand filamentous heterocystous) revealed the presence of genesencoding proteins that, in other organisms, are involved in the laststeps of EPS production. In cyanobacteria those genes, namely wza,wzb, wzc, wzx and wzy are frequently clustered and often occurring asmultiple copies. Using this information a model was proposed for thepolymerization and export of cyanobacterial EPS. Additionally,preliminary results revealed that the transcripts levels of wzb(encoding a tyrosine phosphatase that putatively regulatespolysaccharide chain-length by dephosphorylation of Wzc) and wzy(encoding a polymerase) are higher in cells grown in absence ofcombined nitrogen.

Conclusions: Although several genes encoding proteins putativelyinvolved in the EPS polymerization and export were identified incyanobacterial genomes, their physical organization differs from whatis observed in other microorganisms, where the genes are frequentlyclustered and transcribed as one or two operons [2,3] suggesting thatthe regulation of the synthesis and export of EPS is more complex incyanobacteria.

References:

[1] Pereira et al (2009) FEMS Microbiol Rev (accepted for publication)

[1] De Vuyst L et al (2001) Int Dairy J 11: 687-707

[1] Whitfield C (2006) Annu Rev Biochem 75: 39-68

P.052

IDENTIFICATION OF PROTEINS INVOLVED IN THE CHROMIUMREDUCTION IN SYNECHOCYSTIS DERIVATIVE MK-TR OBTAINEDBY GAMMA IRRADIATION.

Saiqa Razi, Shahida Hasnain, Byung-Gee Kim.

University Of The Punjab, Lahore, Punjab, Pakistan.

Introduction: Synechocystis species, AHZ-HB-MK (DQ 381960)isolated from the local chromium contaminated industrial wastewater,able to reduce 68% toxic hexavalent Chromium (Cr VI), was used inthis study. Cultural characteristics of this strain have been determined(Hameed and Hasnain, 2005) and the plant-microbe interactionexperiments with this strain proved that it is important in thebioremediation of toxic Cr (VI) from the soil and in the improvement ofgrowth parameters of Triticum aestivum grown under Cr (VI) stress(Faisal et al., 2005). To produce mutants of this strain with betterchromate reduction abilities, the strain was irradiated with a 60Cosource in a dose range of 0.5, 1, 2, 5, 10 and 20 Grays (Gy) at differentstages of growth i.e. after 5, 15 and 30 days of incubation. Derivativesreducing < 90% Cr VI were obtained with low dose irradiation incultures irradiated at earlier growth stage (Razi and Hasnain, 2006).Biochemical analysis including the estimation of soluble proteins,peroxidases, carotenoids and auxin showed that low radiation alsocaused stimulatory effects on all of these parameters at earlier stage ofgrowth (Razi and Hasnain, unpublished data). Maximum reductionpotential i.e. 94.93% was obtained at dose 2 Gy in cultures irradiatedafter 5 days of incubation. This derivative was labeled asSynechocystis MK-TR. In the present study, proteomic study of thisselected derivative was done in order to identify the proteins involvedin the chromium reduction.

Methods: After three days of Cr VI exposure, crude extracts fromtreated and untreated cells were compared at the proteome levelusing one-dimensional SDS-polyacryalmide gel electrophoreticapproach. Proteins were extracted from 100 ml of cultures byrepeated freeze-thawing, sonication and vortex mixing (Nayak et al.,2007). From cell extracts, the concentration of proteins was measuredby the Coomassie Blue protein assay at 595 nm utilizing bovine serumalbumin as a standard (Bradford, 1976). Ten out of total twenty proteinbands were upregulated in this derivative under Cr VI stress. These tenprotein bands were cut from 1D-SDS polyacryalmide gels and weresubjected to in-gel trypsin digestion (Shevchenko et al., 1996). Theeluted peptides were dissolved in 0.1% formic acid and loaded on C-18 columns. Samples were analyzed by a combination of anano-HPLC/microelectrospray ionization on a LCQ Deca massspectrometer (ThermoFinnigan, San Jose, CA). MS-MS spectra werematched with the genome sequence of the model strain SynechocystisPCC 6803 CyanoBasehttp://bacteria.kazusa.or.jp/cyanobase/Synechocystis/index.html(Kaneko et al., 1996),

Results: Seventy-four peptides were identified from these ten selectedprotein bands. In the long list of these identified peptides we foundsll0170, a heat shock protein, slr2076 a 60kD chaperonin and sll0416identified as 60kD chaperonin 2 and GroEL2. Whereas sll0170 is also aheat shock protein 70, slr1198 was identified to be an antioxidantprotein and slr1516 was annotated as superoxide dismutase.

Conclusion: This can be assumed from these findings that theseproteins were induced due to radiation-stress and are associated withGeneral Stress-proteins family including Heat-shock proteins (HSP).They helped this strain to cope with the osmotic and heat stress whentreated with gamma radiation. When this strain was subjected to



chromium stress these proteins gave stronger expression and againhelped the strain to overcome the osmotic stress in this case inducedby chromium. The radiation dose of 2 Gy might have causedmutations in the genes of this derivative resulting in theoverproduction of these stress proteins.

P.053

ENHANCEMENT OF GROWTH AND YIELD OF TOMATO BYAPPLICATION OF PURPLE NONSULFUR BACTERIA.

Hong-Gyu Song, Kang-Hyeong Lee.

Kangwon National University, Chuncheon, South Korea.

Purple nonsulfur bacteria were isolated from river sediments and theirgrowth promoting capabilities on tomato were examined. Isolatedstrain Rhodopseudomonas sp. KL9 maximally produced 5.56mM/min/mg protein and 67.2 μM/min/mg protein of indole-3-aceticacid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may beone of the mechanisms of plant growth enhancement. Germinationpercentage of tomato seed, total length and dry weight of germinatedtomato seedling increased by 30.2%, 71.1% and 270.8%, respectivelycompared to those of the uninoculated control 7 days after inoculationof strain KL9. A greenhouse test was also carried out to examine theeffects on tomato growth of application of purple nonsulfur bacteriumRhodopseudomonas sp. The shoot length of tomato plant inoculatedby Rhodopseudomonas sp. KL9 increased by 34.6% compared to thatof control in 8 weeks of cultivation. During the same period, this strainincreased 120.6 and 78.6% of dry weight of shoot and root of tomatoplants, respectively. The formation ratio of tomato fruit from flowerwas also raised by inoculation of KL9. In addition,Rhodopseudomonas sp. KL9 treatment enhanced the fresh weightand lycopene content in the harvested tomato fruits by 98.3 and48.3%, respectively compared to those of the uninoculated control.When the effect on the indigenous bacterial community and fate ofthe inoculated Rhodopseudomonas sp. KL9 were monitored bydenaturing gradient gel electrophoresis analysis, its application didnot affect the native bacterial community in tomato rhizosphere soil,but should be repeated to maintain its population size. This bacterialcapability may be applied as an environment-friendly biofertilizer tocultivation of high quality tomato and other crops including lycopene-containing vegetables and fruits.

P.054

UNUSUAL CAROTENOIDS FROM SOME NEWLY DESCRIBEDPURPLE BACTERIA.

Shinichi Takaichi1, Ch. Sasikala2, Ch. V. Ramana3.1Department of Biology, Nippon Medical School; 2Center forEnvironment, Institute of Science and Technology, J. N. T. University;3Department of Plant Sciences, School of Life Sciences, University ofHyderabad.

Introduction: Anoxgenic phototrophic purple bacteria belong to theProteobacteria, and more than 170 species have been described.These bacteria produce many different carotenoids, which areessential for photoprotection and light-harvesting. Base on thestructures of the carotenoids and the characteristics of thecarotenogenesis enzymes, the normal pathway of carotenogenesis isthe normal spirilloxanthin pathway, and is found from around halfspecies. When one enzyme of the normal spirilloxanthin pathway islacking or is present with reduced activity, the carotenoid compositionof the bacterium will be expected to change (unusual spirilloxanthin,spheroidene, and carotenal pathways) (Takaichi (2009) Distribution and

Biosynthesis of Carotenoids, In The Purple Phototrophic Bacteria,pp.97-117). Further, Ramana group have recently found and describedmore than 20 species of purple bacteria. We found some unusualcarotenoids from these newly described purple bacteria.

Methods: New purple bacteria were described in Int. J. Syst. Evol.Microbiol., and cultured anaerobically under light. Carotenoids wereextracted from the lyophilized cells and purified. They were identifiedbased on absorption spectra, retention time on C18-HPLC, MS, and

1H-NMR analyses.

Results: Phaeospirillum chandramohanii JA145 (Anil Kumar et al.IJSEM in press) contained 10% (mol% of total carotenoids) lycopene,77% hydroxylycopene, 11% hydroxylycopene glucoside and 2%dihydroxylycopene diglucoside; “Phs. oryzae” JA317 contained 12%lycopene, 81% hydroxylycopene and 7% hydroxylycopene glucoside;Phs. molischianum DSM120 contained 17% lycopene, 77%hydroxylycopene, 5% hydroxylycopene glucoside and 1%dihydroxylycopene glucoside; Phs. fuluvum contained 11% lycopene,84% hydroxylycopene, 5% hydroxylycopene glucoside and 1%dihydroxylycopene glucoside. Carotenoid glycosides in purplebacteria are rare, and dihydroxylycopene diglucoside is only foundfrom Halorhodospira. This may be due to no activity of CrtD, andadditionally these have glucosyl transferase.

Roseospira visakhapatnamensis JA131 (Chakravarthy et al. (2007)IJSEM 57: 2453-2457) contained 3% lycopene, 82% 3,4-dehydrorhodopin, 1% anhydrorhodovibrin, 4% hydroxyspirilloxanthinand 10% spirilloxanthin. This may be due to low activity of CrtF. Sinceall of other four species of Roseospira showed almost compatibleabsorption spectra of the cells, the major carotenoid may also be 3,4-dehydrorhodopin.

Conclusion: The main carotenogenesis pathway of purple bacteria isthe normal spirilloxanthin pathway, and unusual spirilloxanthinpathways are also found. We found genus specific unusual carotenoidcompositions, and this might be due to absence or low activity andpresence of certain novel enzyme.

P.055

HYDROGEN PHOTOPRODUCTION BY SUSPENSION ORIMMOBILIZED PURPLE PHOTOSYNTHETIC BACTERIAL CULTURESUSING PRODUCTS OF DARK FERMENTATION OF STARCH ANDPOTATO.

Darya N. Tekucheva1,3, Tatyana V. Laurinavichene1, Maria L. Ghirardi2,Michael Seibert2, Anatoly A. Tsygankov1.1Institute of Basic Biological Problems, Russian Academy of Sciences,Pushchino, Moscow Region, Russia; 2National Renewable EnergyLaboratory, Chemical and Biosciences Center, Golden, CO, USA;3South Federal University, Rostov-on-Don, Russia.

Introduction. Transformation of organic wastes to pure water and H2 isan important societal goal. This project is a preliminary study of thesteps required to integrate dark fermentative waste treatment withlight-dependent H2 production into a single process.

Methods. Fermentative effluents (FEs) after starch and potatotreatment in the dark were characterized and tested as substrates forsubsequent light-dependent H2 production. Before testing, the FEswere neutralized, centrifuged and sterilized. H2 photoproduction andthe efficiency of volatile fatty acid (VFA) utilization by the purplebacteria (PB) were characterized in suspension and immobilizedcultures using different FE dilutions.

Results. Acetate and butyrate were the predominant products of bothstarch and potato fermentations. Concentrated FEs inhibited both



growth and H2 production of PB due mainly to the high VFA content.FEs after potato fermentation contained a variable amount of NH4

+,which is detrimental for short-term H2 photoproduction mediated bythe nitrogenase enzyme. Furthermore, during long-term experimentsin the presence of N compounds, a portion of the VFAs is convertedto biomass, thus lowering the efficiency of H2 production. Moreover, at≥2.5% FE, light limitations occur due to high biomass concentrationpromoted by the availability of N compounds in the FE. In thin-layerphotobioreactors (PhBR) with higher irradiation, a higher percentageof the FE could be treated, and 10% FE provided a volumetric H2

output of approx 2.7 L L-1 PhBR, which corresponded to a potentialvalue of 27 L L-1 FE.

PhBRs containing PB immobilized on a glass-fiber matrix can alsoavoid light limitation. The operational characteristics of such a PhBRunder a continuous H2-production regime were examined usingartificial media with different concentrations of N-sources and electrondonors.

(lactate, acetate, and mixed VFAs) or real FEs (at different dilutions). H2

photoproduction was stable for ~3.5 months. The FEs obtained fromthe fermentation of 400 g L-1 of potato biomass (total VFA ~400 mM)maintained H2 photoproduction at ~60 ml h

-1 L-1 of PhBR volume evenat concentrations of 1.25% FE in water. The potential total volumetricH2 output recalculated for 100% FE was ~36 L L-1 FE. If the darkfermentations were performed at low substrate concentration (i.e., at5% starch) and the accumulated VFA concentration was not high (~27mM total), the photofermentation stage could be successfullyoperated without preliminary FE dilution.

Conclusions. Our results indicate that the integration of darkfermentative treatment of starch or potato biomass and lightdependent H2 production by PB using FEs is possible in principle.However, some critical problems will need to be solved.

This work was supported by the Russian Program of Basic ResearchRAS, RFBR, and the US DOE Hydrogen, Fuel Cell, & InfrastructureTechnologies Program.

P.056

Metabolic Engineering of Glycogen Metabolism to Enhance theProduction of Glucose Derivatives in a CyanobacteriumSynechococcus sp. PCC 7002.

Yu Xu, Donald A. Bryant.

Department of Biochemistry and Molecular Biology, The PennsylvaniaState University, University Park, PA, USA.

Introduction: Glycogen is a major storage form for carbon andreducing power in cyanobacteria. Slower or interrupted glycogenassimilation could provide more glucose or glucose derivatives in anindustrially appealing way for biofuel production. Accumulation ofthese substances could also facilitate the biosynthesis of somevaluable metabolites such as sucrose, lactate, H2 etc., by eitherconverting the carbon product directly or using the reducingequivalents of glucose, or by both ways. Another promisingapplication is to co-cultivate a cyanobacterium with a heterotrophicbacterium, in which system the cyanobacterium provides feedstock forthe heterotroph and the latter produces a valuable end product(s). Inthis report, we use a marine-type cyanobacterium Synechococcus sp.PCC 7002 as the model system to achieve the goals above, byinterrupting several key enzymes involved in glycogen biosynthesis.

Methods: Linearized DNA fragments that contain a suitable antibioticresistance gene marker flanked by upstream and downstreamhomologous regions of the targeted gene were constructed and used

to transform WT cells. Desired deletion mutants were selected andsegregated by selection with specific antibiotics. Glucoseconcentrations in different cell fractions were measured by a glucoseassay kit provided by Sigma. Photosynthetic O2 evolution rates weremeasured with a Clark-type electrode. Cell ultrastructure wasobserved by TEM.

Results: Two glycogen synthase genes glgA1 and glgA2, a 1,4-alpha-glucan branching enzyme gene glgB, and a glucose-1-phosphateadenylyltransferase gene glgC were successfully deleted inSynechococcus sp. PCC 7002 to create the following mutants: glgA1,glgA2, glgB, glgC, glgA1 glgA2, glgA1 glgB, glgA2 glgB and glgA1glgA2 glgB. All mutant alleles segregated completely. High-lightadapted (HLA) strains of each mutant are also selected. Preliminarydata show no obvious difference in O2 evolution rates in those HLAmutants compared to HLA WT cells. HLA mutants of glgA1 glgA2,glgA1 glgA2 glgB, and particularly glgC, were found to have lessinsoluble glycans. They excrete more glucose into the growth mediumthan wild type, although excretion levels were still low. Theultrastructure of HLA glgA1 glgA2 shows noticeably alteredultrastructure, which could result from high levels of glucosederivatives in cells.

Conclusion: These mutants should have comparable photosynthesisrates as the WT, indicating a stable carbon and energy input in thesemutants. However, they are defective in depositing carbon andreducing equivalents into glycogen. Therefore, more glucose orglucose derivatives should accumulate in cells or be excreted. Furtheranalyses are in progress to identify these glucose derivatives andquantify their amounts compared to the WT cells.

P.057

PHOSPHOLIPID FATTY ACID ANALYSIS OF THE EUPHOTICMICROBIAL COMMUNITY OF LAKE TUSCALOOSA.

P.O. Akinwole, E. Lefevre, M.J. Powell, R.H. Findlay.

Department of Biological Sciences, University of Alabama, Tuscaloosa,Alabama, USA.

Introduction: Euphotic microbial communities are metabolicallyimportant in aquatic system, since they carry out primary productionand are critical to the cycling of both organic and inorganic carbon.We studied the seasonal variation of a euphotic microbial communityto determine the factors related to its structural dynamics.

Method: Integrated water samples were collected fortnightly from theeuphotic zone of Lake Tuscaloosa; a man-made system in the west-central Alabama, southeastern USA, during the period February 2008through March 2009. Euphotic microbial biomass and communitystructure were determined using the total phospholipid phosphate(PLP) and fatty acid techniques respectively.

Results: Lake Tuscaloosa is a warm dystrophic monomictic lake thatwas strongly stratified from April to November 2008. Secchi disktransparency depth was inversely correlated with total euphoticmicrobial biomass (r2= 0.6). Euphotic microbial biomass, measured asPLP, ranged from 5.63 (during the period of warm water) to 20.18nmol PLP L−1 (during the period of cold water). Phospholipid fattyacids (PLFA) profiles were dominated by polyenoic fatty acids.Principal component analysis of PLFA profiles showed seasonalpattern of change in the euphotic microbial community. There was asignificant shift from aerobic bacterial and phototrophicmicroeucaryotes during period of cold water (Winter mixing) toanaerobic bacteria and heterotrophic microeucaryotes during theperiod of warm water when the lake was thermally stratified. Maximum



contribution to total microbial biomass by microeucaryotes occurredduring the period of cold water while maximum contribution byprokaryotes occurred during the period of warm water and duringWinter flood events.

Conclusions: This study suggests that euphotic planktonic microbialcommunities show regular variations in structure that are similar tothose previously observed in shallow-water marine and freshwatersediment communities.

P.058

GENE TRACING OF SEASONAL SUCCESSION OFCYANOPROKARYOTES IN MISSISQUOI BAY, LAKE CHAMPLAIN.

Hongmei Jing, David Bird, Nathalie Fortin, Rocio Aranda-Rodriguez,Charles Greer, Serge Paquet, Catalina Gonzalez Rueda.

Université du Québec à Montréal, Montreal, Quebec, Canada.

Introduction: Shallow Missisquoi Bay, in the northern part of LakeChamplain, suffers from serious cyanobacteria blooms. For severalyears now, the public has been advised to stay away from recreationaluse of the bay for most of the summer. Following suggestions fromthe literature, we looked for taxonomic and genotypic changes thatwe could associate with toxin presence, in order to understandsuccessional events and identify driving factors. We followed thetaxonomic and genotypic dynamics, along with changes in nutrientconcentrations and weather variations, to look for linkages.

Methods: Two stations, one pelagic and the other littoral anddownwind, were sampled weekly over the growing season from Mayto November in 2006 and 2007. The seasonal dynamics ofcyanobacterial populations were followed by taxonomic microscopyand denaturing gradient gel electrophoresis (DGGE). Bands wereexcised and sequenced. Two primers were used, one (CYA) thatdetects most Cyanobacteria, and another (MIC) that is more restrictiveand detects mainly toxic species, such as Anabaena, Aphanizomenonand Microcystis.

Results: There was generally a close correspondence betweentaxonomic species richness in the Cyanobacteria, and DGGE bandrichness with the general cyanobacterial primer. DGGE band clusteranalysis revealed two very distinct cyanobacterial communities, oneassociated with summer months (July to September) and the otherdeveloping in fall and reappearing the following spring. We suggestthat this pattern is indicative of 1) niche dynamics and 2) twoecological stategies: species that are constantly present at a low andstable level, and species that appear in summer, bloom and becomedominant, then disappear. Both microscopy and DGGE sequencingshowed that strains of Microcystis were dominant during the summerbloom period in 2006. The greatest number of Microcystis genotypesand morphotypes were detected during the annual radiationmaximum at the summer equinox, before the biomass maximum ofthe bloom was reached. Higher intrageneric diversity of Microcystiswas detected by DGGE than by microscopy. Major storm events werealways associated with important changes in community makeup inthis large shallow lake Although more genotypes of M. elabens, M.viridis and M. wesenbergii were present than were genotypes of thedominant M. aeruginosa, the succession of two major M. aeruginosagenotypes accounted for the observed Microcystis biomass dynamics.The potential toxic Microcystis genotype was present throughout thewhole growing season.

Conclusions: Phytoplankton ecology is in its infancy. Little to nothingis known of the factors that lead to cyanobacterial dominance in agiven lake, nor especially to the dynamics of individual species. This

paper contributes to outlining the ecostrategies of different species,and probes below the species level to show that we have beenattempting to understand the dynamics of a “strain flock” rather thanan individual genotypic organism. Genetic tracing has provided usinsights that were not accessable by microscope alone.

P.059

ADAPTATION REACTIONS OF SIDEROPHILIC CYANOBACTERIATO HIGH AND LOW LEVELS OF ENVIRONMENTAL IRON:IMPLICATIONS FOR BIOSPHERE HISTORY.

I.I. Brown1, D. A. Bryant2, K.L. Thomas-Keprta1, S.A. Sarkisova1, E.E.Foraker1, W.J. Clarke1, G. Shen2, D.H. Garrison1, D.S. McKay1.1NASA Johnson Space Center, Houston, TX; 2The Pennsylvania StateUniversity, University Park, PA; USA.

Introduction: Of all extant environments, iron-depositing hot springsmay constitute the most appropriate natural models (Pierson andParenteau, 2000) for analyzing the ecophysiology of ancientcyanobacteria (CB) that may have emerged in association withhydrothermal activity (Brown et al., 2007) and elevated levels ofenvironmental Fe (Rouxel et al., 2005). Elevated environmental Fe2+

posed a significant challenge to the first oxygenic phototrophsbecause reduced Fe2+ induces toxic Fenton reactions (Wiedenheft etal., 2005). Ancient CB could have also experienced stress duringoccasional migrations from the Fe2+-rich ocean to the basaltic landthat was almost devoid of dissolved Fe2+; therefore, the study ofadaptation reactions of siderophilic CB, which inhabit iron-depositinghot springs, to varying levels of environmental Fe may shed light onthe paleophysiology of ancient oxygenic prokaryotes.

Methods: Siderophilic CB (Brown et al., 2007) were cultivated inmedia with different concentrations of added Fe3+. Basaltic rocks wereused as a source of Fe and trace elements in some cases.Transmission electron microscopy (TEM), scanning electronmicroscopy (SEM), and energy-dispersive spectrometry (EDS)techniques were used for studying Fe mineralization and rockdissolution processes. Fluorescence spectroscopy was used toestimate the contents of chlorophyll-protein complexes.

Results: Five siderophilic isolates (Chroogloeocystis siderophila, JSC-1, JSC-3, JSC-11, and JSC-12) precipitated Fe-bearing phases on theexopolymeric sheaths of their cells when Fe3+ concentration was ~ 400– 600 µM (high Fe). The same Fe3+ concentration was optimal for theculture proliferation rate (Brown et al., 2005; Brown et al., 2007).Higher concentrations of Fe3+ repressed the growth of somesiderophilic CB (Brown et al., 2005). No mineralized Fe3+ wasobserved on the sheath of freshwater isolates Synechocystis sp. PCC6803 and Phormidium aa strain. Scanning TEM and thin-window EDSrevealed intracellular Fe-rich phases within isolates JSC-1, JSC-3, andJSC-11. The elemental composition of the Fe-rich precipitatesindicates P, Fe, and O as the major elements with minor amounts of Aland Ca. The PSI/PSII ratio is higher in JSC-1 and JSC-3 isolates thanin CB without any detectable ability to mineralize Fe.

SEM-EDS studies of the interaction of siderophilic CB with Fe-richminerals and rocks revealed the ability to leach ilmenite, olivine, TiO,FeS, , and ferrosilicates, perhaps because the CB studied can secrete2-oxo-glutarate and malate that possess chelating properties.

The 7.8-Mb draft genome sequence of Cyanobacterium sp. JSC-1 hasbeen determined and is currently being closed and polished; thesedata will hopefully reveal the molecular mechanisms of Femineralization and Fe-rich minerals by siderophilic CB.

Conclusions: The results suggest that colloidal Fe3+ is transported in



the CB cytoplasm most likely through an ABC-type Fe3+ transportsystem (Braun et al., 2004). The prevalence of PSI over PSII in somespecies of siderophilic CB as well as the ability of siderophilic CB tomineralize Fe within their cytoplasm may indirectly support thehypothesis that PSI in CB can be involved in Fe2+oxidation (Cohen,1984; 1989) and could be considered a protective mechanism againstoxidative stress induced by high levels of environmental Fe2+ andultraviolet radiation. The ability to leach Fe-rich minerals could havesupported the expansion of ancient CB onto basaltic land.

P.060

A CLONAL POPULATION OF CHLOROBIUM DOMINATES THEANOXIC BACTERIAL COMMUNITY IN A SWISS MOUNTAIN LAKE.

Raymond P. Cox1, Lea H. Gregersen2, Donald E. Canfield3, KirstenHabicht3, Mette Miller1 and Niels-Ulrik Frigaard2.1Department of Biochemistry and Molecular Biology, University ofSouthern Denmark, Odense, Denmark; 2Department of Biology,University of Copenhagen, Copenhagen, Denmark; 3Institute ofBiology and Nordic Center for Earth Evolution, University of SouthernDenmark, Odense, Denmark.

Introduction: Lake Cadagno is a meromictic lake in the Swiss Alps.The anoxic bottom water contains about 2 mM sulfate and the sulfideproduced by bacterial activity supports a large population ofphototrophic sulfur bacteria at and below the chemocline. Historicallythe dominant anoxygenic phototrophs were purple sulfur bacteria butsince 2000 these have been replaced by green sulfur bacteria (GSB).We have investigated this GSB population using a multigenesequencing approach.

Methods: DNA was extracted from filtered water samples fromdifferent depths and partial gene sequences obtained by PCR, cloningand sequencing. The water samples were also used for turbiditymeasurements and pigment analysis by HPLC with diode-arraydetection.

Results: Analysis of a 16S rRNA clone library obtained with generalbacterial primers showed that at least 50% of the recoveredsequences, and more than 99% of the GSB sequences, wereessentially identical and also identical with 16S rRNA from thesequenced genome of Chlorobium clathratiforme DSM 5477. Thethree 16S rRNA genes on this genome show a 2:1 variation in oneposition, and a similar variation was observed in the recoveredsequences. The clonal nature of the GSB population was confirmed bypartial sequences from a protein-coding gene (fmoA) and from thecsmCA region containing both coding and non-coding regions. Thesequence differences which were observed (less than 1 base variationper 1000 bases) were within the range expected due to sequencingartefacts from cloning of PCR products.. The clonal population waspresent both at the chemocline and deeper in the water columnwhere shading by GSB in the upper layers precludes significantphototrophic growth.

Conclusions: We conclude that the currently dominating GSB hasarisen since 2000 from a single cell or a small clonal population. Thechange might have been due to mutation, invasion or a change in theenvironmental conditions.

P.061

CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUMREVEALS A NEW NICHE FOR CHLOROSOME-CONTAININGPHOTOTROPHS.

Amaya M. Garcia Costas1, Zhenfeng Liu1, Christopher Klatt2, LynnTomsho1, Stephan C. Schuster1, David M. Ward2, Donald A. Bryant1.1Dept. of Biochemistry and Molecular Biology, The Pennsylvania StateUniversity, University Park, PA; 2Dept. of Land Resources andEnvironmental Sciences, Montana State University, Bozeman, MT; USA.

Introduction: Candidatus Chloracidobacterium (Cab.) thermophilum isa thermophilic heterophototroph isolated from the phototrophic matsof Octopus Spring in Yellowstone National Park. It is the only knownphototroph in the bacterial phylum Acidobacteria, and it has not yetbeen isolated from any other environment. The light-harvestingapparatus of Cab. thermophilum resembles that of green sulfurbacteria, and it consists of the vesicle-like chlorosomes as antennaestructures as well as the Fenna-Matthews-Olson (FMO) protein and atype 1, homodimeric reaction center. Unlike the strictly anaerobicgreen sulfur bacteria, Cab. thermophilum grows under oxic conditions.In order to further our understanding of the metabolic capabilities ofCab. thermophilum, as well as the distribution and role of thisorganism in natural environments, we have conducted biochemicalanalyses of its chlorosomes and have complemented these studieswith genomic, metagenomic and metatranscriptomic analyses.

Methods: Chlorosomes of Cab. thermophilum were isolated onsucrose gradients and analyzed by high pressure liquidchromatography (HPLC), absorption and fluorescence spectroscopy,electron microscopy and SDS-PAGE. The Cab. thermophilum genomewas sequenced using 454 technology. Gap closing was done bySanger sequencing of PCR products, and contigs were assembled withPhred/Phrap/Consed. For metatranscriptome analyses, total RNA wasextracted from mat samples that had been collected at four differenttimes during a diel cycle, converted into cDNA, and sequenced bypyrosequencing. RNAs were identified by blastN analyses, initiallyusing the genomes of seven organisms known to occur in the matsand subsequently the GenBank NR database.

Results: The chlorosomes of Cab. thermophilum contain uniquepigments and proteins not present in other chlorosomes. Thebacteriochlorophyll (BChl) c is methylated at the C-8 and C-12positions as occurs in the green sulfur bacteria, but the BChl c isesterified with a mixture of alcohols, of which the major species isoctadecanol. Interestingly, the chlorosomes of Cab. thermophilumexhibit redox-dependent fluorescence emission which suggested thepresence of quinones in the chlorosomes. We have identifiedmenaquinone-8 as the major quinone in chlorosomes. The Cab.thermophilum genome consists of two circular chromosomes, 2.7 and1.1 Mb, respectively, and contains genes involved in phototrophy,photoprotection, and other Acidobacteria-specific genes. Thegenome encodes a complete aerobic respiratory chain, and genesinvolved in carbon fixation appear to be missing. This genecomposition suggests that Cab. thermophilum is a photoheterotroph.Preliminary metatranscriptome analyses have revealed that the genesfor the photosynthetic apparatus are highly expressed at dusk anddawn.

Conclusions: Based on our combined biochemical and genomicsequencing studies we propose that Cab. thermophilum has a uniqueniche in the environment defined by aerophilic/microaerophilicconditions, tolerance of high light, and ability to harvest light in lowlight environments. Traditionally, chlorosomes have been associated



with low-light, anoxic conditions. However, Cab. thermophilumappears to occupy a temporally defined, low-light niche in anotherwise highly oxic and highly illuminated environment.

P.062

PH-DEPENDENT HYDROGEN SULFIDE SPECIES MAY CONTROLCOMPOSITION OF HOT SPRING MICROBIAL MATS.

Zorigto Namsaraev1,2, Bair Namsaraev3,4, Vladimir Gorlenko1.1Winogradsky Institute of Microbiology RAS, Moscow, Russia; 2Centerfor Protein Engineering, University of Liège, Liège, Belgium; 3Instituteof General and Experimental Biology Siberian Branch RAS, Ulan-Ude,Russia; 4Buryat State University, Ulan-Ude, Buryat Republic, Russia.

Introduction. Increasingly more data suggest that early life lived at anenvironmental temperature similar to those of today’s hot springs. Attemperatures lower 73-77 °C and pH higher 4.5 hydrotherms aredominated by microbial mats formed by phototrophic bacteria.Castenholz have shown that with the increase of temperature above~55 °C natural populations of cyanobacteria cannot tolerate highsulfide concentrations. It was concluded that at these temperaturesand concentration of sulfide higher 1 mg/L mats are formedexclusively by anoxygenic phototrophic bacteria (APB).

We studied alkaline (pH up to 9.8) non-mineralized (salinity less 1 g/L)hydrotherms of Baikal rift zone (Southern Siberia, Russia) and foundthat cyanobacteria could develop at much higher concentrations ofsulfide than it was thought before (up to 5.9 mg/L at 62 °C).

Methods. Microscopy, spectrophotometry, radioisotopy, cultivationand cultivation independent methods.

Results. Cyanobacteria belonging to genus Phormidium weredominant and formed so called “translucent” mats up to 13 cm thick.The productivity of mats was relatively high: maximal content ofchlorophyll a is 892 mg m–2, maximal rate of oxygenic photosynthesis3.5 g C m–2d–1. APB Chloroflexus aurantiacus was found and isolatedfrom mats, but it was not dominant.

This fact can be explained by a decrease of sulfide toxicity in increaseof pH. Other sulfidic hydrotherms worldwide follow the same rule. Forexample, at 60 °C and pH lower than 7 the majority of H2S moleculesare not dissociated and are capable of penetrating easily through acell membrane. The community dominated by APB Chloroflexusaurantiacus develops (Iceland, Italy and Yellowstone). Less toxic HS-

ion starts to dominate at pH higher than 7 and cyanobacteria appearin the mat. They form a layer under top layer of APB Chloroflexusaurantiacus which protects cyanobacteria from the influence of sulfidedissolved in water above the mat (Iceland and Kamchatka). At pHhigher than 8.5 all sulfide molecules are transformed to a hydrosulfideion and, as it have been shown by us, cyanobacteria dominate in themicrobial community.

Conclusions. Our results show that distribution of different types ofphototrophic communities in sulfidic hydrotherms can be explained bypH-dependent hydrogen sulfide species.

P.063

ON THE OCCURRENCE OF MICROCYSTIN PRODUCINGCYANOBACTERIA IN MURCHISON BAY OF LAKE VICTORIA,UGANDA.

Sigrid Haande, Thomas Rohrlack, Pål Brettum, Robert Ptacnik, BenteEdvardsen, Anne Lyche-Solheim, Unn-Hilde Refseth, Petter Larsson.

NIVA, Oslo, Norway.

Introduction: One of the major threats to Lake Victoria iseutrophication and an increasing proliferation of cyanobacteria. Themain concern is that many cyanobacterial species have the ability toproduce toxic compounds, among them the commonly reportedhepatotoxic microcystins, which can cause considerable hazards foranimal and human health. Murchison Bay is a 30 km long embaymentin the north western part of Lake Victoria, and the inner part of the bayserves as drinking water supply for the nearby city Kampala, thecapital of Uganda. The bay is also the recipient of both industrial andmunicipal wastes, sewage effluents and surface runoff from the city,and is heavily eutrophicated. This study aimed to examine theabundance of cyanobacteria and microcystins in Murchison Bay and toidentify the main microcystin producing species, as well as to test forpossible correlation between microcystin concentration andenvironmental factors.

Methods: Sampling (n=25) of physical, chemical and biologicalparameters in Murchison Bay was done at two stations, representingthe semi-enclosed innermost part of the bay and the wider outer partof the bay, every second week in the period from April 2003-March2004. Vertical profiles of water temperature, oxygen and electricalconductivity were measured, and the recorded secchi depths wereused to estimate euphotic depth. The nutrients analysed were totalphosphorous, phosphate, total nitrogen, nitrate, total organic carbonand silicon. Clorophyll-a was analysed and phytoplankton countingswere performed. The presence of microcystins was analysed by Liquidchromatography (LC-MS), and the identification of the microcystinproducing strains was performed by real-time PCR, using speciesspecific primers. Correlations between microcystin concentration andvarious environmental parameters were analysed by Spearman’s rankcorrelations.

Results: There was a considerable loading of nutrients to MurchisonBay with high concentrations of total phosphorous (>90µg/L) and totalnitrogen (>1100µg/L) in the inner part of the bay. There was a rapiddecrease in conductivity and nutrient concentrations from theinnermost part of the bay to the outer part of the bay. Thephytoplankton community was dominated by a number ofcyanobacteria, among them known microcystin producing species ofthe genera Microcystis, Anabaena and Anabaenopsis. The proportionof N-fixing species like Anabaena sp. was higher in the outer part ofthe bay whereas Microcystis sp. was more abundant in the inner partof the bay. Microcystins (MC-RR, -LR, -YR) were detected during thewhole study period, on average 1.1 µg L-1 in the inner part of the bayand 0.6 µg L-1 in the outer part of the bay. Based on qPCR analysis,Microcystis aeruginosa was identified as the main microcystinproducer in Murchison Bay. Nutrient availability and light climateseemed to be the most probable influencing environmental factorsinfluencing the microcystin production in Murchison Bay, but themicrocystin concentrations could not be predicted by any givenenvironmental factor alone, by the biovolume of cyanobacteria or of acertain cyanobacterial species.

Conclusions: This study showed that Murchison Bay was heavilyeutrophicated and that there was a presence of microcystin producing



cyanobacteria in the bay. The microcystin concentrations in MurchisonBay were at times higher than the WHO recommended limit of 1 µg L-1

MC for drinking water, possessing a health risk for lake water users.

P.064

DIVERSITY AND NITROGENASE GENE EXPRESSION OFDIAZOTROPHIC CYANOBACTERIA IN THE BALTIC SEA.

Henning Johansen, Kirsten Isensee, Maren Voss, Klaus Jürgens.

Leibniz Institute for Baltic Sea Research, Rostock, Germany.

Introduction: Low phosphorous concentrations in the water columnare important for the development of heterocystous cyanobacteriablooms recurring in summer in the Baltic Sea, a brackish water body inNorthern Europe. These blooms significantly contribute to thenitrogen input into the system and consist mainly of the generaAphanizomenon and Nodularia. While nitrogen fixation ratemeasurements indicate a distinct diel cycle, there are contradictingfindings from the concentration of the nitrogenase enzyme during day.However, there is a general lack of knowledge about nitrogenase geneexpression (nifH) and the coupling between gene activity and nitrogenfixing activity in the Baltic Sea so far. Here we present data on bothgene expression and rate measurements, giving a comprehensiveinsight into regulation of nitrogen fixation of filamentouscyanobacteria.

Methods: Surface water samples (3 m) were taken at a LaGrange driftstation in the Bornholm Basin, Baltic Sea, in July 2008. The diversity ofthe nitrogen fixing community was studied using both cell counts andfingerprint analyses (Denaturing Gradient Gel Electrophoreses) of thenifH gene and its transcripts. Concentrations of gene copies andtranscripts were determined by a quantitative PCR assay using primersspecific for heterocystous cyanobacteria. Rate measurements ofnitrogen fixation and primary production were done using stableisotope labelling (15N2 and

13C).

Results: Sequencing of excised bands from nifH fingerprint analysesshowed highest identity to strains Nodularia sp. KAC13,Aphanizomenon sp. KAC15 and Anabaena sp. PCC7120, being inaccordance with microscopic cell counts. The analyses ofcyanobacterial nifH transcripts indicated the presence and activity ofthe genera Anabaena, Aphanizomenon and Nodularia during acomplete diel cycle. This is in accordance with the nitrogen fixationrate measurements showing a lowered rate but uptake of molecularnitrogen in darkness whereas the pattern of carbon fixation isfollowing the solar radiation pattern. Results of the nifH geneexpression measurements indicate an uncoupling of maximumnitrogen fixation rates and maximum transcripts.

Conclusions: This study is the first to show nifH gene expression innatural bloom conditions in the Baltic Sea throughout the whole dielcycle. Therefore, an uncoupling of nitrogen fixation ratemeasurements and gene expression can be suggested. A tentativeexplanation is the enhanced regeneration of the nitrogenase enzymeduring the night, while nitrogen fixation activity is more closelycoupled to light energy during the day. In case our further studiesconfirm these findings the gene expression may not directly be linkedto nitrogen fixation.

P.065

MAKING SENSE OF MARINE SYNECHOCOCCUS DIVERSITY, THEKEY TO UNDERSTANDING THEIR UBIQUITOUS ECOLOGICALDISTRIBUTION.

Sophie Mazard, Martin Ostrowski, Dave Scanlan.

Department of Biological Sciences, University of Warwick, Gibbet HillRoad, Coventry, United Kingdom.

Introduction: At the global scale the marine photosyntheticcommunity is dominated by two closely related genera ofcyanobacteria, Prochlorococcus and Synechococcus. Their success isunderpinned by a high degree of genetic and genomic variation, suchthat at least 16 different evolutionary lineages of Synechococcus havebeen identified, each occupying distinct spatial and temporal niches inthe ocean. Our overall aim is to understand what factors defineparticular niches (e.g. light, temperature, nutrients, metals) and tocorrelate these factors with community structure. One way to achievethis is by monitoring how the genetic composition of photosyntheticcommunities changes in different ocean regimes linked with targetedmetagenomics using high-throughput sequencing of DNA and RNAextracted from natural populations. Analysis of preliminary resultsrevealed that our ability to interpret the data is limited by the lowtaxonomic resolution and low throughput of community analysesbased on standard markers (16S rRNA and ITS).

Methods: In this work, we describe the development of a fast, reliableand efficient method of assessing community composition at hightaxonomic resolution using the petB locus. In preparation, wedesigned a Multi Locus Sequence Analysis (MLSA) scheme targeting 7housekeeping genes for marine Synechococcus based on fullysequenced genomes. Metagenomics and transcriptomics studies werecarried out on flow-sorted Synechococcus from contrasting stations onoceanographic cruises of the Atlantic (AMT18) and the MediterraneanSea (BOUM).

Results: A high resolution taxonomic framework for marineSynechococcus was produced with our MLSA scheme on more than120 cultured isolates maintained in the Warwick culture collection.Phylogenetic reconstructions based on single and concatenated lociwere congruent and comparable with those obtained with generalphylogenetic makers (16S rDNA, 16S-23S ITS). However, sequencingof the single locus petB allowed us to obtain a resolutiondifferentiating up to 55 coherent taxonomic units as compared to amaximum of 16 for 16S rDNA and ITS. Furthermore, the reliability ofthe petB alignment (cf. ITS) makes this locus suitable for assessingcommunity structure with high-throughput and rapid analyses ofenvironmental libraries. Indeed, preliminary results from environmentallibraries highlighted ecologically important clades and sub-groups at amuch higher resolution than previous studies.

Conclusions: Earlier work performed using classical markersemphasized broad trends in the ecological distribution of marineSynechococcus. The establishment of a high resolution phylogeneticframework helps us to fine tune the geographical partitioning of thisgenus in situ, and will facilitate the uncovering of specific adaptationmechanisms of the numerous phylotypes observed. In concert with in-depth genomic study of this genus, this might prove to be the key tounderstanding its complex but successful ecological distributionthroughout the world’s oceans.



P.066

ALGAECIDAL EFFECT OF RHODOCOCCUS ON MICROCYSTIS.

Chi-Yong Ahn, Young-Ki Lee, Song-Gun Kim, Hee-Sik Kim,Hee-Mock Oh.

Environmental Biotechnology Research Center, KRIBB, Daejeon,Republic of Korea.

Rhodococcus sp. is one of the most common bacteria in the reservoirsof Korea. Rhodococcus sp. is also frequently found with Microcystis, amajor bloom-forming cyanobacterium. Two strains of Microcystis wereisolated in Daechung and Wangsong reservoir, Korea, duringblooming season (from July to October, 2007). Eleven strains of co-exiting bacteria were also isolated from mucilage of cultivatedMicrocystis strains. They were identified by 16S rRNA genesequences. Two of them were designated as Rhodococcus sp. KWB-RH2 and KWB-RH5. Rhodococcus strains exerted an inhibitory effecton the growth of Microcystis aeruginosa NIES 843, UTEX 2388 andisolated M. aeruginosa. The cell-free culture supernatant ofRhodococcus strains also showed prominent algaecidal activity. Toidentify the causal compounds, supernatants of Rhodococcus strainswere dialyzed with a MW 8,000 and MW 2,000 membranes. Thesuspected extracellular substances had molecular weights below2,000 Da. The small molecules were further analyzed by HPLC, LC-MSand enzyme treatments. The extracellular material from Rhodococcusstrains could be a more effective bloom extinguisher, compared togenerally known algaecidal bacteria, because it does not requirebacterial cell itself for bloom control.

P.067

CHARACTERISATION OF CYANOPHAGE RESISTANTANCE IN THEMARINE CYANOBACTERIUM SYNECHOCOCCUS SP. WH7803.

Edward Spence, Nicholas Mann.

University of Warwick, Coventry, United Kingdom.

Introduction: Cyanophage are viruses which specifically infectcyanobacteria. This project focuses on the interaction of thecyanophages S-PM2 and S-RSM42 with their host, the marinecyanobacterium Synechococcus sp. WH7803. The main aim of thisproject is to identify the host receptor(s) and characterise naturallyoccurring phage resistant mutants of Synechococcus sp. WH7803which have been isolated. The phage resistant mutants have beencharacterised in order to determine what physical changes haveoccurred to their cell surface, in order to provide resistance and whatimpact this has on their fitness. Previous research with the freshwatercyanobacterium Anacystis nidulans has shown that thelipopolysaccharide (LPS) component is necessary for the adsorption ofthe cyanophage AS-1.

Methodology: Spontaneous resistant mutants were isolated from re-growth of survivors from completely lysed cultures, followingcyanophage infection. Single colonies were isolated from pour patesand maintained in liquid culture under selection with the addition ofcyanophage lysate. The outer membrane protein component wasisolated by Triton X-100 precipitation and profiled by gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharidewas isolated using hot phenol/water extractions and profiles wereobtained with SDS-PAGE using isolated LPS or using whole celldigests with protease K, visualised with silver staining.

Results: The results indicated that the LPS component of the resistantmutants had an altered profile suggesting changes to theoligosaccharide or variable region. The outer membrane protein

components remained unchanged in the resistant mutants. Theresistant mutants also had a reduction in their growth rates, incomparison to the wild type under standard conditions. An alteredpigment content was also observed suggesting alteration to thephotosynthetic components particularly to the phycobilisome.

Conclusion: It seems likely that the LPS is the major receptor moleculefor the cyanophages S-PM2 and S-RSM42 adhesion to their hostSynechococcus WH7803. Outer membrane proteins do not seem tohave a role in these cases for cyanophage attachment. There is also acost of resistance relating to the growth rate of the resistant mutantsand changes to the pigment content. This suggests that geneticchanges resulting in the alteration of the cell surface receptor may beas a result to changes of a regulatory component. This may explainadditional pleiotrophic phenotypic changes observed, relating topigment composition and a reduction in the growth rate in thecyanophage resistant mutants.

P.068

THE ROLE OF PHOTOTROPHIC MICROORGANISMS INBIOLOGICAL SOIL CRUSTS FROM THE MIDTRE LOVÉN GLACIERFORELAND.

Laura Tiano1,2, Luigi D’Acqui1, Claudio Sili1, Marc Staal3, SilviaTuricchia4, Matthias Zielke5, Stefano Ventura1.1CNR–Institute of Ecosystem Study, Sesto Fiorentino, Italy; 2Universityof Florence, Master in Environmental Biology, Italy; 3Department ofBiology, University of Copenhagen, Denmark; 4University of Siena,PhD School in Polar Science, Siena, Italy; 5Department of MarineBiotechnology, Norway College of Fishery Science, University ofTromsø, Norway.

Introduction: Global change models predict that the speed andmagnitude of climate warming will be greatest at the highest latitudes,with raised mean temperatures, particularly during winter, and alteredpatterns of precipitation (IPCC 2001). These predicted changes areunder way in many parts of the high Arctic, as it can be seen from thedecreasing extent and thickness of the sea ice and the process ofglacial ‘retreat’.

Glacial regression makes new ground surface available to bioticcolonization. Low temperatures, short growing seasons, the effects ofpermafrost, limited moisture and nutrients availability limit the growthof the vegetation, leading to different successions from thoseobserved elsewhere. In these polar environments, pioneeringorganisms such as cyanobacteria, green algae, lichens, mosses andheterotrophic bacteria are typically the first to colonize the substrateas biological soil crusts (BSCs).

Cyanobacteria are often found in great variety in all the high arcticzones, and in several ecosystems

they are the dominant microorganisms in terms of biomass andproductivity (Vincent, 2000). Nitrogen fixation by cyanobacteriacompensates for the lack of nitrogen in arctic soils (Lennihan et al.,1994) and facilitates the subsequent colonization of these habitats byother microorganisms and plants (Bliss and Gold, 1994).

This study describes community assembly on typical high Arcticglacier forelands, with a focus on the phototrophic components of theBSCs, to gain a broader understanding about dynamics anddevelopment of these fragile systems and their rates of recoveryfollowing disturbance.

Methods: Sampling sites were located close to Ny-Ålesund, Svalbard,on the foreland of the Midtre Lovén glacier. Seven locations withdifferent types of crust were chosen, and an integrated approach was



applied, carrying out both morphological and molecular analysis ofenvironmental samples.

A general insight into the phototrophic microorganisms colonizing thecrusts was gained with microscopy observations; a deeperunderstanding of the communities’ structure is expected withmolecular fingerprints (ARISA, T-RFLP).

Also physical, chemical and granulometric characteristics of soil wereanalyzed to investigate the influence of those parameters on thedevelopment of the crusts.

Furthermore, respiration rate, nitrogen fixation and production weremeasured, thus adding more information on the main activitiesperformed by the primary colonizers of these surfaces.

Results: The molecular characterization of the soil crusts is still inprogress, the morphological analysis by microscopy allowed us toidentify several species of cyanobacteria and algae, as Nostoc sp.,Phormidium sp., Leptolyngbya sp., several diatoms, etc.

Nitrogen fixation rate, C/N concentration and granulometriccharacteristics of soil, confirmed the low nutrients availability of thehigh arctic ground and the extremely slow pedogenesis of these soils.

Conclusions: We expect to draw conclusions about the role ofphototrophic microorganisms in these high arctic biological soil crustsas soon as all the results, collected from the different type of analysis,will be matched together and organized under a statistical approach.

P.069

CYANOBACTERIAL DIVERSITY OF THE SÖR RONDANEMOUNTAINS NEAR THE NEW “PRINCESS ELISABETH” STATION(ANTARCTICA).

Zorigto Namsaraev1,2, Rafael Fernandez1, Marie-José Mano1, PatriciaSimon1, Annick Wilmotte1.1Centre for Protein Engineering, University of Liège, Liège, Belgium;2Winogradsky Institute of Microbiology RAS, Moscow, Russia.

Introduction: During austral summer 2008, the new Belgian “PrincessElisabeth” research station has been constructed on a granite ridgenear the Utsteinen nunatak in the Western part of the Sör RondaneMountains. This pristine area has not yet been studied at thebiological level, and exhaustive baseline data are needed to obtaininformation about the initial state of the environment. Moreover, theSör Rondane Mountains could be a possible refuge during the LastGlacial Maximum. Cyanobacterial communities in this area couldharbour species that survived the population bottleneck of glaciations.

Methods. Samples were taken in several locations in the australsummers of 2007 (before the construction start) and of 2009 (BELSPOprojects ANTAR-IMPACT and BELDIVA). A polyphasic approach wasused to study of cyanobacterial diversity. It included a molecular studyusing 16S rRNA gene sequences, an isolation of cyanobacteria inculture and a microscopic observation of environmental samples andisolated cultures.

Results: Preliminary results showed a relatively high biodiversity in thestudied area. Cyanobacteria were mostly associated with gravel and insome cases with rocks, whereas lichens were dominant on rocksurfaces. Molecular studies showed a quite high degree of endemismand genotypes never recorded before. 92% of the studied samplesshared at least one OTU, concordant with a mobility of speciesbetween nearby habitats. Microscopical observations showed thepresence of at least 11 morphotypes of cyanobacteria: Asterocapsasp., Aphanocapsa sp., Chroococcus sp., Coleodesmium sp.,Cyanothece aeruginosa, Leptolyngbya sp., Nostoc commune, Nostoc

sp., Phormidium autumnale, Phormidium priestleyi, Stigonema sp.Cultivation studies showed the importance of the incubationtemperature for the composition of enrichment cultures, with anincreased proportion of unicellular cyanobacteria at lowertemperatures.

Conclusion: Our first results show that the Western part of the SörRondane Moutains harbour a high and unique diversity ofcyanobacteria. Precautions should be taken to avoid introduction ofnon-native species in the area of the research station.

P.070

SYNTHESIS OF BACTERIOCHLOROPHYLL-F.

Hitoshi Tamiaki, Jun Komada, Michio Kunieda, Kazuhiro Fukai, TaichiYoshitomi, Jiro Harada, Tadashi Mizoguchi.

Department of Bioscience and Biotechnology, Kusatsu, Shiga, Japan.

Introduction: Green sulfur bacteria are a family of anaerobicphotoautotrophic bacteria living. These bacteria have elliptic shapedlight-harvesting apparatus, called chlorosomes, that are attached withthe inner side of their cytoplasmic membrane. Chlorosomes areunique photosynthetic antennae, where characteristic chlorophyllouspigments, bacteriochlorophyll(BChl)s-c, d and e self-aggregated toform large oligomers without any assistance of peptides. In contrast,the other antennae are prepared by complexation of pigments withpeptides. In chlorosomal aggregates, specific interaction between the31-OH, Mg and 13-C=O is observed. All the chlorosomal chlorophyllshave such functional groups and three BChls mentioned above aredistinguishable from substituents on the C7- and C20-positions. BChl-c has methyl groups at the C7- and C20-positions, and BChl-d is the20-demethyl form of BChl-c. On the other hand, BChl-e has a formylgroup at the C7-position and a methyl group at the C20-position. The20-demethylated form of BChl-e is named as BChl-f, but it has notbeen detected in any natural bacteria yet. Here we report thesynthesis of BChl-f and its physical properties comparing with BChls-c,d and e extracted from natural photosynthetic green bacteria.

Methods: Naturally occurring chlorophyll(Chl)-b was extracted fromspinach with organic solvents. By modifying Chl-b, methylbacteriopheophorbide-f was obtained according to the previousreport [1]. The methyl ester was transferred to the farnesyl ester andsuccessive magnesium-metallation gave BChl-f possessing 8-ethyl and12-methyl groups. BChls-c, d and e were extracted from culturedgreen bacteria, Chlorobium tepidum, vibrioforme andphaeovibrioides, respectively. BChls-c, d, e and f were analyzed byreverse phase high performance liquid chromatography (RP-HPLC).

Results: RP-HPLC analysis of farnesylated BChls-c, d, e and f indicatedthat they were eluted in the order of BChl-f < BChl-e < BChl-d < BChl-c (31R, 8-Et, 12-Me = R[EM]): ODS column and aqueous MeOH. The20-methylation and 7-reduction retarded their elution. Their visiblespectra showed that the 20-methylation red-shifted both Qy and Soretpeaks in their monomeric states and that the 7-oxidation induced theQy and Soret peaks to move to shorter and longer wavelengths,respectively. Moreover, the ratio of Qy/Soret peak intensitiesdecreased to less than half by the oxidation.

Conclusion: We first synthesized BChl-f from easily available Chl-b.Farnesylated R[EM]BChl-f was eluted more quickly than thecorresponding BChls-e (20-methylation) and d (7-reduction) in RP-HPLC. Based on the analysis, the HPLC peaks of BChl-f were searchedin the extracts from cultured green bacteria, but we could not detect it.

[1] H. Tamiaki, M. Kubo and T. Oba, Tetrahedron, 56, 6245-6257(2000).



P.071

QUORUM SENSING IN CYANOBACTERIAL BLOOM.

Young-Ki Lee, Chi-Yong Ahn, Hee-Sik Kim, Jung-Kee Lee, Won-GonKim, Hee-Mock Oh.

Environmental Biotechnology Research Center, KRIBB, Daejeon,Republic of Korea.

Microcystis aeruginosa is a ubiquitous cyanobacterium that causesecological and economic damage to freshwater ecosystems by bloomformation. Cyanobacterial blooms are visually unpleasant and theyproduce malodorous compounds (geosmin, MIB) and toxicmetabolites (microcystin), which pose more serious problems foranimals and humans. Recent studies focused on cell to cellcommunications in ecogenomics. Cell density-dependentcommunication has been known as “quorum sensing”. To investigatethe relationship between quorum sensing and bloom, we isolatedMicrocystis sp. Mi0601 and Microcystis sp. KW from surface scumsduring bloom season (from July to October, 2007) in Daechungreservoir and Wangsong reservoir, Korea. Both of them were identifiedas M. aeruginosa by 16S rRNA gene and cpc-IGS region.Concentrated cell-free extracts were bioassayed with Agrobacteriumtumefaciens NT1 (traR, tra::lacZ749) and separated by thin-layerchromatography. Two signal molecules were detected in TLC plate butthey were not general signal molecules, such as C4-, C6-, C10-homoserine lactones. Extracted signal molecules were analyzed byLC-MS and NMR. The signal molecule from each strain exhibited thesame molecular weight. NMR analysis indicated that they werecomposed of two unknown materials. A strong correlation was foundbetween Microcystis growth and accumulation of signal molecule. Thisimplies that quorum sensing plays an important role in the onset ofcyanobacterial bloom.

P.072

ISOLATION OF ANOXYGENIC PHOTOTROPHIC SULFURBACTERIA FROM TAMPAMACHOCO LAGOON (VERACRUZ,MÉXICO) USING DIFFERENT ELECTRON DONORS AND AGARPLATE CULTURES.

María Teresa Núñez-Cardona1, Jordi Mas2, Marina Luquin3, MarthaSignoret4.1,4Universidad Autónoma Metropolitana-Xochimilco, 1Laboratory ofEcología Microbiana; 4Plancton y Bioenergética, Distrito Federal,México; 2,3Departament de Genetica i Microbiologia, UniversidadAutónoma de Barcelona, Spain.

Introduction: Phototrohic sulfur bacteria (PSB) usually live in aquaticsystems. Actually there is a limited number of strains described if wecompare with the chemoorganoheterotrophic bacteria. Maybe it isbecause PSB are anaerobic microorganisms, and the use of organiccompounds as carbon and electron donors, for growing is limited. Ithas been shown that PSB are able to use different containing sulfurcompounds as electron donors like H2S (in laboratory cultures asNa2S=S

2-), polysulfides (Sx2-), thiosulfate (S2O3

2-), cysteine, methionine,and acetate (sodium acetate=SA) and also that is possible to incubatethem in gas pack bags. With the aim to isolate and getting purecultures of anoxygenic phototrophic sulfur bacteria it was assayed abasal medium adding different electron donors alone and mixed inagar plate cultures. Fatty acids and pigment composition as well asmorphological properties were used to characterize the strainsisolated from water samples collected at Tampamachoco Lagoon(Veracruz, México)

Methods: Nine collection strains including Allochromatium vinosum(strain DSMZ 183 and DSMZ 185), Lamprocystis roseopersicina (DSMZ229), Thiocapsa roseopersicina (DSMZ 217), Marichromatiumpurpuratum (DSMZ 1591), Thiocystis violaceae (DSMZ 207), Thiocystisminor (DSMZ 178) Chlorobium limicola f. thiosulfatophilum (DSMZ249), were used to test a basal medium specific for purple and greensulfur bacteria proposed by Pfennig and Trüper (1981) and changingthe electron donors like (alone and mixed): a) S2-, b) S2-+S2O3

2-+SA, c)Sx

2- d) Sx2-+SA e) cysteine+SA, f) methionine+SA, g) SA h) cysteine and

i) methionine. Liquid culture media (Na2S as electron donor) specificfor phototrophic sulfur bacteria was enriched with water samples fromTampachachoco Lagoon and positive cultures were used to inoculatePetri dishes containing the next electron donors: a) S2-, b) S2-+S2O3

2-

+SA, c) Sx2- d) Sx

2-+SA. In all cases, anaerobic conditions were gottenusing gas pack bags and the cultures were incubated at roomtemperature and light as energy source (incandescent=IL andfluorescent=FL). Red colonies were isolated in cultures growing inmedium d (Sx

2- and SA). Pure culture of these bacteria were verified byoptical microscopy and their phonotipic identity was done by the useof pigment and fatty acid composition (by gas-chromatography) aswell as cellular morphological properties.

Results: All the collection strains grew well in the medium d (S2-

+S2O32- ) specially on IL (incandescent light) but DSMZ 249 only grew

with FL (fluorescent light) and both strains were able to grow. Acetatealone and mixed with cysteine were used only for strain DSMZ 235and DSMZ 183 (IL and FL); But DSMZ 1591, all purple sulfur bacteriaassayed used Sx

2- (IL). Except DSM235 (IL) and DSMZ 185 (IL and FL) allthe strains were not able to use acetate as electron donator. It waspossible to obtain five pure cultures of phototrophic purple sulfurbacteria using the media d (Sx

2-+SA). The strains were also able togrow well in media b (S2-+S2O3

2-+SA) and d ( Sx2-+SA. Sulfur globules

into the cells, the presence of Bchl a, as well as the octadecenoic(C18:1), hexadecenoic (C16:1) and hexadecanoic (C16:0) as major fattyacids, related all the strains isolated as members of the Chromatiaceafamily.

Conclusions: A combination of electron donors could be analternative to isolate and to obtain pure cultures of marinephototrophic sulfur bacteria in agar plate cultures incubated in gaspack bags.

P.073

TOWARDS TO INTEGRATION OF BACTERIAL LIGHT-DEPENDENTHYDROGEN PRODUCTION AND ELECTRICITY GENERATION.

Evgeny Shastik1,3, Tatyana Laurinavichene1, Evgeny Minakov1, OlegVoronin2, Nikolay Zorin1, Arkady Karyakin2, Anatoly Tsygankov1.1Institute of Basic Biological Problems, Russian Academy of Sciences,Pushchino, Moscow Region; 2Chemical Faculty, Lomonosov MoscowState University, Moscow; 3South Federal University, Rostov-on-Don,Russia.

Introduction. For future H2-based economy many issues should beaddressed. Human-friendly and cheap H2 production andtransformation of H2 energy into electricity are among them. Purplebacteria are promising H2 producers since they use sunlight andsimple organic compounds to evolve H2 gas with high rates. A fuel cell(FC) application to transform hydrogen energy to electrical one seemsto be perspective. Existing FC’s with electrodes based on noble metalcatalysts (e.g. platinum) are not able to work in microbiological media,and an enzymatic catalyst is the best alternative for biological systems.Particularly, hydrogenase (H2ase) from Thiocapsa roseopersicina wasshown to be stable at high temperature, and in the presence of CO,



H2S, and proteases. A direct H2ase bioelectrocatalysis ontechnological carbon materials used in energetic would be optimaland profitable. The aim of this study was to explore a possibility tocombine bacterial H2 production with hydrogen electrode in onespace.

Methods. Reactors with H2 producing bacteria (including immobilizedpurple bacteria), hydrogen enzyme electrodes (carbon textile withpolymerized different promoters) with immobilized H2ases from T.roseopersicina and D. baculatum, and combination of them were usedin this study.

Results. Before the integration of hydrogen producing reactors withbacteria and hydrogen enzyme electrode, some preliminary researchon bacterial reactors and H2 electrode have been done.

It was shown that the photobioreactor with immobilized purplebacteria (Rhodobacter capsulatus and Rba. sphaeroides) undercontinuous synthetic medium flow stably produced H2 with averagerate app. 1.5 ml h-1 ml-1 matrix for more than 5000 hours. Operationalstability of H2 electrodes in K-P buffer saturated by H2 was studied formore than 500 h. pH-dependence of H2 electrode with differentH2ases was studied. Temperature profiles of H2 electrodes withdifferent H2ases with polyviologen and neutral red as promoters wasstudied. It was shown that the temperature dependence of the currentwas dependent on the voltage applied to the electrode. At low (5-20mV) overvoltage the temperature dependence was maximal whereasat high (150-200 mV) overvoltage the temperature dependence wasvery low. The dependence of activation energy on the overvoltage ofthe electrode will be discussed. The stability of H2 electrode in themedium going from the bioreactor was shown to be the same as in K-P buffer. Finally, the stability of H2 electrode integrated into thebioreactors with H2 producing bacteria was studied, detailed resultswill be presented, and problems appeared during the integration ofbacterial H2 production with H2 enzyme electrodes will be discussed.

Conclusion. The integration of H2 producing bacteria with H2 enzymeelectrode in one space is possible. However, many problems shouldbe solved before this system shows its practical efficiency.

This work was supported by the Russian Program of Basic ResearchRAS

P.074

FUNCTIONAL ANALYSIS OF DOMAINS FOR THE SIGNALPERCEPTION OF HIK33 IN SYNECHOCYSTIS SP. PCC 6803.

Yohei Shimura, Satoshi Kimura, Yoshihiro Shiraiwa, Iwane Suzuki.

Graduate School of Life and Environmental Sciences, University ofTsukuba, Japan.

Introduction: Two-component systems, which generally consist of asensory histidine kinase and a response regulator, are major signalingpathways in most of bacteria. In general, histidine kinases possess aconserved transmitter domain associating kinase activity at the C-terminus and a unique signal input domain (SID) at the N-terminus.The SIDs in the histidine kinases are thought to play important roles inperception of the specific stimuli and regulation of the kinase activityof the transmitter domain, however, the actual molecular mechanismsof signal perception are not clarified yet. Hik33, namely NblS orthologin the cyanobacterium Synechocystis sp. PCC 6803, regulates geneexpression under cold, high light, oxidative, high salt andhyperosmotic stress conditions. It has two transmembrane helices, aperiplasmic loop, a HAMP and a PAS domain in its SID. In this study,we examined functions of the subdomains in SID of Hik33 inperception of the stimuli.

Methods: In order to study functions of the subdomains in SID ofHik33 firstly we attempted to replace SID of a phosphate-deficientsensor SphS with the SID from Hik33 to express chimera sensorcontaining SID of Hik33 and transmitter domain of SphS (Hik33n-SphSc). SphS regulates expression of proteins for thephosphate-acquisition, including an alkaline phosphatase and high-affinity phosphate transporters. When SphS was replaced with thechimeric sensor Hik33n-SphSc, it altered expression pattern of theSphS-regulon under the environmental conditions, which might beperceived by the Hik33 but not under the phosphate-deficiency. Then,we deleted the subdomains in the SID of Hik33n-SphSc and examinedeffects on the expression of the phoA gene for alkaline phosphataseto identify the important subdomain to perceive the stimuli.

Results: SphS is activated under the phosphate-deficient conditionand upregulates expression of the phoA gene. However, the strainexpressing Hik33n-SphSc expressed the phoA gene under thestandard growth condition and expression levels of phoA wererepressed under high salt and cold stress conditions, which might beperceived by Hik33. These results indicated that Hik33n-SphSc mightbe active under the standard conditions and once the signals wereaccepted, it might become inactive. Deletion of the periplasmic loopdid not affect the ability of Hik33n-SphSc, i.e., the phoA gene wasexpressed under the standard conditions and repressed under thestressed conditions. However, deletion of PAS domain lost theresponsiveness to the stress, i.e., the phoA gene was constitutivelyexpressed. And simultaneous deletion of HAMP and PAS domains lostactivity of expression of the phoA gene regardless of the growthconditions, suggesting that the HAMP domain might be essential forthe kinase activity.

Conclusions: A chimeric sensor Hik33n-SphSc indicated that SID ofHik33 might activate the transmitter domain under the normal growthconditions. The PAS domain in the SID might play important roles inperception of the stimuli.

P.075

RNASE P FROM THE CHROMATOPHORE OF PAULINELLACHROMATOPHORA.

Agustin Vioque, Pilar Bernal, Leonor Puerto-Galan.

Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevillaand CSIC, Sevilla, Spain.

Introduction: Ribonuclease P (RNase P) is required for the generationof the mature 5’ end of tRNAs from precursors. RNase P is present inall domains of life as a ribonucleoprotein with a catalytic RNA subunit(P RNA). The only exceptions found so far are in human mitochondriaand higher plant chloroplasts, where RNase P seems to be a proteinenzyme lacking an RNA subunit. In several algae the plastid genomecontains a gene homologous to the bacterial P RNA gene (rnpB). Incontrast with the bacterial P RNA, plastids P RNA has no significantactivity in vitro in the absence of protein, and only very weak activitycould be reconstituted with a bacterial protein subunit (P protein) andthe P RNA from Cyanophora paradoxa. Plastid P RNA seems to beunable to fold into a functional structure for pre-tRNA substratebinding.

Paulinella chromatophora is a protist that contains an obligatecyanobacterium endosymbiont (chromatophore) whose genome hasbeen recently sequenced. This genome is highly reduced comparedwith free-living cyanobacteria and could represent an early step in theintegration of a cyanobacterium into a eukaryotic cell, a process that isat the origin of chloroplasts. Therefore, we have characterized thechromatophore RNase P, and compared its structure and function with



both cyanobacterial and plastid RNase P, under the hypothesis that itsproperties could illustrate about the process of P RNA loss of functionin plastid evolution.

Methods: We have identified in the chromatophore genome the rnpA(encoding the P protein) and the rnpB genes and have undertakentheir functional and structural analysis. The rnpB gene has beencloned and the P RNA has been obtained by in vitro transcription. ItsRNase P activity has been analyzed either in the absence or presenceof a cyanobacterial P protein. The structure and folding of the P RNAshas been analyzed by native gel electrophoresis and sensitivity tolead-induced cleavage.

Results: Chromatophore P RNA has a predicted secondary structuresimilar to other cyanobacterial P RNAs. Chromatophore P RNA hasreduced activity in the absence of protein, but it can reconstitute afully functional holoenzyme with cyanobacterial P protein. Comparisonof reaction rates in the presence or absence of the protein subunitindicate that chromatophore RNase P is more dependent on theprotein subunit for activity than free living cyanobacterial RNase P. Thepattern of lead induced cleavage of chromatophore P RNA is similarto the pattern observed with Anabaena 7120 P RNA, but the mobilityof the P RNA on native gel electrophoresis indicates that this RNA hasa reduced ability to fold into a functional native compact structure.

Conclusions: The chromatophore P RNA has functional and structuralproperties that suggest that it might represent an early step in theprocess of increased dependence on protein for folding and activityprevalent in present day plastids.

P.076

THE GENOME SEQUENCE OF THIODICTYON SP. AND ITSSIGNIFICANCE FOR THE CAROTENOGENESIS OF OKENONE.

Kajetan Vogl, Niels-Ulrik Frigaard, Mauro Tonolla, Lynn Thomso, Qi Ji,Stephan C. Schuster,Donald A. Bryant.

Penn State University, University Park, PA, USA.

Introduction: Anoxygenic phototrophic purple bacteria are a majorgroup of phototrophic microorganisms which occur mainly in aquaticenvironments. The group of purple bacteria consists of purplenonsulfur bacteria (alpha- or betaproteobacteria) and purple sulfurbacteria (gammaproteobacteria). Within the group of purple sulfurbacteria, over 25 genera are recognized (Madigan and Jung 2009).These bacteria have two pathways for carotenogenesis, thespirilloxanthin pathway and the okenone pathway. The carotenoidokenone is of special interest, because okenone and its derivativeokenane serve as the only known hydrocarbon biomarker for purplesulfur bacteria (Chromatiaceae) used to reconstruct geologic history(Brocks et al. 2005). However, so far no genome sequence of anokenone-producing purple sulfur bacterium is available and thebiosynthetic pathway of okenone is not understood in detail.

Methods: Thiodictyon sp. strain Cad 16 genomic DNA was isolatedusing standard protocols. Then 454 pyrosequencing was performedand contigs harboring carotenoid genes were identified with BLASTN(Altschul et al. 1997) and annotated manually with MacVector.

Results: The following homologs of carotenogenesis genes arepresent in the okenone producing purple sulfur bacterium:geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase(crtB), phytoene desaturase (crtI), hydroxyneurosporene synthase(crtC), two gene copies of C-3’,4’ desaturase (crtD),hydroxyneurosporene-O-methyltransferase (crtF), gamma-carotenedesaturase (crtU) and lycopene cyclase (crtY), The crtY, crtU, crtD1,crtD2 and crtC are clustered together. CrtE, crtI and crtB are in close

proximity to the gene cluster.

Conclusions: The carotenogenesis gene cluster revealed a so farunknown duplication of the crtD gene. Since this duplication was notobserved in other phototrophic bacteria, it seems to be possible thatthe second crtD gene has another function than desaturating the 3,4and/or 3’,4’ bond (Albrecht et al. 1997), and its function is correlatedwith okenone production. In the past the function of certaincarotenoid genes were successfully examined by heterologousexpression in Eschericha coli (Maresca et al. 2007). In combinationwith the approach of heterologous gene expression in E. coli andChlorobaculum tepidum, the genome sequence of Thiocystis sp. willallow the elucidation of the carotenogenesis pathway for okenone.

References

Albrecht M, Ruther A and Sandmann G (1997) Purification andbiochemical characterization of a hydroxyneurosporene desaturaseinvolved in the biosynthetic pathway of the carotenoid spheroidene inRhodobacter sphaeroides. J Bacteriol 179:7462-7467

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W andLipman DJ (1997) “Gapped BLAST and PSI-BLAST: a new generationof protein database search programs.” Nucleic Acids Res 25:3389-3402

Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA and BowdenSA (2005) Biomarker evidence for green and purple sulphur bacteria ina stratified Palaeoproterozoic sea. Nature 437:866-870

Madigan MT and Jung DO (2009) An Overview of purple bacteria:systematics, physiology, and habitats. In: Hunter CN, Daldal F,Thurnauer MC and Beatty JT (eds) The Purple Phototrophic Bacteria,pp 1-15. Springer Netherlands

Maresca JA, Graham JE, Wu M, Eisen J and Bryant DA (2007)Identification of a fourth type of carotenoid cyclase in photosyntheticorganisms. Proc Natl Acad Sci USA 104:11784-11789

P.077

THYLAKOID MEMBRANE REDUCTION AFFECTS THEPHOTOSYSTEM STOICHIOMETRY IN THE CYANOBACTERIUMSYNECHOCYSTIS SP. PCC 6803.

Eva Fuhrmann, Dirk Schneider.

University Freiburg - Department of Biochemistry and MolecularBiology, Freiburg, Baden-Wurttemberg, Germany.

Introduction: Biogenesis of thylakoid membranes in both chloroplastsand cyanobacteria is incompletely understood today. The “vesicleinducing protein in plastids 1” (Vipp1) was found to be involved inthylakoid membrane formation in chloroplasts and cyanobacteria.Since the exact physiological function of VIPP1 is still mysterious, wegenerated a Synechocystis sp. PCC 6803 Vipp1 depletion strain tocharacterize the physiological effects of the Vipp1 depletion incyanobacteria more in detail.

Methods: To inactivate the Vipp1 gene a kanamycin resistancecassette was introduced into the vipp1 gene (sll0617) fromSynechocystis sp. PCC 6803. Wild type (wt) and mutant cells wereanalyzed by electron microscopy as well as by absorbance and 77 Kfluorescence emission spectroscopy. Photosystem (PS) II and PS Iactivity was determined by oxygen evolution and consumptionmeasurements, respectively. Membrane protein complexes werecharacterized by sucrose density gradient centrifugation as well as byblue native polyacrylamid gelelectrophoresis (BN-PAGE).

Results: Although the vipp1 gene was disrupted in a fewchromosomal copies, no completely segregated strain obtained even



after two years on selective medium. Nevertheless, based onimmunoblot analyses the total Vipp1 content in the mutant strain wassignificantly decreased when compared to wt cells. Electronmicroscopy revealed an average of four to six layers of thylakoidmembranes present in wt cells whereas in the mutant strain only twoto three layers were observed. Under increasing light intensities thechlorophyll content per cell decreases, and, furthermore, it is alwayslower in the merodiploid cells at any light intensity compared to wtcells. Sucrose density gradient centrifugation and BN-PAGE analysesrevealed that the amount of PS I trimers is significantly reduced inmembranes isolated from the mutant cells. Furthermore, the cellularconcentration of chlorophyll binding proteins is significantly lowered inthe merodiploid cells. By 77 K and UV/VIS spectroscopy we observedthat the ratio of PS I to PS II has changed in the mutant strain. Whilethe PS II activity is not significantly altered the activity of PS I wasdetermined to be decreased in the mutant relative to the wt strain.However, the mutant cells still appear to be able to operate theelectron transfer chain at wt rates.

Conclusion: We show that in the cyanobacterium Synechocystis sp.PCC 6803 the synthesis of active PS I depends on the amount ofthylakoid membranes present per cell. Down-regulation of the amountof active PS I appears to be a general adaptation mechanism incyanobacteria and Vipp1 could be directly involved in suchmechanisms, resulting in changes of the PS I to PS II ratio in responseto e.g. increasing light intensities. The depletion of the vipp1 geneproduct resulted in a specific decrease of functional PS I inSynechocystis, whereas the amount of functional PS II was notsignificantly altered. In contrast, a Vipp1 depletion strain ofArabidopsis is deficient in photosynthesis, although the defect couldnot be assigned to a deficiency of a single photosynthetic complexbut appeared to be caused by dysfunction of the entirephotosynthetic electron transfer chain1. Therefore, it has beensuggested that depletion of Vipp1 in Arabidopsis affects thylakoidmembrane formation rather than the assembly of thylakoid membraneprotein complexes2.1 Kroll et al (2001) Proc Natl Acad Sci USA 98: 4238–42422 Aseeva et al (2007) Plant Physiol Biochem 45: 119–128

P.078

THE EVOLUTIONARY PATH TO TERMINAL DIFFERENTIATION ANDDIVISION OF LABOR IN CYANOBACTERIA.

Valentina Rossetti, Bettina E Schirrmeister, Marco V. Bernasconi,Homayoun C Bagheri.

University of Zurich, Zurich, Zurich, Switzerland.

Introduction: The transition from unicellular to multicellular organismsis not a well understood process in evolution. A common trait oftenassociated with multicellularity is cellular differentiation, which is aseparation of tasks through the division of labor.

However, the division of labor does not necessarily have to beconstrained to a multicellular setting. In this study, we focus on thepossible evolutionary paths leading to terminal differentiation incyanobacteria.

Methods:We develop mathematical models for two developmentalstrategies. One, of populations of terminally differentiated single cellssurviving by the exchange of common goods. Second, of populationsexhibiting terminal differentiation in a multicellular setting. We test theevolutionary stability of the two strategies with respect to resistanceagainst disruptive mutations (i.e. “cheater” mutants). We also assessthe effects of selection on the optimization of the ratio of vegetative

(carbon fixing) to heterocystous (nitrogen fixing) cells, which in turnleads to the maximization of the carrying capacity for the populationdensity. In addition we compare the performance of differentiatedpopulations to undifferentiated ones that temporally separate tasks inaccordance to a day/night cycle. We then compare some predictionsof our model with phylogenetic relationships derived from analyzing16S rDNA sequences of different cyanobacterial strains.

Results: In line with studies indicating that group or spatial structureare ways to evolve cooperation and protect against the spread ofcheaters, our work provides theoretical and phylogenetic evidencethat differentiated single-celled populations of cyanobacteria are notstable. The compartmentalization afforded by multicellularity isrequired to maintain the vegetative/heterocyst division and forselection to optimize the carrying capacity.

Conclusions: In cyanobacteria, multicellularity is a necessary conditionfor both the evolutionary stability of terminal differentiation and forthe optimization of the division of labor. Furthermore, in regimes oflong daylight periods, terminally differentiated species perform betterthan undifferentiated species that follow the day/night cycle;indicating that terminal differentiation can be an evolutionaryadvantage in high irradiance regions. Conversely, undifferentiatedspecies have an advantage in regimes of short daylight periods.

P.079

DIVERSITY OF PHOTOTROPHIC BACTERIA IN MICROBIAL MATSOF LAGUNA CHAXA AND LAGUNA TEBENQUICHE AT THESALAR DE ATACAMA, CHILE.

Cristina Dorador, Andrea Gärtner, Yaneisi Vazquez, Carolina Cubillos,Vera Thiel, Johannes F. Imhoff.

Departamento de Acuicultura, Universidad de Antofagasta, AvenidaUniversidad de Antofagasta, Antofagasta, Chile.

Introduction: The Salar de Atacama is located in the Atacama Desert(Chile) at an altitude of 2300 m above sea level. In the interior of thesalar there are several small shallow ponds that exhibit saltconcentrations >100 mS cm-1. Intensive solar radiation and fluctuatingdaily temperatures between day and night are typical features of theseenvironments. It has been proposed that phototrophic bacteria shouldplay an important role in the primary production of saline lakes in theAtacama Desert; however their diversity and functionality are underinvestigated. The diversity of phototrophic bacteria was focused intwo main lakes of the Atacama Desert which exhibit microbial mats ontheir shore.

Methods: We investigated the diversity of specific phototrophicbacterial in samples collected from Laguna Chaxa and LagunaTebenquiche at the Salar de Atacama. DNA was extracted fromhomogenized samples of microbial mats and was amplified usingdescribed specific 16S rRNA gene primers for the groupsChromatiaceae, Chlorobi, Cyanobacteria and Roseobacter clade.Microbial diversity was examined through DGGE and clone libraries ofthe 16S rRNA gene.

Results: Microbial mats were found at the shore of LagunaTebenquiche and Laguna Chaxa. Mats from Laguna Tebenquicheexhibited 1-3 layers compared with mats of Laguna Chaxa which hadbetween 3-4 layers. The target groups were recorded from all samplesanalysed. Analyses of DGGE band patterns and clone librariesrevealed that the lakes supported different microbial communities.

Conclusions: Phototrophic bacteria occupied an important part of themicrobial diversity of inland lakes of Salar de Atacama. TheRoseobacter clade, initially described from marine systems,



represented an important component of the diversity of phototrophicbacteria from inland lakes of the Salar de Atacama. Future studies willinclude activity assays to determine the role of the phototrophicbacteria in the primary productivity of evaporitic basins in northernChile.

P.080

CYANOBACTERIA IN THE PCC: WHO IS RELATED TO WHOM?

Muriel Gugger1, Alexandra Calteau2, Rosmarie Rippka1, ThérèseCoursin1, Thierry Laurent1, Jennifer Tambosco1, Corinne Cruaud2,Frederik Gavory2, Jean Weissenbach2, Nicole Tandeau de Marsac1.1Unité des Cyanobactéries, Institut Pasteur, Paris ; 2CEA / Institut deGénomique / Génoscope / Laboratoire de Génomique Comparative,Evry, France.

Introduction: Cyanobacteria are oxygenic phototrophic prokaryotesthat exhibit a wide range of morphological and physiological featuresand colonize strikingly different ecosystems.

The global picture of cyanobacterial interrelationships is mainly basedon complete or partial 16S rDNA sequences of non-axenic isolates forwhich little information may be available, or on environmental samplesrepresentative of very specific ecosystems. Recently, the evolution ofthis phylum has been re-examined based on cyanobacterial genomesequences, but so far is highly biased towards marinepicocyanobacteria (50 % of the genomes).

Methods: The PCC (Pasteur Culture Collection of Cyanobacteria)presently includes almost 800 axenic strains originating from diverseecosystems and representing about 60 major morphotypes. Theyexhibit widely differing physiological characteristics, and someproduce hepato- or neurotoxins. To overcome the paucity ofinformation associated with many of the available 16S rDNAsequences, either with respect to knowledge of the organisms orbecause of insufficient sequence length, the 16S rRNA genes of 709PCC strains were sequenced over their full length to establish moreprecisely their phylogenetic relationships.

Results: The sequence analyses confirmed the monophyly of theheterocystous cyanobacteria, and the intermixed relationships of theother morphotypes. Although many genera are polyphyletic, severalsustained genetic clusters were recognized that contain PCC referencestrains, previously proposed based on mean DNA base compositionand phenotypic characters. Moreover, PCC strains hithertoinsufficiently characterized can now be assigned to appropriategenetic clusters within the cyanobacterial phylum. In addition,relatives from other culture collections or those only known fromenvironmental sequences could be recognized.

Conclusions: The new 16S rDNA sequence database of the PCCprovides a more complete reference frame for the genetic diversityand interrelationships of cultured and uncultured cyanobacteria, andshould help to place the phenotypic properties of the organisms intoan evolutionary context.

P.081

MOLECULAR BIOLOGICAL AND BIOCHEMICALCHARACTERIZATION OF ODOR PRODUCING CYANOBACTERIAFROM DRINKING WATER RESERVOIRS.

Frank Ludwig, Sabine Hacker, Andy Weiss, Hetvi R. Gandhi.

Isolde Röske Institute of Microbiology, Dresden University ofTechnology, Dresden, Germany.

Introduction: Recently, taste and odor problems (TO problems) couldbe observed frequently. In the most cases the odor is described as anearthy-musty smell, which may be caused by the volatilesesquiterpene geosmin, which has a very low odor thresholdconcentration of 4 ng/l (Young et al. 1996). Besides actinomycetes,cyanobacteria are mostly responsible for the release of odoroussubstances as geosmin and 2-methylisoborneol (2-mib) into the water.Because the drinking water should be free from flavor and smell, TOproblems lead to higher costs. Therefore, we have investigated theoccuring cyanobacteria from three different drinking water reservoirs.

Methods: Material from the phytobenthic mats of the Cranzahl,Klingenberg and Saidenbach (all in Saxony, Germany) drinking waterreservoirs was used to prepare single trichomes of cyanobacteria.From the obtained cultures the isolated dna were used to amplifygenes like the rbcL and geoA. These partial gene sequences andfurthermore the sequence area between the 16S rDNA and 23S rDNAcould be used for the characterization of the isolates. Thedevelopment of degenerated primer pairs for the amplification of thecyanobacterial genes like rbcL and the geoA were the basis for thesuccessful molecular biological determination of the isolated strains.Supplementary we have conducted biochemical analyses withselected cultures for the confirmation that these isolates are capableto produce odorous substances as geosmin or/and 2-mib. Finallyphylogenetic trees were established with the obtained nucleotidesequences for the depiction of the existing diversity.

Results: Through the successful development of a new method toisolate pure cultures of cyanobacteria from very heterogeneousenvironmental samples we were able to obtain a lot of taxonomicallyvery different cyanobacterial cultures. This could be affirmed by thenucleotide sequences of the 16S-23S rDNA, rbcL and the geoAgenes. The biochemical analyses shows also the important potential ofcyanobacteria to produce and to release odor to the ambientbiocenosis. Experiments with different light and nutrient conditionshave demonstrated that the pattern of the produced odor is variable.

Conclusions: The achieved results show the great diversity of theoccurring cyanobacteria in different drinking water reservoirs. Throughthe biochemical analyses it can be confirmed that cyanobacteria are amajor originator of TO problems. It is very interesting that especiallythe light quality has an influence of the synthesized odoroussubstances. Because it is very difficult to cultivate cyanobacteria andto identify factors which affect the odor production, the managementof the drinking water reservoirs is very interested to find outparameters which can indicate that odorous substances will bereleased into the water at a great scale.



P.082

THE TWIN ARGININE TRANSLOCATION SYSTEM INSYNECHOCYSTIS PCC6803.

Anja Nenninger1,3, Gemma Warren2, Conrad W. Mullineaux1 and ColinRobinson3.1Queen Mary University of London, London; 2MOAC, DoctoralTraining Centre, University of Warwick, Coventry; 3University ofWarwick, Coventry; United Kingdom

Introduction: Targeting and translocation of newly synthesisedproteins is a crucial process in living calls, using a range of mechanismat different membrane systems. The transport mechanism studied hereis the twin arginine translocation (Tat) system. Discovered in the early1990s in plant thylakoids it has since been shown to operate in theplasma membrane of a wide variety bacteria.

Cyanobacteria represent a unique subject for Tat studies. They areconsidered to be the progenitors of plant chloroplasts with an oxygen-evolving photosynthetic apparatus very similar to higher plants at anabundant internal network of thylakoid membranes; and as Gram-negative bacteria they have a cell envelope consisting of an outermembrane, a periplasmic compartment, and a plasma membrane.Sequencing of the Synechocystis PCC6803 genome has shown that asingle set of tat genes is present, with a homologue of cptatC/tatCand two paralogues of tha4/tatA.

Methods: The plasmid described in Spence et al. (2003) was used astemplate for QuikChange® Site-directed Mutagenesis and thefollowing mutants of the twin arginine motif were obtained: RK, KR,KK. Translocation of GFP in Synechocystis was observed using aconfocal microscopy. (Spence E. et al., 2003, Mol. Micro. 48, 1481-89)

Results: In Synechocystis GFP tagged with a Tat signal sequence (fromE. coli TMAO reductase; torA-GFP) is found exclusively in theperiplasm. GFP fluorescence can be seen contiguous to the thylakoidmembrane system corresponding with exported periplasmic GFP.Disrupting the twin arginine motif leads to accumulation of clearlyvisible GFP aggregates inside the plasma membrane. The analysis ofnumerous cells for the different mutations has shown that theseaggregates are primarily located in the thylakoid region, but they canas well be found in the cytoplasm.

Conclusions: Proteomic studies on Synechocystis identified severalpotential Tat substrates. The twin arginine is part of the signalsequence in most Tat substrates in plants and bacteria. However, theimportance of this motif varies between the two systems. In plantssubstitution of either arginine led to a complete block in translocation,whilst bacterial Tat system only shows a full block in translocationwhen both arginine residues were replaced.

Substitution of the arginine residues in Synechocystis shows anextreme level of stringency very similar to the situation observed inplants. This absolute requirement for an intact twin arginine motif isunique among bacteria studied to date. With the endosymbiotictheory now widely accepted for the origin of plastids in higher plantsand phylogenetic studies clustering the cyanobacterial tatC (sll0194)with the chloroplast cptatC the results described here emphasise therelationship of cyanobacteria and plant chloroplast on a functionallevel for the first time.

P.083

POLYFACETIC STUDY OF NON PURPLE SULFUR BACTERIAISOLATED FROM THE SOUTHERN GULF OF MEXICO WATERSAMPLES.

María Teresa Núñez-Cardona1, Verónica Pacheco-González2,Jaime García-Mena3, Martha Signoret4.

Universidad Autónoma Metropolitana-Xochimilco, 1,2Laboratory ofEcología Microbiana; 4Plancton and Bioenergética; 3Centro deInvestigación y de Estudios Avanzados-Zacatenco, Departamento deGenética y Biología Molecular, Distrito Federal, México.

Introduction: Studies about diversity of phototrophic bacteria (PB)using laboratory cultures are scarce if we compared with theapplication of molecular techniques like nucleic acids hybridization insitu and the gene bank. These molecular tools have lead to theconclusion that the most abundant marine microbial groups are as yetuncultivated and probably they play a significant role in marinebiogeochemical cycling. Nevertheless pure laboratory cultures areused as reference and as models too. The goal of this investigationwas to isolate and to identify nonpurple sulfur bacteria from a 100 mdepth water sample collected in the Gulf of Mexico.

Methods: Conventional and molecular analyses were used tocharacterize and to identify the isolated strains. Water samples werecollected at 100 m depth and were used to enrich glass vialscontaining van Niel medium, specific for growth of nonpurple sulfurbacteria. Strains were purified by the agar shake technique. All thecultures were incubated at room temperature (20-28 °C) and exposedto light (1.3-1.8 Klux). Morphological, pigment composition (in vivoanalysis) and the ability to use different chemical compounds aselectron donors in van Niel basal medium, were used to characterizethe isolated strains. They were identified by the analysis of the 16SrDNA. This one was achieved and amplified by PCR technique and theuse of Unifor and Unirev primers. Results were analyzed byelectrophoresis in an agarose gel (1.0%) and the DNA sequences weregotten.

Results: Pure cultures (13 strains) of nonsulfur bacteria were obtainedafter serial dilutions on semisolid agar. Cell morphology of thesecorresponded to small budding Gram negative rods, with a littledifference in size (2-3 μm). In vivo pigments analyses revealed thepresence of bacteriochlorophyll a and spirilloxanthin as majoraccessory pigment in all wild strains. It was assayed 20 differentcompounds to know the capability of the strains to use them aselectron donors. Sodium and magnesium acetate as well as glycerolhave been used for all the strains assayed, 12 used yeast extract andeleven to the pyruvate and propionic acid, sucrose, maltose, lactose,by other hand, succinate. methionin, mannitol and glycine were usedonly by 5, 6 and 7, strains, respectively. A BLAST and phylogeneticanalyses based on 16S rDNA gene sequence revealed that four strainswere related with Rhodobium bactotapetarum, three toRhodopseudomonas spp and other three were identified as membersof Rhodopseudomonas julia. It was observed differences in the use ofthe electron donors among the strains of the same specie.

Conclusions: With the enrichment technique used it was possible todetect, isolate and to get pure cultures of phototrophic bacteria frommarine water samples. The presence of bacteriocholorophyll a and the16S rDNA analysis identify them as members of purple non sulfurbacteria



P.084

DETECTION OF CYANOBACTERIA WITH CLASSICAL MOLECULARMETHODS IN THE SEDIMENT OF A DRINKING WATERRESERVOIR.

Susanne Schumann1, Heidemarie Horn2, Tim Köhler3, Frank Ludwig1,Isolde Röske1, Kerstin Röske2.1Institute of Microbiology, Dresden University of Technology, Dresden;2Taxony Academy of Sciences, Leipzig; 3Max Planck Institute forTerrestrial Microbiology, Karl-von-Frisch-Straße, Marburg; Germany.

Introduction: Cyanobacteria were able to colonize almost all habitatsand are special important in the water because they have the ability todo photosynthesis. So they make a great contribution to primaryproduction (Martínez-Alonso et al., 2004). Through formation of matson the water surface, cyanobacteria can be able to reduce thedevelopment of organisms in deeper water layers.

Methods: Sediment samples of different sampling sites of the dinkingwater reservoir Saidenbach (Saxony, Germany) and the pre-reservoirForchheim were examined to follow up the deposition ofcyanobacteria. To analyze the seasonal changes of the cyanobacteriacomposition in this habitat cultivation methods as well as PCR withestablished primer sets for cyanobacteria were applied. These partlywell-known primer were combined with Cya564F, a new one tosequence the 16S rDNA to 23S rDNA of the cyanobacteria cultureswhich provides the basis of an alignment and cluster analysis. Thisresults were compared with a phylogenetic tree which was establishedon the basis of the sequencing of the cyanobacterial phycocyaninegene (cpc). In addition a new primer system specific for species of thegenus Synechococcus was developed and tested by PCR and cloning.

Results: With the applied methods it was possible to detectdifferences and parallels in the emerging of cyanobacteria during theexamination period. The amplification with the new primer systemdesignated ProCR demonstrated clear variation in the occurrence ofspecies of the genus Synechococcus. In spring, all samples werepositive but in the further examination period it showed partlynegative results. These findings could be confirmed by microscopyand cultivation. The alignment of the whole 16S rDNA of about 30cyanobacterial pure cultures corroborates the belief that there is anincrease of differences in the rear sequence sections.

Conclusions: The received results show seasonal changes in thedeposition of cyanobacteria of the examined drinking water reservoir.Furthermore differences between the sampling sites Saidenbach andForchheim could be demonstrated. The first molecular biologicalanalysis show the necessity of the development of new primer sets tosequence the whole 16S rDNA. So a reliable analysis of the isolatedcyanobacterial cultures becomes possible.

P.085

ECOLOGICAL AND PHYSIOLOGICAL STUDIES ON PURPLESULFUR BACTERIA (CHROMATIACEAE) AT ASWAN HIGH DAMLAKE.

Mohamed S. A. Shabeb 1, Nahla S. Abd-El Azim1,Ahmed A. M. A. Shoreit 2.1Botany Department, Faculty of Science, Aswan South ValleyUniversity, Aswan, Egypt; 2Botany Department, Faculty of Science,Assiut University, Assiut, Egypt

322 isolates of purple sulfur bacteria were isolated from differentlocalities of study in Aswan High Dam Lake. These comprise five

genera of purple sulphur bacteria (Chromatiaceae). Allochromatiumwas the most common genus 161 (50%) followed by Thiocystis 50(15.53%), Thiocapsa 40 (12.42%), Thiodictyon 38 (11.8%) andLamprocystis 33 (I0.25%) from the total number of isolates during thewhole period of study. These genera represented by six specesAllochromatium vinosum and A. warmingii; Thiocystis violacea;Thiocapsa roseopersicina; Thiodictyon elegans and Lamprocystisroseopersicina. Physico–chemical parameters of water samples weredetermined.

P.086

FUNCTIONAL GENE APPROACH ON COMMUNITIES OFPHOTOTROPHIC PROKARYOTES IN HIGH ALTITUDE WETLANDSIN NORTHERN CHILE.

Vera Thiel, Marcus Tank, Cristina Dorador, Johannes F. Imhoff.

IFM-GEOMAR, Leibniz Institute of Marine Sciences (at the Universityof Kiel), Kiel, Schlewig-Holstein, Germany.

Introduction: The Chilean Altiplano located 2300 m above sea levelharbours a set of extreme habitats characterized by high UV-radiation,partly extreme saline conditions and high diurnal temperaturevariations. These unexpectedly quite productive habitats aredominated by microbial life and phototrophic bacteria are assumed tohave a major impact. However, key processes of primary productionhave not yet been identified and little is known about the diversity andcomposition of the phototrophic prokaryotic communities.

To gain insight into the diversity of assumed key players involved inprimary production in Chilean Altiplano salares, we studied thephototrophic bacterial communities of different locations around theSalar de Atacama in the Chilean Altiplano characterized by salinitiesvarying from 54 mS cm-1 to 261 mS cm-1.

Due to the polyphyletic character of the physiological groups ofphototrophic bacteria, 16S rRNA gene based molecular studies oftenfail to cover the diversity of the phototrophic community. Thus, inorder to specifically study the phototrophic prokaryotes of thehabitats, we used molecular genetic analyses with group specificprimers for functional genes in addition to isolation methods.

Methods: We sampled several colourful microbial mats along twolakes of the Salar de Atacama (Laguna Chaxa and LagunaTebenquiche). Different liquid media were inoculated for enrichmentand isolation of phototrophic bacteria. Pure cultures were obtained byserial dilution in agar shakes and identified by 16S rRNA genesequencing. For the molecular approach genomic DNA was extractedand the specific functional genes fmoA, pufLM and bchY amplifiedusing PCR method. Clone libraries were constructed and thecommunity composition was established on the basis of the functionalgene sequences.

Results: Enrichment and purification in different media lead to theisolation and identification of representatives of the green sulfur andpurple sulfur bacteria as well as purple nonsulfur bacteria.

Using functional genes members of different phototrophic bacterialgroups were detected in all samples. The clone libraries lead to anestimate of the diversity of phototrophic prokaryotes in the differentsamples. The diversity and composition of the phototrophicprokaryotic communities varied between the different samples andbetween the two lakes.

Conclusion: This study is the first report on the diversity ofphototrophic prokaryotes in the Chilean Altiplano salares determinedby functional gene approach and isolation methods. We demonstratethe suitability of the used functional genes, fmoA, pufLM and bchY for



studying phototrophic communities as well as the uniquephototrophic communities of the different habitats around the Salarde Atacama.

P.087

EVOLUTION OF THE CYANOBACTERIUM NOSTOC –PLANTSYMBIOSES.

Akiko Tomitani.

Institute of Biogeosciences, Japan Agency for Marine-Earth Science &Technology, Yokosuka, Kanagawa, Japan.

Cyanobacteria are oxygenic photosynthetic bacteria, many of whichhave ability to fix nitrogen. Filamentous cyanobacteria of the genusNostoc produce differentiated cells such as heterocysts (specializedcells for nitrogen fixation) and hormogonia (transient motile filements),and many of them are known to establish symbiotic association withvarious plants (bryophytes, pteridophytes, gymnosperms,angiosperms) and algae and supply fixed nitrogen to the hosts.

To investigate how Nostoc has evolved the capability to infect a widerange of plants, molecular-phylogenetic analyses together with co-culturing experiments were performed. Phylogenetic analyses offree-living and symbiotic Nostoc species suggest that symbioticcompetency may have evoloved polyphyletically within the genus.Co-culturing assays using 10 Nostoc strains and a host bryophyteindicate that their ability of hormogonia formation may notcorrespond to plant-infection efficiency.

Continued molecular-biological and physiological study of diverseNostoc species will provide a clue to understand mechanisms andevolution of the cyanobacterium-plant symbioses.

P.088

BIOGEOGRAPHICAL DISTRIBUTION AND DIVERSITY OFCYANOBACTERIA IN ANTARCTICA, A POLYPHASIC APPROACH.

Annick Wilmotte1, Pedro De Caralho Maalouf1, Frédéric Zakhia1,Arnaud Taton1, Rafael Fernandez-Carazo1, Aaike De Wever2, ElieVerleyen2.1Centre d’Ingénierie des Protéines, B6, Université de Liège, Liège;2Department of Biology, Research group of Protistology and AquaticEcology (PAE), Gent University, Gent; Belgium.

Introduction: In high latitude ecosystems, cyanobacteria are ofparticular interest because they often represent the predominantphototrophs. The cyanobacterial diversity from microbial mats inantarctic lakes was investigated using microscopic and molecularapproaches. This enables us to assess the relative importance ofecological versus historical factors in explaining the geographicaldistribution of cyanobacteria in Antarctica.

Methods: Samples from a wide geographic scale were chosen inorder to represent a range of environmental conditions and toevaluate the influence of lake characteristics on the cyanobacterialdiversity. In the framework of the AMBIO project (“Antarctic MicrobialBIOdiversity: the importance of geographical and ecological factors”,www.ambio.ulg.ac.be), microscopic identification and morphologicalcharacterization were complemented with molecular tools (PolymeraseChain Reaction, Denaturating Gradient Gel Electrophoresis,sequencing, clone libraries). This polyphasic approach aims to revealthe cultivated and non-cultivated diversity of microorganisms.

Results: Preliminary results from the 16S rDNA sequence analysisallowed the construction of phylogenetic tree and revealed thepresence of 23 Operational Taxonomic Units (OTU). Five OTUs are

endemic to Antarctica and three others constitute a previouslyundiscovered diversity. A multivariate analysis run with data fromvarious samples revealed that the OTU composition is geographicallystructured as each region has its more or less unique flora. Moreover,salinity seems to play an important role on the composition of thecyanobacterial communities in these samples.

Conclusions: The three new OTUs included sequences from theBelgian Base, Transantarctic Mountains and East Antarctica whichsuggests a flux of microorganisms between these regions. On theother hand, differences in cyanobacterial composition betweengeographically close lakes were observed. These might be underlainby several reasons, such as differences in limnological properties(inferring different histories and ecological processes betweenregions), or rather the result from dispersal limitation amongcyanobacteria. Also, samples originating from coastal lakes seem tobe more diverse (average number of OTUs) than others, this may bedue to the difference in the meteorological conditions (oceanic versuscontinental climates).

More detailed studies and refined molecular analysis (Real TimeQuantitative PCR, clone libraries) should allow us to assess the realcyanobacterial diversity in antarctic lakes and the importance ofgeographical and environmental factors shaping the microbialcommunities.

P.089

IN THE FRAME OF THE BELSPO PROJECT B-BLOOMS2, WE HAVEOBSERVED THE PRESENCE OF MICROCYSTINS BY ELISA INBLOOMS OF WORONICHINIA IN ONE BRUSSELS POND.

Annick Wilmotte, Yannick Lara, Alexandre Lambion, AnatolyPeretyatko, Ludwig Triest, Geoffrey Codd.

University of Liege, Liege, Belgium.

So far, Woronichinia naegliana was isolated only two times accordingto the literature (Rajaniemi et al., 2005; Willame et al., 2006) and theisolates were not toxic. However, the cultures were quickly lost,illustrating the difficulties in obtaining and keeping strains of thisgenus. Therefore, there is an important lack of knowledge andmolecular data. As an alternative, we propose to analyze genotypes ofenvironmental single colonies.

Methods: Woronichinia were directly isolated under a binocular froma fresh sample. As the DNA content of one single colony limits thenumber of PCR reactions that can be carried out, we have developeda new approach using Whole Genome Amplification with Phi29polymerase to allow for the Multi Locus Sequences Analysis of a singlecolony. Subsequent PCR reactions were performed with cyanbacterialspecific primers.

Results: For the first time, we have obtained the sequences of rpoC1,rbcLX and rRNA-ITS from 4 single colonies of the genus Woronichinia(identified by microscopy). About 12 PCR reactions were successfullyperformed on one single colony. mcyE genes were not detected byPCR.

Conclusion: This approach allows to work with a small amount ofDNA, and represents a concrete answer to the lack of molecular dataon non-cultivable or difficult to isolate cyanobacteria.



P.090

HOW DOES CYANOBACTERIAL PHOTOSYNTHESIS CHANGEWITH SALINITY AND TEMPERATURE IN HYPERSALINE MICROBIALMATS LOCATED AT DIFFERENT INTERTIDAL POSITIONS?

Raeid M. M. Abed.

College of Science, Biology Department, Sultan Qaboos University, AlKhoud, Sultanate of Oman.

Intertidal cyanobacterial mats of the Arabian Gulf are exposed tocontinuous fluctuation in temperature and salinity, daily andseasonally. The temperature ranges between 18-55°C and salinityvaries between 4-25% depending on the mat’s tidal position. Usingmicrosensors, we studied the short-term temperature effects and theeffects of salinity fluctuation on gross photosynthesis (GP), netphotosynthesis (NP) and light respiration (LR) in three submerged matsfrom a transect on an intertidal flat. Areal rates of GP and NPincreased with temperature and maximum rates were detected at45°C. The photosynthetic zone decreased from 3mm to 1.75mm withincreasing temperature. Above 50°C, photosynthesis was completelyinhibited, probably due to high sulfide concentrations. The areal LRrates between 25°C and 45°C did not change significantly but showeda decreasing trend in the photosynthetic zone. This trend suggestedthat the coupling between photosynthesis and LR was apparentlybroken. The response of GP, NP and LR in lower, middle and uppertidal mats to various salinities (65, 100, 150 and 200‰) was compared.GP and LR at the ambient salinities of the mats decreased from thelower to the upper tidal zone. All mats, regardless of their tidallocation, exhibited a decrease in areal GP and LR rates at salinities>100‰. The extent of inhibition of these processes at higher salinitiessuggests an increase in salt adaptation of the mats microorganismswith distance from the low water line. We conclude that the resilienceof microbial mats towards different temperatures and salinity regimeson intertidal flats is accompanied by adjustment of the diversity andfunction of their microbial communities.

P.091

DISCOVERING THE METABOLIC BOTTLENECKS OF FUELPRODUCTION: LIQUID CHROMATOGRAPHY MASSSPECTROMETRY (LC-MS) ANALYSIS OF THE FERMENTATIVEMETABOLOME OF THE HYDROGEN-EVOLVINGCYANOBACTRIUM SYNECHOCOCCUS SP. PCC 7002.

Nick Bennette, Kelsey McNeely, John Eng, G. Charles Dismukes.

Princeton University, Princeton, NJ, USA.

Introduction: Many cyanobacteria possess the capability to evolvehydrogen fermentatively, deriving the requisite protons and electronsfrom water in a potential method of clean and renewable energyproduction. Unfortunately, much of the research regarding theproduction of hydrogen from cyanobacteria has dealt with internalcellular metabolism as a “black box” rather than a complex network ofindividual biochemical reactions in which electron and proton flux tohydrogen production may be bottlenecked at many different points. Inorder to resolve the internal cellular dynamics leading to hydrogenevolution, detection of the involved metabolite pools as well asresolution of their intervening fluxes is required. The objective of thisresearch is to quantify the time-dependent intracellular metabolicresponse of a model hydrogen-evolving cyanobacterium,Synechococcus Sp PCC 7002, to effectors of H2 rate and yield,specifically the effect of extracellular nitrate.

Methods: Genetic analysis of Synechococcus was performed using

the KEGG pathway database (www.genome.jp/kegg/pathway.html)and NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). Analysis of auto-fermentation of Synechococcus was conducted by re-suspension ofcells in the appropriate medium, sealing to light & air, purging withargon gas, and performing an optimized chemical extraction methodon samples in triplicate at selecting time points post-anaerobisis. Achemical assay for total internal reducing sugars was performed onremaining cell material, and extracellular nitrate and metabolic endproducts were measured by chemical assay and NMR of thefermentation medium, respectively. Analysis of metabolite extractswas conducted using reversed-phase ion-pairing chromatographycoupled directly to multiple-reaction monitoring (MRM) detectionusing an Agilent 6410 Triple Quadrupole mass spectrometer.

Results: A hypothetical fermentative reaction network was constructedfor Synechococcus based on whole genome information. An analysismethod for these individual metabolites was developed using onlinechromatography coupled to mass-spectrometry, in which compound-specific mass decomposition channels were optimized for each targetmetabolite and chromatography examined for optimal resolution andsensitivity of the suite of target compounds. Using this method, ananaerobic extraction protocol for Synechococcous samples employingfast cell filtration and cold chemical extraction was optimized for thetarget metabolome. This chemical/analytical procedure was appliedto monitor the metabolic response of Synechococcous during auto-fermentation. As predicted, direct LC-MS detection of pyridinenucleotides and adenosine phosphates indicate increased redox poiseand decreased cellular energy charge during fermentation. Thedifferential effect of nitrate availability on metabolite pools duringauto-fermentation was subsequently tested, and the roles of nitrogenas a both an electron sink (and competitor with hydrogenase) as wellas a precursor to biosynthesis examined.

Conclusions: A robust method for the metabolomic analysis ofcyanobacterial fermentation has been developed using the modelorganism Synechococcus sp. PCC 7002. This approach has beenemployed to quantitatively monitor the intracellular response of thisphototroph to auto-fermentative conditions, as well as the specificeffect of nitrate on hydrogen evolution and fermentative metabolism.

P.092

EXAMINATION OF ALLOPHYCOCYANIN AND PHYCOERYTHRINSUBUNIT BIOSYNTHESIS.

Avijit Biswas, Shervonda Williams, Monica Kronfel, Yasmin Vasquez,Richard Alvey, Donald Bryant, Wendy Schluchter.

Univeristy of New Orleans, New Orleans, Louisiana, USA.

Introduction: Cyanobacterial phycobilisomes are composed of thebrilliantly-colored, water-soluble, highly fluorescent phycobiliproteinsin addition to the linker proteins which hold the structure together.Each phycobiliprotein is composed of two subunits, α and β, and eachsubunit has between one and three linear tetrapyrrole prostheticgroup(s) called bilin(s) attached at Cys residues via a thioether bond.In cyanobacteria, there are four different naturally occurring bilins withthe most prevalent being phycocyanobilin (PCB) and phycoerythrobilin(PEB). For most phycobiliproteins, enzymes called bilin lyases areresponsible for catalyzing the attachment of the bilin to theappropriate Cys residue. This study determined the bilin lyasesinvolved in allophycocyanin subunit biosynthesis, evaluated thesubstrate specificity of two different types of bilin lyase; CpcE/F andCpcSU toward addition of PEB instead of PCB, and expressed putativebilin lyases to be tested for attachment of bilins to Phycoerythrin II(PEII) subunits.



Methods: An in vivo heterologous expression system using E. coli wasdeveloped by co-expression of genes encoding the phycobiliproteinsubstrate, the biosynthetic enzymes of the prosthetic group, and theenzymes which catalyze the attachment of bilin. The phycobiliproteinsubunit genes were fused downstream of a gene encoding a tag (e.g.Histidine tag or Glutathione S-transferase tag). The purified productswere evaluated by absorbance and fluorescence spectroscopy, andthe protein and bilin content were assessed after SDS-PAGE. Forsome experiments, in vitro assays were used to test activity where allrecombinant proteins were expressed separately.

Results: A multi-plasmid heterologous system was developed andused to create holo-allophycocyanin in E. coli cells. We also used thissystem to show that the heterodimeric CpcS/CpcU fromSynechococcus sp. PCC 7002 is required for attachment of PCB to theallophycocyanin subunits ApcF and ApcD. For the ApcE linker protein(LCM

99), we show that the amino-terminal allophycocyanin-like domainhas autocatalytic bilin lyase activity and is also similar in sequence toCpcS and CpcU proteins. We also show that the CpcEF bilin lyase canefficiently attach PEB to CpcA using the in vivo heterologous system,enabling us to create unique phycobiliproteins. The CpcSU bilin lyasewas not as efficient at attaching PEB over PCB to CpcB in this system.We have successfully overproduced soluble Synechococcus WH8020HT-MpeB (β-phycoerythrin II) and HT-MpeA (α-phycoerythrin II) by co-expression with the E. coli GroES chaperone. The putative bilin lyasesubunits MpeU, MpeV, MpeY, CpeU, and CpeS have beenoverproduced separately. Most of them are insoluble, but we areattempting to renature them from inclusion bodies. Results fromthese studies will be discussed.

Conclusions: This in vivo heterologous system was developed in orderto facilitate to test the activity of many different bilin lyases, to test theability of known bilin lyases to attach different chromophores to createunique phycobiliproteins, and to produce large amounts of holo-phycobiliproteins. For some bilin lyases that are insoluble in E. coli,we may be able to renature them and test for bilin lyase activity invitro.

P.093

GENETIC ANALYSIS OF CHLOROBACULUM TEPIDUM SULFURISLANDS.

Ernest O. Bonsu1,2, Lisa Waidner2, Thomas E. Hanson1,2,3 .

Department of Biological Sciences1, Delaware Biotechnology Institute2

and College of Marine and Earth Studies3, University of Delaware,Newark, DE, USA.

Introduction: Chlorobaculum tepidum is an anaerobic phototrophicbacterium that utilizes reduced sulfur compounds such as sulfide,elemental sulfur and thiosulfate as electron donors during anoxygenicphotosynthesis. Chlorobaculum tepidum is a model system for thestudy of anaerobic sulfur oxidation pathways as it grows rapidly, isgenetically amenable, and its genome has been completelysequenced. In the C. tepidum genome sequence many predictedsulfur oxidation genes are grouped in clusters called Sulfur Islands.The overarching goal of this project is to refine models of anaerobicsulfur oxidation in C. tepidum by the genetic analysis of the SulfurIslands. The analysis of two specific mutant strains will be discussed.

Methods: In-vitro transposon mutagenesis (IVTM) was used togenerate mutations in regions of the C. tepidum chromosomecontaining the sulfur islands. Mutant C. tepidum strains showingdistinct growth or sulfur oxidation phenotypes were then characterizedin detail to determine what genes were affected by the transposoninsertion. Pulsed field gel electrophoresis, PCR, RT-PCR, DNA

sequencing and rescue cloning of DNA flanking transposon insertionshave all been utilized to define the genotypes of interesting mutantstrains. Further characterization of the genes affected is oftennecessary by creating mutant strains carrying a single geneinactivation.

Results: Two strains are the focus of the current study. Mutant strainC5 was incapable of growth with thiosulfate as the sole electrondonor. The genotype of strain C5 (∆CT0867CT0876::TnOGm) iscomplex, a deletion-insertion event that eliminated all or part of ninegenes in a section of Sulfur Island I. By comparison of the deletedregion with other anaerobic sulfur oxidizers, gene CT0872 washypothesized to be responsible for the thiosulfate growth defect. Thephenotype of strain CT0872::TnOGm is consistent with this predictionwhile the inactivation of CT0873 and CT0874 had no significant effect.Strain C3 grows slowly under all conditions tested and accumulatesextremely high levels of extracellular sulfur globules during growth.PFGE data indicated that mutant strain C3 carries two TnOGminsertions localized in two distinct regions of the chromosome that areconsistent with insertions in Sulfur Islands I and II, both of which carrycopies of the dsr genes that are crucial for elemental sulfur oxidationin purple sulfur phototrophic bacteria.

Conclusion: Detailed analysis of genes deleted in strain C5 hasimplicated the CT0872 gene product in thiosulfate oxidation. Theidentification of genes inactivated in strain C3 will provide importantclues to the specific gene products required for elemental sulfuroxidation in C. tepidum.

P.094

THE STOMATIN, PROHIBITIN, FLOTILIN AND HFLK/C DOMAINHOMOLOGUE SLR1768 IS REQUIRED FOR THYLAKOIDBIOGENESIS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP.PCC 6803.

Samantha Bryan, Edward Spence, Conrad Mullineaux.

Queen Mary, University of London, London, United Kingdom.

Introduction: Nearly all cyanobacteria possess thylakoid membranes,the single known exception being the atypical cyanobacteriumGloeobacter, which houses all its photosynthetic complexes in theplasma membrane. Thylakoids are intricate internal membranesystems, where all the photosynthetic complexes that catalyse thelight induced oxidation of water to molecular oxygen are contained.Biogenesis and regulation of the thylakoids is still an open problem,with only two known regulators, Vipp1 and Alb3 indentified to date.

Methods: A slr1768 knockout was generated using the pGEM T-easyvector and REDIRECT technology. Whole cells were scanned forabsorption in a UV 500 spectrophotometer. Photochemical efficiencywas determined using 77K fluorescence emission spectra, andchlorophyll a fluorescence. Maximum photochemical efficiency wascalculated as (FM`dark – F0)/ FM`dark. Both wild-type Synechocystis sp. PCC6803 and ∆slr1768 strains were collected and fixed, ultrathin sectionswere then negative stained and viewed using a JEOL 1220transmission electron microscope.

Results: By comparing growth and the photosynthetic efficiency of the�slr1768 mutant with the wild-type, we found that �slr1768 has aconditional phenotype; specifically under highlight conditions (130µmol m-2 s-1) thylakoid biogenesis is disrupted leading to cell deathon a scale of days. The thylakoids show considerable disruption, withloss of both structure and density. The chlorophyll a pigment contentdecreases with the loss of thylakoids, although the photosyntheticefficiency is unaffected.



Conclusions: This is the first example of a gene that affects thylakoidbiogenesis with a phenotype conditional on light intensity. Our resultsdemonstrate that Slr768 has a leading role in acclimatisation, linkinglight damage with maintenance and biogenesis of the thylakoids.

P.095

LIGHT VARIABILITY ILLUMINATES NICHE-PARTITIONING AMONGMARINE PICOCYANOBACTERIA.

Douglas Campbell, Zoe Finkel, Andrew Irwin, Christophe Six.

Mount Allison University, Sackville, NB, Canada.

Prochlorococcus and Synechococcus picocyanobacteria are dominantcontributors to marine primary production over large areas of theocean. Phytoplankton cells are entrained in the water column and arethus often exposed to rapid changes in irradiance within the uppermixed layer of the ocean. An upward fluctuation in irradiance canresult in photosystem II photoinactivation exceeding counteractingrepair rates through protein turnover, thereby leading to netphotoinhibition of primary productivity, and potentially to cell death.We show that the effective cross-section for photosystem IIphotoinactivation is conserved across the picocyanobacteria, but thattheir photosystem II repair capacity and protein-specific photosystemII light capture are negatively correlated, and vary widely across thestrains. The differences in repair rate correspond to the light andnutrient conditions that characterize the site of origin of theProchlorococcus and Synechococcus isolates, and determine theupward fluctuation in irradiance they can tolerate, indicating thatphotoinhibition due to transient high-light exposure influences theirdistribution in the ocean.

P.096

CHROMATIC PHOTOACCLIMATION EXTENDS UTILISABLEPHOTOSYNTHETICALLY ACTIVE RADIATION IN THECHLOROPHYLL D-CONTAINING CYANOBACTERIUM,ACARYOCHLORIS MARINA.

Zane Duxbury, Martin Schliep, Raymond J. Ritchie, Anthony W. D.Larkum, Min Chen.

School of Biological Sciences, University of Sydney, Australia.

Chromatic photoacclimation and photosynthesis were examined intwo strains of Acaryochloris marina (MBIC11017 and CCMEE5410) andin Synechococcus PCC7942. Acaryochloris contains Chl d, which hasan absorption peak at ca 710 nm in vivo. Cultures were grown in oneof three wavelengths (525 nm, 625 nm and 720 nm) of light fromnarrow-band photodiodes to determine the effects on pigmentcomposition, growth rate and photosynthesis. No growth occurred in525 nm light. Synechococcus did not grow in 720 nm light becauseChl a does not absorb effectively at this long wavelength.Acaryochloris did grow in 720 nm light, although strain MBIC11017showed a decrease in phycobilins over time. Synechococcus andAcaryochloris MBIC11017 showed a dramatic increase in phycobilinswhen grown in 625 nm light. The various photopigments wereacclimated in response to the light spectral conditions.Photoacclimation and the Qy peak of Chl d can be understood interms of the ecological niche of Acaryochloris, which grows in habitatsenriched in near infra-red radiation.

P.097

ROLE OF RUBISCO AND RUBISCO LIKE PROTEINS IN SULFURMETABOLISM IN RHODOSPIRULLUM RUBRUM.

Swati Dey, Jaya Singh, F. Robert Tabita.

Departments of Microbiology, Environmental Sciences GraduateProgram and the Plant Cellular Molecular Biology, The Ohio StateUniversity, Columbus, Ohio, USA.

Introduction: With the discovery of the type IV RubisCOs or theRubisCO- like proteins (RLP), a new family of proteins was discoveredthat was found to be structurally similar to the large subunit ofRubisCO, though RLP was unable to catalyze CO2 fixation likeRubisCO. RLPs from organisms like Bacillus subtilis, Geobacilluskaustophilus and Microcystis aeruginosa have been shown to beinvolved in an enolase reaction of a methionine sulfur salvagepathway. We have been studying the functional role of RLP in variousorganisms such as Chlorobium tepidum, Rhodopseudomonaspalustris, and Rhodospriullum rubrum and its relation to RubisCO. Itwas shown that RLP from R. rubrum utilizes methylthioadenosine(MTA, an intermediate of known methionine salvage pathways) as solesulfur source. The RLP from R. rubrum catalyzes a novel isomerizationreaction distinct from that catalyzed by the B. subtilis-type RLP of themethionine salvage pathway. Interestingly R. rubrum RubisCO wasalso shown to weakly catalyze an enolase reaction similar to the RLP ofB. subtilis. In the present study, we found that R. rubrum RubisCO isinvolved in the sulfur salvage pathways under anaerobic conditionapart from its normal CO2 fixing ability.

Methods: Using MTA as a sole source of sulfur, growth experimentswere performed under both aerobic and anaerobic conditions toillustrate the function of RLP and the dual function of RubisCO in R.rubrum after inactivation of the RLP gene. Complementation studieswere used to confirm these results. In addition, RubisCO activityassays and western immunoblots also confirmed the presence ofRubisCO in mutant strains lacking RLP that were capable ofsimultaneously fixing CO2 and using MTA as a sole source of sulfur.

Results: Studies show that R. rubrum strains with an inactivated RLPgene were able to grow with MTA as a sole source of sulfur underanaerobic conditions. R. rubrum RLP gene deletion strains wereunable to grow under aerobic conditions. Complementation of RLPgene supported aerobic growth in these strains. Western blotsshowed the expression of RLP under aerobic conditions and RubisCOunder anaerobic conditions in various mutant strains.

Conclusion: These studies showed that under aerobic growthconditions RLP is involved in the salvage of sulfur in R. rubrum. Adifferent and novel substrate is utilized by RLP under aerobic growthconditions when MTA is used as a sulfur source for growth. However,under anaerobic conditions, using MTA as sole sulfur source, it is notRLP, but RubisCO that catalyzes a B. subtilis type reaction using 2,3-diketo-5-methylthiopentanyl -1- phosphate as substrate. In addition,this work illustrates the dual role of RubisCO as an enzyme requiredfor CO2 fixation as well as sulfur salvage. Hence, the RubisCO familynot only is involved in carbon metabolism but this enzyme may beused simultaneously to catalyze an important reaction of sulfurmetabolism in vivo.



P.098

CHARACTERIZATION OF BILIN LYASES FOR R-PHYCOCYANIN INSYNECHOCOCCUS SP. WH8020

Tierna Dragomani, Avijit Biswas, Wendy M. Schluchter.

Department of Biological Sciences, University of New Orleans, NewOrleans, LA, USA.

Introduction: Cyanobacteria utilize phycobilisomes composedprimarily of phycobiliproteins to maximize capture of light energy. Inmarine Synechococcus, various strains exhibit distinctivephycobiliprotein compositions adapted to capitalize on thewavelengths of available light. Specific attachment of phycobilinsendows each phycobiliprotein with characteristic capacities for lightabsorption and emission. Rarely, this attachment may be autocatalytic,but more often it is catalyzed by bilin lyases that attach phycobilins atconserved cysteine residues on the phycobiliprotein subunit. The E/Ftype bilin lyase was the first one characterized; each CpcE and CpcFsubunit contains conserved “E-Z” motifs, and together, theheterodimer catalyzes bilin attachment at Cys-84 of the α-subunit ofthe phycobiliprotein, phycocyanin (PC). The rpcE and rpcF genes aresimilar to cpcE and cpcF, respectively and are located downstream ofthe rpcA gene in Synechococcus sp. WH8020. Recently, RpcG fromSynechococcus sp. WH8102, a fusion of RpcE and RpcF, was shown tohave phycoerythrobilin (PEB) lyase and isomerase activity (Blot et al.,2009; J. Biol. Chem. 284: 9290-9298). The purpose of this study is totest whether rpcE and rpcF in strain WH8020 encode an E/Fheterodimeric lyase that catalyzes attachment of PEB to Cys-84 of α-R-phycocyanin (R-PC).

Methods: In Synechococcus sp. WH8020, the cpe/ mpe operonsencoding phycoerythrin and R-Phycocyanin genes have beensequenced (Wilbanks and Glazer, 1993; J. Biol. Chem. 268:1226-1235). Oligonucleotide primers were developed to amplify the rpcAgene (α-R-PC) from WH8020 chromosomal DNA, and this product wasligated into the pET Duet vector so that its sequence was fused to thereading frame encoding a Hexa-histidine tag. The rpcE and rpcFgenes were cloned into the pCDF Duet vector (individually andtogether). A pACYC Duet plasmid contained genes (ho1, pebS)encoding enzymes known to convert heme to biliverdin and ultimatelyto PEB (generous gift of Dr. Frankenberg-Dinkel). Plasmids will beintroduced into chemically competent E. coli strain BL21(DE3) andplated onto Luria-Brutani agar containing the appropriate antibiotics.Transformants will be cultured and protein production will be inducedwith IPTG. Each plasmid will be tested individually in cells for proteinproduction to determine optimal growth temperature conditions foreach set of proteins.

Results: The rpcA plasmid construct resulted in the production ofsoluble His-tagged RpcA which was purified by metal affinitychromatography and verified by SDS-PAGE. We have successfullydemonstrated that CpcE and CpcF can attach PEB to HT-RpcA.However, we are currently testing conditions for expression of solubleRpcE and RpcF. Results from these studies will be presented. Inaddition, we will test whether RpcE and RpcF form a heterodimer.

Conclusions: Soluble expression of RpcA was achieved, and it was asuitable substrate for bilin addition by another closely related bilinlyase CpcE/CpcF (from Synechocystis sp. PCC 6803). Assays withRpcA, RpcE and RpcF will be performed if soluble protein can beobtained.

P.099

MOLECULAR COMMUNICATION BETWEEN CYANOBACTERIAAND THEIR HOST PLANTS: ROLE OF CHEMOTAXIS IN SYMBIOTICCOMPETENCY.

Paula Duggan, Dave Adams.

Faculty of Biological Sciences, Leeds, Yorkshire, United Kingdom.

Introduction: Cyanobacteria, such as Nostoc punctiforme, are able tolive in symbiotic association with a wide range of host plants.Prerequisites for successful infection of the host tissues include theformation of motile filaments known as hormogonia and theproduction of a plant-derived chemical(s) that attracts these filamentstowards the sites of infection within the host plant. There is evidenceof two-way communication between plants and their prokaryoticpartners regulating the infection process and the events involved inthe establishment of a functional dintrogen-fixing association.Moreover there is increasing evidence to suggest that chemotaxis-likesignal transduction systems may be central to these processes. Thegenome of N. punctiforme contains five loci of genes encodingelements resembling chemotaxis genes of other bacteria. We havefocused our attention on one locus, which appears to have beenacquired from a horizontal transfer event and is unique amongst theother clusters in that it contains a putative CheR methyltransferase andCheB methylesterase, which are central to chemotactic adaptation inother bacterial systems and enable the bacterium to respond to newstimuli in a background containing constant levels of chemoattractantsand/or chemorepellents.

Methods : Nostoc genes NpR0244 (cheB) and NpR0248 (cheR) wereinactivated by insertional mutagenesis and recombinant strains werecharacterised in terms of chemotactic motility and their ability to infectthe host bryophyte Blasia pusilla. Electron microscopy was used toexamine the levels of cell surface piliation of hormogoniadifferentiated by the mutant strains compared with those of the wild-type.

Results: Both the NpR0244 (cheB) and NpR0248 (cheR) mutants failedto infect the host plant even after protracted periods of co-culture withthe host B. pusilla. Chemotaxis assays revealed that the mutant strainslack the wild-type positive chemotactic behaviour towards exudatesreleased from nitrogen-starved B. pusilla tissue. Wild-type piliationphenotypes were observed with both the cheB-like NpR0244 and thecheR-like NpR0248 mutants implying that, unlike some other bacterialtaxis systems, these genes are not involved in the biosynthesis of thepilus-like appendages present on the surface of hormogonia.

Conclusions: Our data provide molecular evidence that thechemotaxis-like elements studied here function in the establishment ofthe Nostoc-Blasia symbiosis, most probably through the disruption ofthe normal chemotaxis responses that occur towards host-derivedsignals and that cyanobacteria, like other bacteria, also exhibit anadaptation response.



P.100

RESPONSES OF CHLOROBACULUM TEPIDUM TO ELEVATEDSULFIDE.

Brian J. Eddie, Leong-Keat Chan and Thomas E. Hanson.

College of Marine and Earth Studies and DBI, Newark, DE, USA.

Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is agreen sulfur bacterium (GSB) that has become a model system forphototrophic sulfur oxidation due to the relative ease of culture andgenetic manipulation. While it preferentially uses sulfide as anelectron donor, C. tepidum is inhibited by sulfide concentrations inexcess of 8 mM, a much higher sulfide tolerance than originallyreported for this strain. To understand mechanisms of sulfidetolerance, we seek to identify sulfide regulated genes and assess theirrole(s) in C. tepidum‘s physiology

Methods: C. tepidum was grown either on thiosulfate as the soleelectron donor or with varying concentrations of sulfide followed by acomparison of cellular properties and the proteome of cultures by 1DSDS-PAGE. In separate experiments, C. tepidum actively growing onthiosulfate as the sole electron donor was provided with varying dosesof sulfide and the adaptation of the cultures to this shock wasmonitored by quantifying sulfur compound turnover, cellularproperties and changes in the proteome. In addition, samples weretaken for subsequent analysis by a modified version of the mRNA-Seqprotocol normally applied to larger sized eukaryotic genomes. Thisprotocol has been developed to allow inexpensive multiplexing ofsamples from prokaryotic genomes to scale the power of nextgeneration sequencing techniques to smaller genome sizes.

Results: C. tepidum cultures grown with varying sulfide concentrationsshowed a clear alteration in the major in vivo absorption peak of Bchlc and several polypeptides were significantly more abundant in highsulfide cultures. A high concentration sulfide shock (8 mM) resulted incomplete growth inhibition, but it is not known whether this was dueto sulfide poisoning or pH inhibition. Milder doses of neutralizedsulfide (1.6mM) did not result in growth inhibition or cause significantalterations in the protein profile of the culture. Two independentsamples from this experiment were analyzed by the mRNA-seqprotocol and produced millions of sequences that could be aligned tothe C. tepidum TLS genome. Analysis of the reproducibility of themodified mRNA-seq protocol and multiplexing technique will bepresented as will cell property and protein data from additional sulfideshock experiments.

Conclusions: This study is the first to investigate the response of C.tepidum TLS to sulfide and is one of the first to use mRNA-seq toinvestigate a prokaryotic transcriptome. Preliminary results indicatethat the modified mRNA-seq protocol applied to C. tepidum identifiesactively transcribed regions of the genome in a less annotationdependent fashion than can be achieved with current microarraytechnology. This included the identification of non-coding RNA’s,which are receiving increased attention as regulatory elements inprokaryotes.

P.101

IN VITRO ASSEMBLY OF THE CARBOXYSOMAL BICARBONATEDEHYDRATION COMPLEX.

Charlotte De Araujo, Swan Cot, George Espie.

University of Toronto, Mississauga, ON, Canada.

Introduction: Cyanobacteria possess a carbon dioxide concentrating

mechanism (CCM) that greatly enhances photosynthetic efficiency.This adaptive mechanism minimally consists of multiple membranetransport systems for inorganic carbon and intracellular inclusionsknown as carboxysomes, where Rubisco is located. The structural andfunctional basis through which carboxysomes enhance CO2 fixationremains unclear. Based on yeast two-hybrid analysis Cot et al. (J.Bacteriol. 2008,190: 936) hypothesized that the proteins, CcaA, CcmMand CcmN interact to form a multiprotein bicarbonate dehydrationcomplex (BDC) within the carboxysome which vectorially channels CO2

to Rubisco, thereby enhancing fixation.

Methods: ccaA, ccmM and ccmN (slr1347, sll1031 & slr1032) fromSynechocystis sp. PCC6803 were cloned and over-expressed usingstandard techniques. Carbonic anhydrase activity was determined bymass spectrometry.

Results: Mass spectrometric assays revealed that neither recombinantCcmM nor CcmN possessed HCO3

- dehydration activity alone. Over-expression of CcaA using the pET-15b E.coli expression systemproduced active protein capable of the catalytic interconversion ofHCO3

- and CO2. To examine the role of the BDC in the supply of CO2,attempts were made to reconstitute it in vitro using affinity purificationtechniques. We found that N-terminal 6xHis-tag-CcaA retainedcatalytic activity when bound alone to an affinity matrix. In contrast, acomplex of matrix-bound T7-tag-CcmM + 6x-His-CcaA wascatalytically inactive as was the T7-tag-CcmM + flag-CcmN + 6x-His-CcaA. Western blot analysis verified the presence of all 3 proteinswithin the affinity matrix. However, matrix-bound 6x His-CcaA + T7-CcmM complexes and His-CcaA + T7-CcmM + flag-CcmN exhibitedCA activity, suggesting that the orientation of CcaA was an importantdeterminant in maintaining catalytic activity. In buffered solution at pH8, CcaA activity was reduced by 20 % in the presence of CcmM, butnot BSA.

Conclusions: Initial results show that CcmM-CcmN-CcaA complexesare formed by affinity tag binding to a matrix and subsequentprotein/protein interactions. The orientations of the proteins inrelationship to one another are critical in maintaining CA activity.When the CcaA active site at the N-terminus of the protein is exposedto CcmM, activity was eliminated, suggesting that the binding ofCcmM modulates CcaA activity. Potentially, small differences in thenature of the CcmM – CcaA interaction within carboxysomes may playa role in controlling the supply of HCO3

- to CcaA and in regulatingCcaA dehydration activity.

P.102

THE CYANOBACTERIAL RIBOSOMAL-ASSOCIATED PROTEIN LrtAMODULATES TRANSLATION IN SYNECHOCYSTIS SP. PCC 6803.

Carla V. Galmozzi, Francisco J. Florencio Bellido, M. Isabel MuroPastor.

Instituto De Bioquímica Vegetal Y Fotosíntesis. Universidad De Sevilla-Csic, Seville, Seville, Spain.

LrtA is a protein encoded by a light-repressed transcript incyanobacteria. The gene coding for LrtA was identified inSynechococcus sp. PCC 7002 and showed expression dependent ondarkness (Samartzidou y Widger, 1998; Tan et al., 1994). LrtA is relatedto a family of proteins present in several bacteria. In Escherichia colitwo proteins belonging to this family, termed YfiA and YhbH, havebeen shown to be associated with the 30S ribosome subunit(Agafonov et al., 1999; Maki et al., 2000). YfiA has also been shown toinhibit translation in vitro (Agafonov et al., 2001) suggesting a role ofthis family of proteins in translation control related to stressadaptation. However, in vivo data that demonstrate a function of these



proteins in translation have not yet been reported. The lrtA geneproduct also displays sequence similarity to a spinach plastid-specificribosomal protein (PSRP-1), which is present in the chloroplast stromaeither unbound or associated with the 30S ribosomal subunit(Yamaguchi et al., 2000). Genes homologous to lrtA are present in allthe cyanobacterial sequenced genomes, but their function remainsunknown. The analysis of the Synechocystis sp. PCC 6803 genomeshowed the existence of an open reading frame (sll0947), the productof which displayed strong similarity to the previously identifiedSynechococcus sp. LrtA protein. In Synechocystis it has been shownthat the amount of SigB sigma factor increased in darkness andcontributes significantly to the up-regulation of lrtA in these conditions(Imamura et al., 2003). In addition, proteomic analysis of salt-stressproteins in the plasma membrane of Synechocystis resulted in theidentification of LrtA protein induced under these conditions (Huanget al., 2006).

We analyzed the transcript levels of the Synechocystis lrtA gene andthe LrtA protein. In agreement with the Synechococcus homologousgene, the level of the Synechocystis lrtA transcript is up-regulatedunder dark conditions. However, the level of LrtA protein remainsconstant upon light-dark transitions. Using extracts from Synechocystisfractionated by sucrose gradient we show that LrtA is a ribosomeassociated protein present in 30S and 70S ribosomal particles,suggesting a role of this protein in translation control. We haveconstructed an lrtA-deleted Synechocystis strain, which was shown tobe viable in laboratory conditions, indicating that lrtA is a dispensablegene in this cyanobacterium. Furthermore, lrtA mutants presentdecreased sensitivity to tylosin and erythromycin, two inhibitors ofprotein synthesis, again connecting the role of LrtA with translation. Inaddition, we analyzed in vivo incorporation of [35S]methionine intoproteins in both wild-type and ∆lrtA strains. We found that at least twoproteins were diferentially synthesized in WT versus ∆lrtA cells. We willcarry out proteomic analysis comparing wild-type and ∆lrtA strains, indifferent culture conditions or stresses, to identify changes provokedby the absence of the LrtA protein. We expect to identify proteinswhose synthesis is in some way regulated by LrtA. These results mayallow us to establish a role of this protein in translational adaptation.

References:

Agafonov, D.E., Kolb, V.A., Nazimov, I.V., y Spirin, A.S. (1999) Aprotein residing at the subunit interface of the bacterial ribosome.Proc Natl Acad Sci U S A 96: 12345-12349.

Agafonov, D.E., Kolb, V.A., y Spirin, A.S. (2001) Ribosome-associated protein that inhibits translation at the aminoacyl-tRNAbinding stage. EMBO Rep 2: 399-402.

Huang, F., Fulda, S., Hagemann, M., y Norling, B. (2006) Proteomicscreening of salt-stress-induced changes in plasma membranes ofSynechocystis sp. strain PCC 6803. Proteomics 6: 910-920.

Imamura, S., Asayama, M., Takahashi, H., Tanaka, K., Takahashi, H.,y Shirai, M. (2003) Antagonistic dark/light-induced SigB/SigD, group2 sigma factors, expression through redox potential and their roles incyanobacteria. FEBS Lett 554: 357-362.

Maki, Y., Yoshida, H., y Wada, A. (2000) Two proteins, YfiA and YhbH,associated with resting ribosomes in stationary phase Escherichia coli.Genes Cells 5: 965-974.

Samartzidou, H., y Widger, W.R. (1998) Transcriptional andposttranscriptional control of mRNA from lrtA, a light-repressedtranscript in Synechococcus sp. PCC 7002. Plant Physiol 117: 225-234.

Tan, X., Varughese, M., y Widger, W.R. (1994) A light-repressedtranscript found in Synechococcus PCC 7002 is similar to achloroplast-specific small subunit ribosomal protein and to a

transcription modulator protein associated with sigma 54. J Biol Chem269: 20905-20912.

Yamaguchi, K., von Knoblauch, K., y Subramanian, A.R. (2000) Theplastid ribosomal proteins. Identification of all the proteins in the 30 Ssubunit of an organelle ribosome (chloroplast). J Biol Chem 275:28455-28465.

P.103

INVESTIGATING THE MOLECULAR BASIS FOR CYANOBACTERIALEXCAVATION IN A BORING CYANOBACTERIUM(MASTIGOCOLEUS/FISCHERELLA).

Q. Gao, E. Ramirez-Reinat, F. Garcia-Pichel.

School of Life Sciences, Arizona State University, Tempe, AZ, USA.

Introduction: Microorganisms are the most common, widespread andenvironmentally significant among carbonate borers. Boringcyanobacteria, as photosynthetic microbes, have been a part of thegeological reworking of carbonates since at least 1500 Myr ago. Theiraction has played important roles in geological cycles, and they areimportant to the fisheries industry, as they can be a pest of mullusks.Despite this, the boring mechanism of any of these cyanobacteriaremains unknown. Concurrent physiological experiments in ourlaboratory have recently shown that active pumping of calcium ions(through cellular uptake, trans-cellular transport and extrusionprocesses) is at the base of the boring ability, likely involving P-typeATPases. To rigorously identify the putative cellular mechanism for thisunique phenomenon, we attempted to find genes encoding forcalcium-transporting P-type ATPases in a model cyanobacterium(Mastigocoleus/ Fischerella), so that expression analyses could becarried out. Our study provides the first glance at the molecularmechanism for microbial excavation.

Methods: The genes sequences for calcium related P-type ATPasesfrom a variety of cyanobacteria were retrieved from the NCBIdatabase and Cyanobase. 40 such putative calcium-related P-typeATPase genes were identified as having both a calcium binding motifand an ATP-binding motif, and classified into two groups according toindependently obtained consensus phylogeny of the cyanobacteriafrom which the genes were obtained. Group-specific sequencealignments were used to design degenerate primers matchingconserved sequence regions corresponding to the phosphorylatingand ATP-binding motifs. These primers were used to clone calcium-transporting P-type ATPases from Fischerella/Mastigocoleus genomethrough PCR amplification of genomic DNA. The Purified PCRproducts were cloned into pCR®4 vector through invitrogen TOPOTA Cloning system. Six individual clones were sequenced to verifyeach PCR product. The corresponding, deduced protein sequenceswere compared with known proteins in the databases by availableblast methods.

Results: Using genomic DNA from two known calcium P-type ATPase-containing strains, Synechocystis sp. PCC 6803 and Nostocpunctiforme, we could show that our primers amplify P-type ATPasesfrom cyanobacteria, and were specific for P-type ATPase genes, sincethey yielded a single product of the predicted size. Using genomicDNA of boring Mastigocoleus/Fischerella as template, each primerpair yielded product of the expected size, about 155 bp and 1098 bp,resepctively. Cloning and sequencing of the two products, provedthat they belonged to two distinct P-type ATPase genes, oneresembling most that of Nostoc punctiforme and another one mostclosey related to one from Synechocystis sp. The sequence analysis ofthe two PCR products indicates there exist at least two putativecalcium-binding P-type ATPases in Mastigocladus/Fischerella genome.



Conclusions: Our experiments confirmed the feasibility of thegenomic scanning by PCR method in the detection of calcium relatedP-type ATPases genes. This result, more importantly, providesmolecular evidence for the existence of multiple putative calciumtransporter genes in Fischerella, which could support our proposedmechanism for boring activity. Although this experiment is a first stepin our exploration, cloning of calcium P-type ATPase paves the way forthe further investigation of the molecular mechanism of cyanobacterialexcavation. Ongoing studies attempt to address the expression ofeach of these genes under boring vs. free-living conditions, and thelocalization of this activity in these morphologically complexcyanobacteria.

P.104

THE GLOBAL RESPONSE TO UVA STRESS IN NOSTOCPUNCTIFORME ATCC 29133 ASSESSED BY A TEMPORAL DNAMICROARRAY STUDY.

F. Garcia-Pichel1, Q. Gao1, J. C. Meeks2, V. Stout1, T. Soule1,3.1School of Life Sciences, Arizona State University, Tempe, AZ; 2Sectionof Microbiology, University of California, Davis, CA; 3EnvironmentalBiotechnology, Savannah River National Laboratory, Aiken, SC; USA.

Introduction: UVA radiation (320-400 nm) is an important componentof the solar spectrum reaching the ground. Unlike short-wavelengthultraviolet, UVA does not directly damage biological materials. Itsharmful effects come about through photosensitizers, molecules thatcreate reactive compounds upon photons absorption and in thepresence of oxygen. Cyanobacteria, because they contain high levelof photosensitizing pigments, and are exposed to internally generatedoxygen, are particularly prone to UVA-mediated damage. Despite this,gene expression studies have focused in the response to UVA havenot been presented. To identify some of the cellular mechanisms usedto cope with this part of solar radiation, we examined the temporalglobal stress response to an up-shift in UVA the cyanobacteriumNostoc punctiforme ATCC 29133 as a model organism using wholegenome DNA microarrays. Our study provides the first global UVAstress response analysis for any phototroph.

Methods: Nostoc cultures grown under white light were split into 2series, one kept under white light, and the second exposed tosupplemental UVA radiation, over the course of four days. At 24 hrintervals, triplicate cultures were harvested from each treatment. TotalRNA was extracted, reverse-transcribed, and labeled with fluorescentdyes. Labeled cDNAs were hybridized to microarray slides and rawimages were processed for analysis. Genes with at least a statisticallysignificant 2-fold differential expression level between the UVA-treated and untreated samples were considered responsive to UVAand were used to construct gene lists for analysis.

Results: The study detected statistically robust signals for differentialregulation in response to UVA in 573 out of the 6903 genes probed onthe array. Most of the up-regulated genes, 299, were uncategorized,and 8% (82) of all of the genes known to be dedicated to adaptivemetabolism were upregulated. The most common category of down-regulated genes was those associated with light-harvesting pigmentbiosynthesis, likely to minimize photosensitizer density. Among theexpected responses, we could detect the up-regulation of severalantioxidant enzymes, likely to help the cells cope with reactive oxygenspecies, and the up-regulation of genes for the biosynthetic pathwayof the UVA sunscreen, scytonemin. Most intriguingly, we found that byfar, the strongest and most sustained up-regulation involved a groupof contiguous genes of unassigned metabolism, but widely distributedamong cyanobacteria, that on plasmid A.

Conclusions: Our experiments confirmed the importance of severalwell known adaptive strategies to cope with UV: decrease inphotosensitizers, increase in antioxidant systems, and synthesis of UVAsunscreens. It also revealed that we know virtually nothing about thevast majority of genes involved in this response. Overall, our resultssuggest that UVA plays an important role in the physiology of Nostoc,but also that the UVA cyanobacterial stress response is obviously farfrom being well understood. Some of the genes identified hereprovide new avenues for exploration.

P.105

THE BRANCHPOINT FOR BACTERIOPHEOPHYTIN BIOSYNTHESISIN RHODOSPIRILLUM RUBRUM.

Robin Ghosh1, Hartmut Grammel2, Jörg Hammel1, Rudolf Saegesser1,and Khaled Abou-Aisha1.1Dept. of Bioenergetics, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany, 2Max-Planck Institute for the Dynamics ofComplex Technical Systems, Magdeburg, Germany.

Introduction: The pathway for bacteriochlorophyll a (BChla)biosynthesis has now been well-characterized in purple photosyntheticbacteria, particularly in the species Rhodobacter capsulatus andRhodobacter sphaeroides. By contrast, the biosynthesis ofbacteriopheophytin a (BPha) appears to have been largely ignored. InR. rubrum, the chemical structure of BPha is useful for tracing thepathway of biosynthesis, as BChla contains a geranyl-geranyl chain,wheras BPha contains a phytyl chain. In this study, by a combination oforganic separation and spectroscopy we have deduced the probablybranch point of BPha biosynthesis from the main BChla pathway.

Methods and Results: A number of photosynthetically incompetentmutants in genes for pigment synthesis in R. rubrum were generatedby both random as well as Tn5 mutagenesis. The lesions wereidentified by both Southern hybridization as well as complementationanalysis. Cells were grown semi-aerobically in the dark using a uniquemedium (M2SF) which allows up-regulation of photosynthetic genes tolevels normally only observed under low light photoheterotrophicconditions. Growth in this medium causes R. rubrum to produce verylarge quantities of pigments, thereby simplifying subsequent chemicalanalysis. The pigments were separated in organic solvents andanalyzed by absorption, fluorescence and mass spectroscopy. Thisanalysis allowed us to deduce the probable locus of the BPha shunt.

Conclusions: The removal of Mg2+ from the BChla-precursor occurs atan unexpected locus.

P.106

HGDD, A TOLC-LIKE OMF INVOLVED IN HETEROCYSTDEVELOPMENT.

Alexander Hahn, Peter Staron, Iris Maldener, Enrico Schleiff.

Goethe Universitat, Cluster of Excellence, Center of MembraneProteomics, Frankfurt, Hessen, Germany.

Protein secretion of the type I system and multi-drug efflux in Gram-negative bacteria are based on a tripartite secretion complex. Thiscomplex usually contains an inner membrane ABC-transporter or aproton driven efflux pump, a membrane fusion protein (MFP) and anouter membrane factor (OMF) of the TolC-family. Together they form asingle channel tunnel to secret proteins and toxic substances from thecytoplasm or the inter membrane space. The cyanobacteriumAnabaena sp. PCC 7120 has only one TolC–like protein. We identifiedits expression in all cells of the filament, but it was found to be



indispensable only for heterocyst development. The phenotype of thedeletion strain parallels that of the knock out of the devBCA operonencoding for an ABC-transporter. Hence, it is concluded that DevBCAtogether with the identified TolC-like protein is involved in theassembly of the heterocyst specific glycolipid-layer (HGL-layer) and istherefore termed HgdD. As HgdD is the only TolC present in the outermembrane of Anabaena sp. PCC 7120 an interaction with themultitude of inner membrane located ABC-transporter is expected, ahypothesis which is currently investigated.

P.107

CYANOBACTERIOCHROME CCAS REGULATES PHYCOERYTHRINACCUMULATION IN NOSTOC PUNCTIFORME THAT PERFORMSGROUP II CHROMATIC ADAPTATION.

Yuu Hirose1, Mitsunori Katayama2, Rei Narikawa3,Masahiko Ikeuchi1,3.1Department of Biological Science, Graduate School of Sciences, theUniversity of Tokyo; 2College of Industrial Technology, NihonUniversity; 3Department of Life Sciences (Biology), the University ofTokyo; Japan.

Introduction: Cyanobacteriochromes are unique phytochrome-relatedphotoreceptors that undergoes reversible conversion betweenviolet/yellow, blue/green or green/red absorbing forms withphotoisomerization of a covalently bound linear tetrapyrrole (Ikeuchiand Ishizuka, 2008). Cyanobacteria utilize phycobilisome (PBS) as lightharvesting antenna. Typical light harvesting proteins of PBS arephycoerythrin (PE) and phycocyanin (PC), which absorb green light(GL) and red light (RL), respectively. Certain cyanobacterial species areable to raise PE/PC ratio under GL and reduce under RL, which hasbeen called “complementary chromatic adaptation”. Some acclimateonly PE accumulation (group II) while the others acclimate both PE andPC accumulation (group III). In Fremyella diplosiphon that performsgroup III chromatic adaptation, a cyanobacteriochrome gene, rcaE, issuggested to induce the expression of PC genes under RL, althoughanother GL receptor was implicated to regulate the expression of PEgenes (Kehoe and Gutu, 2006). Our recent studies suggested thatanother cyanobacteriochrome CcaS regulates the expression of PBSlinker gene, cpcG2, in Synechocystis sp. PCC 6803 that possesses PCbut not PE. CcaS shows reversible photoconversion between GL andRL-absorbing forms and GL-activated phosphorylation to the cognateresponse regulator CcaR (Hirose et al, 2008). Interestingly, CcaS andCcaR orthologs are clustered with PE genes (cpeC, cpcG2 and cpeR)in the genome of Nostoc punctiforme ATCC 29133 that performsgroup II chromatic adaptation. In this study, we disrupted ccaS, ccaRand the clustered PE genes in N. punctiforme and examined theirresponse to GL and RL.

Material and methods: N. punctiforme cells were grown in liquidBG11 medium under 20 μE m-2s-1 of GL (peaked at 530 nm) or RL (650nm). Absorption spectra of fully acclimated cells were measured byspectrophotometer with an end-on photomultiplier. To create ccaS,ccaR and PE gene mutants, each gene was replaced with a neomycincassette from pSCR9 as described (Cohen and Meeks, 1997). RNA wasextracted by acidic phenol. Northern blotting analysis was performedaccording to standard protocol using radio-labeled probes.

Results: We found that PE was hardly detected in wild type cellsgrown under RL, while PE content was high in cells grown under GL. Ina ∆ccaS mutant, some amounts of PE accumulated under both GL andRL, suggesting that ccaS is essential not only for PE accumulationunder GL but also for repression under RL. In a ∆ccaR mutant, PEhardly accumulated under both GL and RL, suggesting that CcaR istranscriptional activator for PE accumulation. Northern blotting

analysis indicated that the clustered PE genes, cpeC, cpcG2, andcpeR, are co-transcribed under GL but not RL in wild type.∆cpeC/∆cpeG2/∆cpeR triple mutant was also defective in PEaccumulation under both GL and RL, suggesting that the induction ofthese genes are essential for PE accumulation. From these results, weconclude that CcaS regulates PE accumulation by the transcriptionalregulation of cpeC, cpcG2, and cpeR, via GL-activatedphosphorylation and probably RL-activated dephosphorylation ofCcaR.

Conclusion: In complementary chromatic adaptation, the green lightreceptor for PE accumulation has not yet been identified. In this study,we found that cyanobacteriochrome CcaS and response regulatorCcaR regulates PE accumulation via the transcriptional regulation ofthe clustered PE genes in N. punctiforme.

P.108

CIRCADIAN TRANSCRIPTIONAL REGULATION IN THE DARKWITHOUT CYCLIC KAI GENE EXPRESSION IN SYNECHOCOCCUS.

N. Hosokawa1, T. Hatakeyama1, H. Iwasaki1,2.1Department of Electrical Engineering and Bioscience, WasedaUniversity; 2PRESTO, Japan Science and Technology Agency,Wakamatsu, Shinjuku, Tokyo; Japan.

Most of organisms exhibit daily cycles, driven by endogenouscircadian clocks. The unicellular cyanobacterium, Synechococcuselongatus PCC7942 is an obligate photoautotroph and known as thesimplest model organism for circadian biology. In S.elongatus, most ofgenes exhibit circadian expression rhythms, which are regulated bythree clock genes, kaiA, kaiB and kaiC, under continuous light (LL)conditions. Interestingly, when cells are transferred to continuous dark(DD) conditions from hour 12 in the light, kaiA and kaiBC genes arerapidly downregulated to the zero level, while the KaiCphosphorylation cycle persists in the dark even in the presence ofexcess transcription/translation inhibitors. Thus, the basic oscillation isgenerated via post-translational process (Tomita et al., 2005).

When we performed DNA microarray analysis, expression of most ofgenes on the genome was also dramatically and rapidly suppressed inthe dark, whereas a minor subset of genes was upregulated. Thesedark-induced genes required de novo transcription under DD. Then,we examined if such gene expression profiles under DD were affectedby the Kai-based clock even in the absence of de novo kai-geneexpression. We found the magnitude of dark-induction in some geneswas dependent on time when the cells were transferred from light toDD. Moreover, expression profiles of such genes in DD weredramatically altered in the kaiABC-null mutant strain. Thus, in contrastto our previous model, the Synechococcus clock regulatestranscriptional outputs even in the dark.

P.109

CHARACTERIZATION OF UNIQUE PHOTOCHEMICAL PROPERTIESOF CYANOBACTERIOCHROME TePixJ.

T. Ishizuka1, A. Kamiya2, H. Suzuki3, T. Noguchi3, T. Kohchi4, K.Inomata2, M. Ikeuchi1.1Department of Life Sciences (Biology), The university of Tokyo;2Division of Material Sciences, Graduate School of Natural Scienceand Technology, Kanazawa University; 3Institute of Materials Science,University of Tsukuba; 4Graduate school of Biostudies, KyotoUniversity; Japan.

Introduction:Cyanobacteria harbor many putative GAF-containing



photoreceptors that may bind a linear tetrapyrrole as a chromophore(cyanobacteriochromes) in addition to typical phytochromes. One ofthem, TePixJ of Thermosynechococcus elongatus BP-1 is essential forphototaxis. Previously, we reported novel properties of the GAFdomain of TePixJ (denoted TePixJ_GAF) that was expressed inSynechocystis PCC 6803. Purified TePixJ_GAF showed reversiblephotoconversion between the 433nm (Pb form) and 531nm-absorbingforms (Pg form). In contrast with Pr and Pfr forms of Synechocystisphytochrome Cph1 carrying phycocyanobilin (PCB), Pb and Pg ofTePixJ_GAF are extremely blue-shifted (approximately 150~200nm).Here, we studied the unique photochemical properties of TePixJ.

Methods: in vitro reconstitution: synthetic PCB was added to areconstitution buffer containing TePixJ_GAF apoprotein prepared fromE. coli. The mixture was incubated in the dark at 50ºC, which is closeto the optimum growth temperature of T. elongatus. in vivo co-expression: TePixJ_GAF apoprotein and two enzymes (Ho1, PcyA),which synthesize PCB from heme, were co-expressed in E. coli cells toallow in vivo assembly of the holoprotein. Denaturation analysis: ThePb*, Pb, Pg* and Pg forms of TePixJ_GAF and Pr and Pfr of Cph1 weredenatured with 8 M urea/HCl pH 2.0 at room temperature in the dark.Native holo-TePixJ_GAF (~20 mg/ml) was subjected to FTIR analysis.

Results: Previously, we reported that molecular mass of thechromophore of TePixJ was identical to PCB but was different inspectral properties. Moreover, we carefully compared TePixJ withCph1 after denaturation with acidic urea. The spectral properties ofTePixJ chromophore were clearly different from those of Cph1 PCB,but were very similar to those of phycoviolobilin (PVB), an isomer ofPCB. In the early phase of in vitro reconstitution, free PCB wascovalently incorporated with concomitant accumulation ofphotoconvertible holoprotein between Pb-like blue-absorbing form(Pb* form) and Pg-like green-absorbing form (Pg* form). Furtherincubation at 50ºC resulted in conversion to spectral properties similarto the native TePixJ. Concomitantly, PCB was isomerized to PVB asrevealed by denaturation analysis. The holocomplex prepared from invivo co-expression included both PCB and PVB as a chromophore.When this holocomplex was further incubated, additionalisomerization from PCB to PVB was detected.

There is conserved cysteine residue (Cys494: TePixJ numbering)among blue-green photoreversible cyanobacteriochromes. Mutationof Cys494 resulted in assembly of a red-absorbing form which did notexhibit photoconversion. Moreover, FTIR spectroscopy of the nativeTePixJ_GAF revealed green light-induced crosslinking of a free SHgroup. These results suggest that the light-induced crosslinking of acycteine residue (probably Cys494) to the PVB takes place in theassembly of Pb that is extremely blue-shifted compared with thetypical phytochrome.

Conclusions: As a first step, TePixJ apoprotein and PCB form anintermediary photoactive Pb*. Secondly, subsequent isomerizationfrom PCB to PVB proceeds during in vitro reconstitution. The extremeblue shift of TePixJ can be explained by reversible adduct formationbetween the chromophore and Cys494. Thus, we can conclude thatthe GAF domain of TePixJ is sufficient for both lyase, isomerase andphotoconversion activities.

P.110

CELL LINEAGE ANALYSIS ON HETEROCYST PATTERNFORMATION IN ANABAENA SP. PCC 7120.

S. Iwamori1, H. Asai1, K. Kawai1, S. Shoji1, H. Iwasaki1,2.1Department of Electrical Engineering and Bioscience, WasedaUniversity, Japan; 2PRESTO, Japan Science and Technology Agency,

Wakamatsu, Shinjuku, Tokyo; Japan.

hetR and patS genes have been reported as an activator and arepressor of heterocyst formation, respectively, in the filamentouscyanobacterium, Anabaena sp. PCC 7120. It is proposed that hetRactivates its own transcription and patS gene expression, while thePatS peptide inhibits the HetR function. This type of combination ofthe negative and positive feedback loops with a possible diffusibleinhibitor is reminiscent of the Turing instability dynamics, while itspossibility has not yet been well validated experimentally. For betterunderstanding of spatio-temporal dynamics underlying the heterocystpatterning, we have developed a monitoring/culturing system thatenabled us to observe morphological changes, hetR expressionprofile, and chlorophyll fluorescence from individual filaments duringthe course of heterocyst development. Moreover, micro-liquidchamber arrays have been developed to analyze multiple Anabaenafilaments simultaneously. Cell lineage analyses demonstrated thatinitial distributions of hetR::gfp signals and chlorophyll activity signalsat nitrogen-step-down were not correlated to the resulting distributionof developed heterocysts, supporting a random and non-deterministicselection of initial heterocyst positions. We also observed cells thatdifferentiated into heterocysts without cell division after nitrogen-step-down, suggesting that cell division of mother cells is not an essentialrequirement for heterocyst differentiation. We will also report that thehetR expression profile exhibits a complicated, transient and possiblycompeting dynamics even before determination of initial heterocystpositions.

P.111

THE PHYSIOLOGICAL ROLE OF THE CBBRRS TWO –COMPONENT SYSTEM IN RHODOPSEUDOMONAS PALUSTRIS.

Gauri. S. Joshi, F. Robert Tabita.

Department of Microbiology, The Ohio State University, Columbus,OH, USA.

Introduction: Rhodopseudomonas palustris is a metabolically versatilenonsulfur purple photosynthetic bacterium. The unique feature of theR. palustris cbbICO2 fixation regulon is the presence of a two-component system (referred to as the CbbbRRS system) between themaster transcriptional regulator CbbR and genes encoding form IRubisCO (cbbLS). CbbR belongs to the LysR family of regulators. TheCbbRRS system is an atypical two-component system consisting of asensor kinase and two response regulators with no apparent DNAbinding domains on any of these proteins. Recent physiologicalstudies have shown that the CbbRRS system plays a regulatory role inmodulating the expression and activity of form I RubisCO only duringphotoautotrophic (CO2) growth and not during photoheterotrophic(benzoate) growth. The distinctive presence of this two componentsystem and its role in modulating form I RubisCO expression is uniqueto R. palustris as similar systems have not been described in closelyrelated NSP bacteria, Rhodobacter spaheroides, Rhodobactercapsulatus, and Rhodospirillum rubrum. The observations stemmingfrom the physiological studies with photoautotrophically grown cellsled to the hypothesis that the CbbRRS proteins (especially theresponse regulators CbbRR1 and CbbRR2) influence the interactionsof CbbR at the cbbI promoter and thereby influence form I RubisCOexpression.

Methods: A bacterial two hybrid analysis (qualitative and quantitative)was performed to test whether the response regulators (CbbRR1 andCbbRR2) interact with CbbR in vivo. In this Escherichia.coli-basedsystem, interaction between a pair of proteins results in transcriptionalactivation of a selectable (His3- aadA)/quantitative (lacZ) reporter gene



by recruitment of RNA polymerase at the promoter. The strength ofthe interaction between the protein pair determines the magnitude oftranscriptional activation. In vitro gel shift assays and chemicalcrosslinking are additional approaches adopted to demonstrate theDNA – protein and protein – protein interactions.

Results: The bacterial two hybrid system enabled the identification ofa protein – protein interaction between the transcriptional regulatorCbbR and CbbRR1, response regulator 1 of the CbbRRS system. Sitedirected mutagenesis of the phosphoacceptor residues of CbbRR1(D54N and H171D) did not affect its interaction with CbbR significantlysuggesting that phosphorylation of CbbRR1 may not be important forthe interaction. Gel mobility shift analyses revealed that the affinity ofCbbR for the cbbI promoter was specifically enhanced in the presenceof CbbRR1, while the presence of CbbRR2 in the complex decreasedthe mobility of the DNA – protein complex. Chemical crosslinking ofCbbR with the response regulators CbbRR1 and CbbRR2 is underwayto further support the in vivo protein – protein interaction data.

Conclusions: The results of the bacterial two hybrid analysis indicatethat the CbbRRS two component system potentially exerts itsregulatory effect on the cbbI promoter (form I RubisCO expression) byinteracting with the master transcriptional regulator CbbR. Thebinding of CbbR to the cbbI promoter is stabilized in the presence ofCbbRR1 as observed in gel shift assays, further strengthening the invivo data. The interactions of the response regulators with CbbRrepresent additional transcriptional control beyond that provided byCbbR alone, for fine tuning the expression of form I RubisCO in R.palustris.

P.112

PHOTOTROPISM OF CYANOBACTERIA.

Mitsunori Katayama1, Mari Kobayashi2, Masahiko Ikeuchi3.1Department of Liberal Arts and Basic Sciences, Nihon University;2Tokyo Institute of Technology, School and Graduate School ofBioscience and Biotechnology; 3Department of Life Sciences (Biology),The University of Tokyo; Japan.

Introduction. Phototropism is directional growth in response todirectional light, which is widely observed in eukaryotes such as fungi,algae, and higher plants. In eukaryotes, blue light commonly inducesphototropic response. On the contrary, phototropism was barelyreported for prokaryotes. We recently found out phototropic responsein filamentous cyanobacterium Rivularia sp. IAM M-261 (Rivularia M-261). To characterize phototropism in cyanobacteria, its spectraldependence and the distribution among cyanobacteria wereinvestigated.

Methods. Microscopic examination of filamentous cyanobacteriacollected from environmental samples and culture collections wasmade to survey species exhibiting phototropsm. Spectral dependenceof the phototropic response was examined in the three Calothrixspecies and in the one Fischerella species by irradiation with series ofmonochromatic light (400 nm to 750 nm) provided by light emittingdiode. Phototropic response was quantified by measurement ofaverage angles of curvature of multicellular filaments.

Results. Phototropism was observed in some species that belong toCalothrix, Tolypothrix, Scytonema and Fischerella. Rivularia M-261 wasthought to be classified into Calothrix based on the 16S rDNAsequence. As for spectral dependence of the phototropic response,Rivularia M-261, Calothrix sp. PCC 7715 and Calothrix sp. PCC 7102exhibited positive phototropic response to blue light (460 nm). On theother hand, one Fischerella sp. exhibited positive phototropic

response to blue light (460 nm) and far-red light (750 nm).

Conclusions. Cyanobacteria belongs to diverse group (subsection IVand subsection V) were shown to exhibit phototropic response. Bluelight commonly induced phototropic response in Calothrix andFischerella likewise eukaryotes. In addition to this, one Fischerellaspecies exhibited phototropic response to far-red light. Most ofspecies exhibiting phototropism grow on surface of soil or rocksuggesting that phototropism is advantageous for terrestrial growth incyanobacteria.

P.113

EFFECTS OF GROWTH CONDITIONS AND THE COMPOSITION OFGAS PHASE ON PHOTOBIOLOGICAL HYDROGENACCUMULATION IN THE HYDROGENASE MUTANT OF NOSTOCSP. PCC 7422.

Masaharu Kitashima, Hajime Masukawa, Hidehiro Sakurai, KazuhitoInoue.

Department of Biological Sciences, Kanagawa University, Hiratsuka,Kanagawa, Japan.

Introduction: We are aiming at developing nitrogenase-basedphotobiological hydrogen production utilizing cyanobacteria withwater as the terminal electron donor. In the previous report (Yoshino etal., 2007), we reported that the uptake hydrogenase gene disrupted(∆hup) mutant of Nostoc sp. PCC 7422 had high hydrogen productionactivity and accumulated hydrogen to about 30% (v/v) under a startinggas phase of Ar + 5% CO2. In order to attain a higher level ofaccumulation of hydrogen concentration, we have studied the effectsof nitrogen and CO2 gas concentration of the first (heterocystinduction) and the second (hydrogen production period) gas phase onhydrogen accumulation.

Methods: The culture media were either BG11 or BG110 (NaNO3

omitted) medium. The starting culture of the ∆hup mutant was grownphotoautotrophically at 26°C by bubbling air under the 12 hour light-12 hour dark cycle. In the light period, cultures were illuminated byfluorescent light at photosynthetically active radiation (PAR) of about100 µmol photons m-2 s-1. The cells in the exponential growth phasewere washed with and suspended in BG110 medium at a chlorophyll αconcentration of about 3 µg ml-1, and 15 ml samples were transferredinto 25-ml Fernbach flasks equipped with butyl rubber stoppers. Gascomposition of the first phase was Ar + 5% CO2 + variedconcentration of N2 (1 - 80%). After photoautotrophically cultivatingfor 2 to 4 days, the gas phase was changed to Ar + 5% CO2 + variedconcentration of N2 (0.5 - 80%) for the second phase. Fordetermination of accumulated hydrogen and oxygen, 50 µl each ofgas sample was withdrawn from flasks at about the middle of the lightphase and analyzed by gas chromatography (Molecular Sieve 13Xcolumn). Nitrogenase activity was assayed by reduction of acetyleneto ethylene.

Results: When the duration of in the first phase was two days, thenitrogen concentration at 1-20% did not greatly affect the hydrogenaccumulation in the second phase, but 80% nitrogen significantlydecreased the accumulation. With the first phase of three days, thehydrogen accumulation by 5% nitrogen culture was lower than that by1% nitrogen culture. After two days under 1% nitrogen, the nitrogenconcentration was changed (the second phase). As for the hydrogenaccumulation of the culture (0-13 days), 0.5% nitrogen gave slightlyhigher concentration than 1%. At 5% and 20% nitrogen, theaccumulation was much lower than the formers. After about 8 daysunder 0.5% nitrogen, the hydrogen concentration became about 50%(v/v) with oxygen concentration of about 20-25%. The profiles of



nitrogenase activity could largely account for the hydrogenaccumulation profiles. These results indicate that a large part ofhydrogen was produced with water as the terminal electron donorunder the experimental conditions used.

Conclusions: By optimizing the gas composition of the culture, thegenetically improved mutant strain of cyanobacterium cells are able toaccumulate hydrogen to about 50% catalyzed by nitrogenase reaction,mostly using water as the terminal electron donor.

Reference: F. Yoshino, H. Ikeda, H. Masukawa, H. Sakurai, Highphotobiological hydrogen production activity of a Nostoc sp. PCC7422 uptake hydrogenase-deficient mutant with high nitrogenaseactivity, Mar. Biotechnol., 9: 101-112 (2007)

P.114

CHARACTERIZING THE PRIMARY METABOLISM OFSYNECHOCYSTIS SP. PCC6803: NETWORK RECONSTRUCTION,THERMODYNAMIC CONSTRAINTS AND FLUX-BALANCEANALYSIS.

H. Knoop, Y. Zilliges, W. Lockau and R. Steuer.

Institut fuer Biologie, Humboldt Universitaet Berlin, Germany.

Introduction: Cyanobacteria, are the only prokaryotes which possessthe capability to carry out photosynthesis, similar to higher plants.Therefor it is not surprising, that cyanobacteria attract growingattention in various areas of research and for economic purposes.Among the diverse strains, Synechocystis sp. PCC6803 is a widelyused model organism for the analysis of regulatory networks,metabolic pathways as well as photosynthetic processes. With a richcompendium of genomic, biochemical and physiological dataavailable, the cyanobacterium Synechocystis sp. Strain PCC 6803 canserve as an ideal example for the reconstruction of an integrativecomputational model that describes the relevant metabolic processesin the cell.

Methods and Results:We present a draft reconstruction of theprimary metabolism of Synechocystis sp. PCC6803. Our reconstructionis based on multiple data sources and extensive manual curation.Integrating information from the genome database (CyanoBase),several pathway databases, as well as information from the primarybiochemical literature. Our reconstruction comprises the mainpathways of central and intermediate metabolism, including the Calvincycle, glycolysis, the TCA cycle, the pentose phosphate pathway,oxidative phosphorylation and amino-acid synthesis. Based on thereconstruction, for which also thermodynamic constraints were takeninto account, we systematically evaluate and describe the metaboliccapabilities of Synechocystis sp. PCC6803 during phototrophicgrowth. In more detail, we used flux balance analysis to calculate therobustness of the model and to check the sensitivity of specificpathways. Additionally essential genes, correlated reaction sets andpotential metabolic bottlenecks were identified, as well as severalgaps and inconsistencies in current annotations. In addition, kineticparameters were added to the model at interesting metabolic branchpoints, which could play a key role for biotechnological engineering.

Conclusion: Owing to the complexity of even comparatively simplemetabolic maps, a detailed and systematic evaluation of theproperties and metabolic capabilities of organisms necessitates acomputational approach. Our reconstruction of the primarymetabolism of Synechocystis sp. PCC6803 allows for such asystematic computational evaluation of metabolic routes. Thereconstruction significantly improves upon the current genomeannotation and provides a resource for targeted manipulation of

metabolism.

Our results will be supplemented with and tested against high-qualitydata, aiming to iteratively improve and refine the current annotation.Our stoichiometric model is intended to serve as a first step towards acomprehensive computational description of cellular metabolism inunicellular autotrophs and can be extended to a genome-scale level infurther studies.

P.115

CIRCADIAN SYSTEM IN THE HETEROCYST-FORMINGCYANOBACTERIUM, ANABAENA SP. PCC 7120.

H. Kushige1, M. Matsuoka1, H. Iwasaki1,2.1Department of Electrical Engineering and Bioscience, WasedaUniversity, Japan; 2PRESTO, Japan Science and Technology Agency, 2-2 Wakamatsu, Shinjuku, Tokyo, Japan.

Cyanobacteria are the simplest organisms to exhibit circadian rhythms,while studies on the mechanism and physical properties of the clockregulation have been exclusively performed in the unicellularSynechococcus. Since Anabaena is one of the simplest multicellularorganisms which harbor both pattern formation with celldifferentiation and circadian rhythms, it provides an excellent modelsystem to analyze circadian functions and mechanisms which cannotbe addressed in unicellular species. i) Is there any difference in thekaiABC clock gene functions between Synechococcus and Anabaena?Note that KaiA homologs in filamentous species lack two-third of theprotein that are well conserved among unicellular species. ii) Is thereany difference between the clock systems in vegetative cells andheterocysts? iii) Are clocks in neighboring cells synchronized to eachother? Note that in unicellular Synechococcus cell-cell communicationhas been validated to be negligible in terms of circadiansynchronization. iv) Is heterocyst differentiation/patterning modified bythe circadian clock? As an initial step to address these questions, weperformed DNA microarray analysis to reveal the genome-widecircadian expression profile. Surprisingly, none of Anabaena kai genesshow significant expression rhythms, while we found ~600 clock-controlled genes. In contrast, in Synechococcus the kaiBC operonexpression shows the highest amplitude cycle. Moreover, althoughclock-controlled genes in Synechococcus peaked exclusively atsubjective dawn or dusk, that in Anabaena peaked more widelythroughout the circadian cycle. Thus, there is striking difference incircadian outputs as well as the clock gene expression profile betweenthe two species. For better understanding the clock function, we havedisrupted the kai genes in Anabaena. Transcriptomic and physiologicalproperties in the mutants will be also reported.

P.116

EXAMINING THE ROLE OF THE CBBX PROTEIN INRHODOBACTER SPHAEROIDES.

Amanda K. Luther, Rick A. Laguna, F. R. Tabita.

Department of Microbiology, The Ohio State University, Columbus,OH; USA.

Introduction: Carbon dioxide is fixed into organic carbon via theCalvin-Benson-Bassham (CBB) reductive pentose phosphate pathwayduring photoautotrophic and chemoautotrophic growth of nonsulfurpurple photosynthetic bacteria. However during photoheterotrophicgrowth, the primary function of CO2 fixation is to maintain cellularredox balance. Rhodobacter sphaeroides belongs to the alphasubdivision of proteobacteria. R. sphaeroides possesses two distinctcbb operons, cbbI and cbbII. Each operon encodes enzymes for the



CBB cycle, including ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO), the enzyme that catalyzes the reduction of CO2.These operons are located on two distinct genetic elements and theregulation of the cbbI and cbbII operon differs upon carbon availability.During photoautotrophic growth, both operons are maximallyexpressed, although the cbbI operon attains a greater level ofexpression as compared to the cbbII operon. When conditions arechanged to photoheterotrophic growth, down-regulation of eachoperon occurs. However, under this condition cbbII expression isgreater than cbbI expression.

In R. sphaeroides the cbbXYZ operon is located immediatelydownstream of the cbbI operon. With the exception of CbbZ (whichfunctions as a phosphoglycolate phosphatase), the physiologicalfunctions of proteins encoded by genes of this operon have yet to bedetermined in R. sphaeroides. However, a deletion of the cbbX generesulted in a long lag during photoautotrophic growth conditionscompared to wild-type. It was recently shown that Cyanidioschyzonmerolae, a unicellular red alga, encodes a CbbX protein that functionsas a transcriptional regulator of the cbbLS gene that encodesRubisCO. We are currently examining whether the CbbX proteinencoded by R. sphaeroides possesses such function.

Methods: To gain a greater understanding of CbbX protein function inR. sphaeroides, the following experiments were performed: Wecompared the R. sphaeroides CbbX protein in terms of genomiccontext, primary amino acid sequence, and domain architecture to theCbbX protein of C. merolae. We examined the RubisCO activity andexpression of the cbbI and cbbII operons in the cbbX gene deletionstrain of R. sphaeroides as compared to the wild-type strain underdifferent growth conditions and carbon sources. RubisCO activity wasdetermined by a standard RubisCO assay, and cbbI and cbbII

expression were determined by immunoblot analysis of the RubisCOproteins encoded by each operon.

Results: The R sphaeroides cbbX gene deletion strain appeared toincrease RubisCO activity as compared to the wild-type strain underphotoautotrophic growth conditions. The increased activity appearedto be a result of increased RubisCO protein as determined byimmunoblot analysis. Under photoheterotrophic growth conditionswith malate as the carbon source, RubisCO activity was the same asthe wild-type. However, when acetate was used as the carbon source,there was an apparent loss of regulation of the cbbII operon in thecbbX gene deletion strain, as compared to the wild-type.

Conclusion: Based on the results thus far attained, it appears that theCbbX protein in R. sphaeroides may function as a regulator of one orboth of the cbb operons. Further experiments are under way todetermine the exact role that this protein may play in regulating theseoperons in R. sphaeroides.

P.117

CHARACTERIZATION OF CAROTENOID CLEAVAGEDIOXYGENASE IN ANOXYGENIC PHOTOTROPHIC BACTERIUM,RHODOPSEUDOMONAS PALUSTRIS.

Isamu Maeda, Atsushi Inaba, Kazuyuki Yoshida.

Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.

Introduction: Carotenoid oxygenases catalyze formation ofapocarotenoids such as retinoids and the precursors of somephytohormones through oxidative cleavage at specific double bondsof carotenoids. Carotenoid cleavage dioxygenases (CCDs) belong to asubgroup of carotenoid oxygenases, and have been found in severalhigher plants and two cyanobacterial species. Although their substrate

specificities and cleavage patterns have been characterized in detail,none of CCDs in anoxygenic phototrophic bacteria have beenexperimentally characterized. In this study, therefore,Rhodopseudomonas palustris CCD (RpsCCD) was chosen to analyzethe activity of anoxygenic phototrophic bacterial enzyme. The in vivoand in vitro cleavages of carotenoids by RpsCCD were analyzed usinglycopene, produced in a recombinant E. coli strain, and b-apo-8’-carotenal as the substrate, respectively.

Methods: Pantoea ananatis crtE, crtB, and Pantoea agglomerans crtIwere introduced to E. coli JM109, resulting in lycopene-producing E.coli (pHYcrtEIB). Rps. palustris ccd was inserted in pMG103 toconstruct pMG-Rpsccd. Apocarotenoid production was performedwith cultivation of E. coli (pHYcrtEIB, pMG-Rpsccd) and E. coli(pHYcrtEIB, pMG103) for 24 hours. RpsCCD produced in E. coli BL21(DE3, pMG-Rpsccd) was used in the in vitro analysis. The crude proteinextract was incubated with all-trans-b-apo-8’-carotenal (96% purity,Fluka) in the presence of 0.1% (v/v) Triton X-100. Apocarotenoids weredirectly extracted with hexane/diethyl ether (1:4, v/v). The organicphase was analyzed with GC-MS.

Results: A specific peak was detected in the chromatogram of E. coli(pHYcrtEIB, pMG-Rpsccd) extract. The mass spectrum showed theparent-ion mass of 154, andcorresponded to that of citrol, thereduced form of citral. The datasuggests that citral is producedthrough the 7,8 (7’,8’) double bondcleavage of lycopene by RpsCCD andthen reduced to citrol in E. coli cells.The in vitro analysis using b-apo-8’-carotenal showed two specific peakswith the parent-ion masses of 284 and164. The mass spectra correspondedto those of retinal (m/z 284) and apo-8’,15’-apocarotene-dial (m/z164). The data demonstrates the 15,15’ double bond cleavage of b-apo-8’-carotenal by RpsCCD.

Conclusions: Dioxygenase activity was confirmed using recombinantRpsCCD protein. This is the first report that has identified cleavagepatterns of an anoxygenic phototrophic bacterial enzyme.

P.118

ANABAENA SP. STRAIN PCC 7120 GENE ALL0187 ISDEVELOPMENTALLY REGULATED AND ESSENTIAL FORDIAZOTROPHIC GROWTH AND HETEROCYST MORPHOGENESIS.

Rodrigo A. Mella-Herrera*, M. Ramona Neunuebel, James W. Golden*

Department of Biology, Texas A&M University, College Station, TX;*Current address: Division of Biological Sciences, University ofCalifornia, San Diego, CA, USA.

Introduction: Under diazotrophic growth conditions, the filamentouscyanobacterium Anabaena sp. strain PCC 7120 undergoesdevelopment to form a pattern of about one heterocyst every ten totwenty vegetative cells along filaments. The heterocyst differentiationprocess involves genetic, physiological, and morphological changes toproduce the micro-oxic environment necessary for nitrogen fixation.These changes include remodeling of the cell wall and deposition oftwo heterocyst-specific extracellular envelope layers. The all0187 geneis predicted to encode a protein containing a LytR domain, which isknown to be associated with regulation of cell wall maintenance. Weinvestigated all0187 to determine if it is involved in heterocystdifferentiation or in diazotrophic growth.



Methods: Anabaena (Nostoc) sp. strain PCC 7120 and its derivativeswere grown in BG-11 or BG-110 (lacking sodium nitrate) medium at30°C with white-light illumination of approximately 75 µM photonsm−2 s−1. Standard protocols were used for cloning, E. colitransformation, PCR, and northern RNA blot analysis. Microarraysexperiments were performed using Anabaena PCC 7120 RNA sampleshybridized to a custom microarray slide containing 768 probes. Lightand fluorescence microscopy were performed on a Zeiss Axioplan IImicroscope with a 40´ objective and green fluorescent protein (GFP)-specific emission (518 ± 13 nm) filter sets. Nitrogenase activity wasmeasured with an acetylene reduction assay.

Results: Insertional inactivation of all0187 caused a partial cell divisiondefect of vegetative cells grown in nitrate-containing medium. Innitrogen-free medium, mutant filaments formed abnormally longheterocysts and were unable to grow diazotrophically, but vegetativecell septation appeared normal. Septum formation betweenheterocysts and their flanking vegetative cells was incomplete, leavingone or both poles of the heterocysts partially opened. Weinvestigated whether these morphological defects led to inactivationof nitrogenase due to lessened protection against oxygen. Acetylenereduction assays for nitrogenase activity showed that the mutant strainretained approximately seventy percent of the wild-type activity; thisobservation shows that heterocysts of the all0187 mutant strain arepartially functional. Northern RNA blot analysis showed that all0187expression was upregulated by 8 h after nitrogen step-down andfluorescence microscopy of a Pall0187-gfp reporter strain revealedincreased GFP fluorescence in proheterocysts and heterocysts by 9 hafter nitrogen step-down, whereas vegetative cells maintained a lowerlevel of fluorescence.

Conclusions: We hypothesize that the diazotrophic growth defect ofthe all0187 mutant is caused by the inability of the heterocysts totransport fixed nitrogen to the neighboring vegetative cells.

P.119

FUNCTIONAL ANALYSIS OF THE ISCR-LIKE TRANSCRIPTIONFACTOR, INVOLVED IN EXPRESSION OF THE PSAAB TRANSCRIPTIN SYNECHOCYSTIS SP. PCC 6803.

T. Midorikawa1, K. Matsumoto1, R. Narikawa2, M. Ikeuchi1,2.1Department of Biological Science, Graduate School of Sciences;2Graduate School of Arts and Sciences; University of Tokyo, Japan.

Introduction: Since the light environment varies depending onlocation, time, and weather, it is important for the oxygenicphototrophs to acclimate to these environmental changes. It has beenestablished that the regulated accumulation of photosystem I (PSI) iscritical for long term acclimation to the light conditions incyanobacteria. To this end, expression of psaA and psaB, whichencode the PSI reaction center subunits, is tightly regulated in theacclimation processes. In the course of our screening of transcriptionalregulators in Synechocystis, we found that an iscR-like regulatorSlr0846 is critical for maintenance of normal chlorophyll accumulation.Here we studied the target genes of Slr0846 by DNA microarrayanalysis, gel shift assay and primer extension analysis. We also studiedPSI/PSII ratio of the slr0846 disruptant under various light conditions.

Methods: The original motile strain of Synechocystis sp. PCC 6803showing positive phototaxis was used as wild type. The slr0846 genewas disrupted by insertion of the chloramphenicol resistance gene.The low temperature chlorophyll fluorescence at 77 K was monitoredfor estimation of the PSI/PSII ratio. DNA microarray analysis was done

with CyanoCHIP version 1.6. The N-terminal poly histidine-taggedSlr0846 prepared from E. coli was used for gel shift assay.

Result: Full segregation of the ∆slr0846 mutant was achieved. It issuggested that Slr0846 is not essential for this strain. DNA microarrayanalysis revealed that the expression level of psaAB was downshiftedin the ∆slr0846 mutant, which was grown under the standard lightconditions. Direct binding of Slr0846 protein to a promoter of psaABwas studied by gel shift assay. Further dissection revealed that Slr0846bound to a far upstream region in the promoter. Primer extensionanalysis showed that transcription from the two known start sites wasalmost equally affected in the mutant. These results suggest thatSlr0846 is a transcriptional activator for psaAB. The ∆slr0846 mutantexhibited much lower chlorophyll contents and PSI/PSII ratio than wildtype. The mutant also showed the growth sensitive to high light.Notably, the difference in the PSI/PSII ratio between wild type and themutant was attenuated under the PSI light conditions. These findingssuggest that the activity of Slr0846 may respond to various lightconditions.

Conclusion: In this study, we demonstrated that the Slr0846 binds to afar upstream region in a promoter of psaAB and acts as atranscriptional activator. It may contribute to the optimal regulation ofthe psaAB expression under various light conditions.

P.120

ALR2269 – THE OMP85 IN ANABAENA SP. PCC 7120.

Kerstin Nicolaisen, Constance Vollmer, Iwo Tews, Enrique Flores,Enrico Schleiff.

Goethe University Frankfurt, Frankfurt, Germany.

The outer membrane of gram-negative bacteria protects the organismagainst environmental influences and allows interactions with itssurrounding. Proteins of the Omp85 class embedded in the outermembrane function as “chaperones” for the insertion of the outermembrane proteome required for the function of this barrier. Omp85are polypeptide transporting β-barrel proteins (1), which are alsofound to transport polypeptides across the outer envelope ofchloroplasts (1). It is thereby an essential class of proteins inendosymbiotically derived organelles and bacteria. In Anabaena sp.PCC 7120, the closest relative to the ancestor of chloroplasts (2),Alr2269 functions as Omp85-like protein (3). Therefore we studied itsrole as transporter (3), analyzed its properties in protein recognition (5)and are investigating the possibilities to complement other proteins ofthe Omp85 class with Alr2269 and vice versa (6).

References:

1. Schleiff, E., Soll, J. (2005) EMBO Rep. 6, 1023-1027

2. Bredemeier, R. et al. (2007) J.Biol.Chem. 282,1882-1890

3. Ertel, F. et al. (2005) J.Biol.Chem. 31, 28281-28289

4. Nicolaisen, K. et al. (2009) Mol. Microbiol. submitted

5. Wunder, T. et al. (2007) BMC Evol. Biol. 7: 236

6. Wunder, T. et al. (2009) Endocyt. Cell Res.



P.121

ANALYSIS OF BIOSYNTHETIC PATHWAY OF CHL A ESTRIFIEDWITH ∆∆2,6-PHYTADIENOL IN CHLOROBIUM TEPIDUM BYCONSTRUCTING DISRUPTION MUTANTS OF CT1232 AND CT2256GENES.

Jiro�Harada1,�Syohei�Miyago2,�Tadashi�Mizoguchi1,�Hitoshi�Tamiaki1,Hirozo�Oh-oka2.1Department�of�Bioscience�and�Biotechnology,�Faculty�of�Science�andEngineering, Ritsumeikan�University;�2Department�of�BiologicalSciences,�Graduate�School�of�Science,�Osaka�University;�Japan.

The�green�sulfur�bacteria contain three�kinds�of�(bacterio)chlorophyll[(B)Chl]�pigments,�BChl aP,�Chl�aPD and�BChl�cF.�BChls�cF are�located�inlarge�light-harvesting�apparatuses,�chlorosomes,�where�light�energy�iscaptured�and�migrated�to�FMO�protein�via�baseplate�and�finallyconverged�on�P840,�a�special�dimer�of�BChl�aP,�in�the�reaction�center.BChls�aP also�serve�as�antenna�pigments�associated�with�CsmA�proteinin�baseplate,�FMO�protein�and�the�reaction�center�complex.��Withinthe�reaction�center�complex,�Chls�aPD are�supposed�to�function�as�aprimary�electron�acceptor�as�well�as�accessory�pigments.��Chl�aPD hasthe�same�chlorin�π-system as�Chl�aP in�higher�plants�and�cyanobacteriaexcept�for�a�long�hydrocarbon�chain�at�the�C-17�position; the�formeris�esterified�with�∆2,6-phytadienol�and�the�latter�is�with�phytol�[1].��Aprecursor�of�phytol�is�geranylgeraniol,�which�is�stepwisely�reduced�bygeranylgeranyl�reductase�(GGR)�[2].��However,�the�reaction�process�toproduce�∆2,6-phytadienyl�ester�is�still�unknown.��In�the�genome�ofChlorobium tepidum,�two�paralogous�genes,�CT1232 and�CT2256,exhibit�sequence�similarity�to�GGR�genes�of�cyanobacteria�and/orpurple�bacteria,�and�are�presumed�to�be�involved�in�hydrogenation�ofgeranylgeranyl�group.

In�order�to�clarify�the�biosynthetic�process of�∆2,6-phytadienyl�ester,we�constructed�disruption�mutants�of these�genes�using an insertionalinactivation�method�and�analyzed�their�pigment�compositions andphotosynthetic�competences [3,�4].��The�CT2256 mutant�accumulatedChl�aGG and�BChl�aGG esterified�with�geranylgeraniol,�indicating�thatCT2256�was involved�in�the�production of�both ∆2,6-phytadienyl�andphytyl�groups.��The�mutant�still�grew�photosynthetically�although�itsgrowth�rate�drastically�decreased�to�less�than�a�half�compared�to�thatof�the�wild�type.��This�could�be�explained�by�some�hindrance�of�theenergy�transfer�from�chlorosomes�to�the�reaction�center�complex,because�the�relatively�high�fluorescence�emission�from�chlorosomes�inthe�mutant�was�observed.��However,�the�CT1232 mutant�showed�noapparent�phenotype compared�to�the�wild type.��The purplebacterium�Rhodobacter capsulatus mutant�defective�in�the�bchP genewas�partially�complemented�with�the�CT2256 gene by�a�conventionalconjugation�method; BChl�aP was�synthesized�in�the�mutant�inaddition�to�accumulating�other�intermediates. Furthermore,cyanobacterial�and�purple�bacterial�GGR�genes,�which�are�chlP andbchP genes,�respectively,�were�incorporated�into�the�CT2256 mutantgenome�by�replacing�the�disrupted�site�of�the�relevant�gene.� The�twostrains,�thus�obtained,�partially�restored�a�reduction�activity�toproduce�phytol�and�other�intermediates�from�geranylgeraniol.��All�ofthe�above�data�suggest�that�the�CT2256�might�discriminate�thedifference�of�ring�structures�between�chlorin and�bacteriochlorin andproduce�BChl�aP and�Chl�aPD from�BChl�aGG and�Chl�aGG,�respectively.

References

[1]�M.�Kobayashi,�H.�Oh-oka,�S.�Akutus,�M.�Akiyama,�K.�Tominaga,�H.Kise,�F.�Nishida,�T.�Watanabe,�J.�Amesz,�M.�Koizumi,�N.�Ishida�and�H.Kano,�Photosynth. Res.,�63:�269-280�(2000).

[2]�T.�Mizoguchi,�J.�Harada�and�H.�Tamiaki,�FEBS Lett.,�580:�6644-6648(2006).

[3]�J.�Harada,�S.�Miyago,�T.�Mizoguchi,�C.�Azai,�K.�Inoue,�H.�Tamiakiand�H.�Oh-oka,�Photochem. Photobiol. Sci. 7:�1179-1187�(2008).

[4]�T.�Mizoguchi,�M.�Isaji,�J.�Harada,�K.�Watabe�and�H.�Tamiaki,�J.Porphyr. Phthal. (in press).

P.122

ACID STRESS RESPONSIVE GENES, SLR0967 AND SLL0939, AREDIRECTLY INVOLVED IN LOW-PH TOLERANCE OFCYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.

Hisataka�Ohta1,2,�Atsushi�Moriyama1,�Yuko�Kubo1,�Yousuke�Shibata1,Youhei�Haseyama1,�Yuka�Yoshino1,�Takehiro�Suzuki1,�Masahiko�Ikeuchi3,Isao�Enami1,�Shusei�Sato4,�Yasukazu�Nakamura4,�Satoshi�Tabata4�.1Department of�Biology,�Faculty of�Science,�Tokyo�University ofScience, Shinjuku; 2Tissue�Engineering�Research�Center,�TokyoUniversity�of�Science,�Noda;�3Department of�Life�Science,�University�ofTokyo,�Meguro,�Tokyo;�4Kazusa�DNA�Research�Institute,�Kisarazu,Chiba;�Japan.

Introduction: Dating�from�the�Precambrian�era,�cyanobacteria�have�along�history�of�adapting�to�the�Earth’s�environment.�By�evolvingoxygen�via�photosynthetic�reactions�similar�to�those�of�plants�andgreen�algae,�these�prokaryotes�were�essential�to�the�evolution�of�thepresent�biosphere.� They�continue�to�make�a�large�contribution�to�theequilibrium�of�the�Earth’s�atmosphere�by�producing�oxygen�andremoving�carbon�dioxide. To�survive�in�extreme�or�variableenvironments,�cyanobacteria�have�developed�specific�regulatorysystems,�in�addition�to�more�general�mechanisms�equivalent�to�thoseof�other�prokaryotes�or�photosynthesis�bacteria (1). Several�species�ofcyanobacteria�serve�as�model�organisms�for�elucidating�bothfunctional�and�regulatory�aspects�of�photosynthesis (2). Above�all,Synechocystis sp.�PCC�6803�was�the�first�photosynthetic�organism�forwhich�a�complete�genome�sequence�became�available�(3), and�DNAmicroarrays�have�been�used�to�examine�gene�expression�in�responseto�various�kinds�of�stress�such�as�osmotic,�salinity, and�high�light�stress(4-7).

Acid�rain�is one�of�the�most�serious�of�environmental�stresses.��Itcauses�acidification�of�lakes�and�streams�and�contributes�to�damageof�plants, algae, and�cyanobacteria in�many�parts�of�the�world.Rhizotoxicity�in�acid�soil,�which�involves�the�action�of�Al3+ has�beenwell�investigated�(8). Nevertheless, little�has�been�done�to�elucidatethe�basic�set�of�adaptations�necessary�for�acid�tolerance�in�plants,algae, or�cyanobacteria.

DNA�microarray�analysis�of�Synechocystis sp.�PCC�6803 cells revealedthat acid�stress�induced�the�expression�of putative�stress-relatedproteins,�such�as�chaperones (slr0093�[dnaJ],�sll1514�[hspA],�andsll0170�[dnaK)]), regulatory�factors�(sll0306�[sigB] and�sll2012 [sigD)]),and�proteins�of unknown�function�(9).�Among�the�up-regulated�geneswith�unknown�function,�slr0967 and�sll0939 continuously�increase�7and�16-fold�after�4�h�of�acid�stress�and�are�upregulated by�osmoticand�salt�stresses�(6).��Interestingly,�these�two�genes�are�locatedadjacently�on�the�Synechocystis sp. PCC�6803 genome�(Fig.�1).

In�this�study,�we�examined the�physiological�function�of�cyanobacteriagenes using mutant cells�in�which�each�gene�was�disrupted�by�akanamycin-resistance cartridge�gene. Based�on�phenotypes of�themutants�and�real-time quantitative�RTPCR analysis of�the�transcripts ofthe�two�genes, the�expression�profile�of�the�slr0967 deletion�mutanton�acid�stress�was�compared�with�that�of�wild-type�cells.



Methods: Culture conditions- Synechocystis sp.�PCC�6803�wascultivated�in BG-11�medium�(10) buffered�with�10�mM�TES�NaOH,�pH8.0, or�in�10�mM�MES�NaOH�pH�6.0�(long-term�acid�stress�condition),at�30°C.� Cultures�were�bubbled�with�3%�CO2-containing�air andilluminated�with�30�µmol�photons�m−2 s−1 from�incandescent bulbs.Cell�growth�was�measured�using�a�Pharmacia�spectrophotometer�at730�nm (Amersham�Pharmacia�Biotech,�Piscataway,�NJ,�USA).

Generation of insertion mutants- Mutants�impaired in�selected�geneswere�generated�by�reverse genetics.�The�coding�sequences�andneighboring sequences�were�amplified�by�PCR.�Approximately 2�kb�ofPCR�products�were�cloned�into�pUC19 (Toyobo,�Osaka,�Japan).�Theprimers�for�amplification�were designed�using�the�complete�genomesequence�of Synechocystis (3).�Sequences�that contained�appropriaterestriction�sites�were�selected to�improve�cloning�of fragments.� Thekanamycin�(Km) resistance gene�(kmr)�isolated�from plasmid�pUC4K(Amersham�Pharmacia)�was�inserted�into unique�restriction�sites�of�theencoding�sequences. Transformants�were�initially�selected�on�amedium containing�10�μg�Km�mL−1 (Wako�Pure�Chemical,�Osaka,Japan),�whereas�the segregation�of�clones�was�performed�byrestreaking�(at�least�three�transfers)�of�primary clones�on�platessupplemented�with�50�μg�Km mL−1. During�the�cultivation�of�mutants,Km�was�added�to�the�liquid�media.

Generation of overexpressing mutants-�Overexpressing�mutants�trc-slr0967 and�trc-sll0939 of�Synechocystis 6803 were�generated�usingpTrc-slr0967�and�pTrc-sll0939 plasmids.�The�plasmids were�introducedinto�the cmr gene from pLysS (Novagen,�Gibbstown,�NJ,�USA) andthe trc�promoter�was�introduced�from�pTrcHis�B (Invitrogen,�Carlsbad,CA,�USA) into�the�upstream�of�the�respective�genes�as�described�byKamei�et�al.�(11).

Short-term acid stress conditions-�Exponentially-growing cells�wereacid-stressed�by�centrifuging�the�cell cultures�and�resuspending�thecell�pellets�in�a�pH adjusted BG-11�medium�using�10�mM�glycine�(pH3.0)�buffers instead�of�TES�buffer for�8�h.� Serial�dilutions�of�the�cellswere�spotted onto�normal�BG-11 plates after�treatment and�culturedfor�7 days at�30°C.�Experiments�were performed�in�duplicate�at�leastthree�times.

RNA isolation and quantitative real-time RTPCR-�Total�RNA�wasisolated�using�the�RNeasy Mini�kit�(Qiagen,�Hilden,�Germany)�asdescribed by�Hihara�et�al. (7).�For�the�reverse�transcriptase (RT)reaction,�100�ng�RNA�was�incubated�with�a mixture�of�PCR�reverseprimers�for�10�min�at�70°C prior�to�adding�100�U�Superscript�II�RT(Gibco-BRL,�Carlsbad,�CA,�USA). The�RT�reaction�was�performed�at42°C�for�1�h�and terminated�by�incubating�the�cells�at�72°C�for�10min. A�Perfect�Real�Time�kit�(Takara�Bio,�Otsu,�Shiga,�Japan)�was�usedaccording to�the�manufacturer’s�instructions.

DNA microarray analysis- Cells�were�harvested�from�10�mL�of�cultureby�centrifugation�at�4,000�×�g for�5�min�at�25°C�and�then�brokenimmediately�with�a�Mini-Bead�Beater�(Biospec,�Bartlesville,�OK,�USA).Total�RNA�was�isolated�using�the�RNeasy�Mini�kit�(Qiagen)�asdescribed�by Hihara�et�al. (7). A�Synechocystis DNA�microarray(CyanoCHIP)�was�obtained�from�Takara�(Kyoto,�Japan).�This�microarraycovered�3,079�of�the�3,168�open�reading�frames�(ORFs) ofSynechocystis,�excluding�ORFs�transposases.�Conditions�for�thesynthesis�of�Cy3-labeled�and�Cy5-labeled�cDNAs,�hybridization,�andwashing�were�as�previously�described�(Hihara�et�al.�2001). Imageacquisition�with�a�ScanArray�4000�(GSI�Lumonics,Watertown,�MA,USA)�was�performed�with�the�autobalance-autorange feature.� Withthis�feature,�the�sensitivity�of�the�instrument can�be�automaticallyadjusted�by�changing�the�laser�power�andphotomultiplier�gainsettings�so�that�the�signal�is�within�90%of�maximum�to�preventsaturation.�The�raw�data�obtained�with the�ScanArray�4000�were

analyzed�with�QuantArray�version�2.0 software�(GSI�Lumonics,�Tokyo,Japan). The�fluorescence�intensity�of�each spot�for�both�Cy3�and�Cy5images�was�quantified,�and�local�background fluorescence�levels�weresubtracted. Cy3�and�Cy5�images�were normalized�by�adjusting�thetotal�signal�intensities�of�the two�images. The�results�shown�areaverages�of�4–6 biologically�independent�experiments.

Results: Characterization of the deletion mutants of slr0967 andsll0939 Wild-type�Synechocystis sp.�PCC�6803�was�transformed withslr0967 and�sll0939 that had�been�interrupted�with�cassette�conferringresistance to�kanamycin. To�examine�segregation of�these�geneswithin�the�Synechocystis genome, we performed�a�PCR�analysis�usingDNA�of�wild-type�and the�∆slr0967 and�∆sll0939 mutant�cells.�PCRwith genomic DNA�of�wild-type�cells as�a�template�amplifiedrespective 0.4 kb�DNA�fragments�for�slr0967 and�sll0939,�whereasPCR�with�DNA�from�∆slr0967 and�∆sll0939 mutant�cells�yielded�afragment�of 1.6 kb�(results not�shown). These�results�indicated�thatslr0967 and�sll0939 genes�in�∆slr0967 and�∆sll0939 mutant�cells�hadbeen�disrupted�by�the�insertion of�the�kmr gene.

In�normal�BG-11�medium�at�pH�8.0,�all�strains�exhibited�a�similarphotoautotrophic�doubling�time�(Fig.�2A),�suggesting that�deletion�ofthese�genes�did�not�affect their�growth�in�normal�conditions. Incontrast, in�the�acid�stressed condition�at�pH�6.0,�the�growth�of�allmutant cells was slightly�but�significantly�inhibited compared�with�thatof�wild-type�cells�(Fig.�2B�and�2C). The�sll0939 and�the�double�slr0967and�sll0939 mutants were more�sensitive to�acid�stress�than�the∆slr0967 cells�(Fig.�2B) indicating�that�slr0967 and�sll0939 are�involvedin�acid�tolerance�of�Synechocystis cells.�

Overexpression of slr0967 and sll0939 Overexpression�of�these�genesfailed�to�enhance�the�acid�tolerance�after culturing�at�pH�6.0�for�7days�(Fig. 3A).�We�then�characterized the�expression�of�these�twogenes�at�the�level�of�the�transcript�by�quantitative�RT-PCR using�totalRNA�isolated�from�acid�treated�wild-type�cells�and�trc-slr0967�and�trc-sll0939 mutant�cells. The�relative�expression�of slr0967 and sll0939promoted�by�trc was�lower�than�that�of�wild-type�cells�during�a 4�hacid�stress�but�higher�than�that�from�non-treated�wild-type�cells�(Fig.3C�and�D).�Accordingly,�survivability of�the�mutants�by�acid�stresstreatment�at�pH�3.0�for�1–12�h�was�tested (data�not�shown).�As�aresult,�we�found�a significant�difference�in survival�between�wild-typeand�trc-mutants�that were�treated�for 8�h�(Fig. 3B).�The�trc-mutantsbecame more�tolerant�to�acid�stress�than�wild-type�cells.�

Quantitative RT-PCR analysis of slr0967 and sll0939 in mutual deletionmutants. These�two�genes are�located�adjacent to�the operon�and�arecoded�by�opposite strands on�the�Synechocystis genome.�To�examinethe�regulatory�relationship�between�these�two�genes whose�deletionmutants were�sensitive�to�acid�stress�compared�with�the�wild-type,�theabundance�of�mutual genes in�the�deletion�mutants was measured innormal�growth�and�acid�stress conditions by culturing them�at�pH 3.0for�30�min. As�shown in�Fig. 3,�there�was�increased�expression�ofslr0967 and�sll0939 in�wild-type�cells�by�acid�stress�after�30 min;however,�there�was no�significant alteration�in�slr0967 expressionevident�in�the �sll0939 mutant.�There�was�a�slight�up-regulation�ofsll0939 in the normal�growth�condition,�but deletion�of�sll0939 did�notaffect�slr0967 expression�in�the acid�stressed condition.�In�contrast,sll0939 expression�was�considerably affected�by�deletion�of�slr0967 inboth�normal�and�acid�stressed conditions.�The�relative�levels�of�thesll0939 transcript�were�only�3%�and�4%�of�wild-type�cells, respectively.These�results�indicate�that�slr0967 regulates�sll0939 expression�inSynechocystis in�both�normal�and�acid�stressed conditions.�

DNA micro array analysis of ∆slr0967 mutant. We�examined�thegenome-wide�expression�of�genes�in�∆slr0967 mutant�cells�using�DNAmicroarrays�to�determine�whether slr0967 is�involved in�the�expression



of�other�acid�stress�responsive�genes. Table�1�shows�genes that�were3-fold�up-regulated�by�slr0967 deletion compared�to wild-type�cells.These�genes�can�be�classified�into two�groups.�1) Genes�that�were�notrepressed in�mutant�cells�but�were�3-fold�or�more�repressed in�wild-type�cells�under acid�stress,�and�2)�Genes�that�were�up-regulatedmore�than�3-fold�in�mutant�cells�but�not�in�wild-type�cells. The�firstgroup�contains�genes�for�proteins involved�in�CO2 uptake, CO2

fixation, and�bicarbonate�transport�(sll1732 [ndhF3],�sll1733 [ndhD3],sll1734 [cupA],�slr0040 [cmpA], slr0041 [cmpB], slr0042, slr0043,slr0044, and�slr1512 [sbtA]) (12-16). As�previously�described�(9),�thebicarbonate�transport�system�might�have�become�redundant�for�wild-type�cells�under�acid�stress�because�most�of�the�bicarbonate�exists�inthe�form�of�CO2 that�diffuses�through�the�cell�membranes�freely. Itmay�be�that�these�genes�were�not�up-regulated�in�mutant�cells�forsurvival�but�the�repression�machinery�of�these�genes�was�virtuallyhalted�due�to�lack�of�slr0967. The�second�group�contains�genesencoding�stress�regulated�proteins�(ssl0452 [nblA],�sll0247 [isiA],slr0373,�slr0374, slr0376, and slr0228 [ftsH]).�Degradation�of�thecyanobacterial�phycobilisome�light-harvesting�antenna�is�a�generalacclimation�response�that�is�observed�under�various�stress�conditionssuch�as�N-,�S-,�and�P-starvation�(17-20). In�the�Synechocystis strainPCC�6803�genome,�two�tandem�copies�of�nblA are�present.��Theexpression�of�ssl0453 (nblA2), which�is�located�downstream�of�ssl0452(nblA1),�was�not�affected�by�slr0967 deletion�(data�not�shown). TheisiA gene�encodes�a�protein�that�is�similar�to�the�photosystem�IIchlorophyll-binding�protein�CP43�and�is�induced�under�iron-stressedconditions�(21,�22).�The�slr0373,�slr0374, and slr0376 genes�constitutean operon that�is�responsive�to�various�environmental�stresses�buttheir�functions�are�unknown�(23,�24).��An�FtsH protease�plays�anessential�role�in�the�turnover�of�the�reaction�center�D1�protein�inSynechocystis PCC�6803�under�heat�stress as�well�as�light�stressconditions�(25,�26).�SLR0967 deletion�increases�death�due�to�acidstress,�resulting�in�increase�in�the�number�of�stress�responsive�genesbeing�up-regulated�in�mutant�cells.�Genes whose�expression�were up-regulated�in�wild-type�cell�by�acid�stress�were�almost�all�downregulated by�deletion�of�slr0967 (data�not�shown).

Table�2�shows�genes�that�were�3-fold�or�more�repressed�by�slr0967deletion�than�those�in�wild-type�cells. These�genes�were�also�dividedinto�two�groups:�1) Genes�whose�repression�was�observed in�mutantcells�but�not�in�wild-type�cells�by�acid�stress,�and�2)�Genes�whose�up-regulation�decreased�in�mutant�cells�but were 3-fold�or�moreup-regulated�in�wild-type�cells.�Genes�encoding�the�phycobilisomeproteins�(sll1577 [cpcB], sll1578 [cpcA], sll1579 [cpcC2], sll1580[cpcC1], slr0335 [apcE], slr1986 [apcB], slr2067 [apcA], slr2051[cpcG1]),�photosystem�I�subunits�(slr1834 [psaA], ssl0563 [psaC],slr0737 [psaD], sll0819 [psaF], and slr1655 [psaL]) and�chaperoneproteins�(slr2075 [GroES] and�slr2076 [GroEL1]) that�were�repressed�bydeletion�of�slr0967.�Allakhverdiev�et�al.�(27,�28).��demonstrated�thatsalt�stress�and�hyperosmotic�stress�have�inhibitory�effects�on�theelectron-transfer�activity�of�photosystem�I.�Repressed�expression�ofgenes�for�photosystem�I�and�phycobilisomes�might�be�important�formaintenance�of�a�certain�level�of�photosynthetic�activity.�Moreover,�itseems�possible�that decreased�abundance�of�Fe-requiringphotosystem�I�components�is�accompanied�by�iron�deficiency,�whichmay�be�caused�by�acid�stress.�It�is�unknown�why�chaperone�expressionwas�repressesed�as�these�genes�were�only�marginally up-regulated�byacid�stress�in�wild-type�cells.� High-light�inducible�protein�ssr2595(HilB) (29)�and�the�two�component�response�regulator�slr1214 (rre15)showed remarkably�decreased�expression�following slr0967deletion.

Conclusions: In�this�study,�we�found�that�acid�responsible�genesslr0967 and�sll0939 are�directly�involved�in�acid�tolerance�of�thecyanobacteria�Synechocystis sp.�PCC 6803, and�slr0967 may�regulate

the�expression�of�genes�that�respond�to�environmental�stresses. Thephysiological�functions�of�these�genes�have�still�not�been�identified.Sequence�analysis�revealed�that�sll0939�has�two�transmembranesegments�and�slr0967�has�tow�pentapeptide�sequences sequences.Interestingly,�preliminary�data�from�a�two�hybrid�yeast�analysisrevealed�that�sll0939�interacts�with�slr1596�(pxcA), which�is�a�protein�inthe�cytoplasmic�membrane�involved�in�light-induced�proton�extrusion(data�not�shown).��The�deletion�mutant�of�slr1596 is�unable�to�grow�atacidic�pH�(30). These�data�suggest�that�sll0939 may�also�function�withslr1596�for�light-induced�proton�extrusion.��However,�deletion�ofsll0939 and�slr0967 is�not�affected�by�light-induced�proton�extrusion(data�not�shown). Up-regulation�of�slr1214 (rre15) considerablydecreased by deletion�of�slr0967. It�was�thought that�slr1214 affectedacid�stress�tolerance,�but�deletion�of�the�gene�did not�affect�growth�atpH�6.0�(31).�Therefore,�we�are�now�intensively�studying�therelationship�of�these�proteins. To�further�understand�the�function�ofslr0967�and�sll0939,�we�have�investigated the�protein�expression�ofslr0967 and�sll0939 in�E. coli cells�using�pET�vectors,�but�their�functionis�still�unknown.�IPTG�induction�of�these�genes�in�E. coli cause�celllyses�and�the�cells�are�unable�to�recover�these�proteins.�We�areinterested�in�the�regulatory�mechanisms�of�acid-responsible�genes;therefore,�analysis�of�the�promoter�region�of�slr0967 and�sll0939should�be�investigated�intensively.

P.123

CELL-SPECIFIC TRANSCRIPTOME ANALYSIS IN ANABAENAVARIABILIS ATCC 29413 GROWN PHOTOTROPHICALLY,PHOTOHETEROTROPHICALLY, AND HETEROTROPHICALLY.

Jeong-Jin�Park1,�Sigal�Lechno-Yossef2,�Hajime�Masukawa1,�C.�Peter�Wolk2,3,�Claire�Vieille1.1Department�of�Microbiology�and�Molecular�Genetics;�2MSU-DOEPlant�Research�Laboratory;�3Department�of�Plant�Biology,�MichiganState�University,�East�Lansing,�MI,�USA.

Introduction: Anabaena variabilis ATCC�29413�is�a�potentialcandidate�for�the�production�of�hydrogen�(H2)�as�a�clean�renewableenergy�commodity.�A. variabilis filaments�are�composed�of�vegetativecells�that�perform�oxygenic�photosynthesis,�and�of�heterocysts�that�fixnitrogen�(N2)�in�an�oxic�environment.�A. variabilis produces�H2 as�a�by-product�of�N2 fixation.�To�understand�how�electrons�are�channeled�toH2 production�in�A. variabilis under�different�growth�conditions,�weare�comparing�gene�expression�in�phototrophic�(in�the�presence�oflight),�photoheterotrophic�(in�the�presence�of�light�plus�fructose),�andheterotrophic�(with�fructose�in�the�dark)�cultures�using�RNA�extractedseparately�from�vegetative�cells,�heterocysts,�and�whole�filaments.These�studies�should�help�us�identify�genes�whose�roles�in�H2

production�are�affected�by�carbon�and�energy�sources,�and�regulatorymechanisms�that�inter-relate�those�genes.

Methods: A.variabilis ATCC�29413�was�grown�in�AA/8�medium�at30°C�in�Fernbach�flasks�under�white�fluorescent�light�or�in�the�dark.Heterotrophic�cultures�were�supplemented�with�5�mM�fructose.Heterocysts�and�vegetative�cells�were�isolated�as�described�(Petersonand�Wolk,�PNAS,�1978,�75:6271),�with�variations.�RNA�was�extractedfrom�each�type�of�cell�using�Ambion�RiboPure”!-Bacteria�kits.�RT-qPCRreactions�were�performed�with�primers�specific�to�the�A. variabilis 16srRNA,�rnpB,�rbcL, ftsZ, nifK,�and�fdxN genes,�using�16s�rRNA�or�rnpBas�internal�controls�for�data�normalization.�cDNA�was�prepared�byusing�Superscript�II�reverse�transcriptase�and�random�primers.�RT-qPCR�was�performed�using�SYBR�green�(Du�and�Arvidson.�Infect.Immun. 2006,�74:2767).�Microarray�experiments�are�being�performedwith�Roche�NimbleGen�60-mer�arrays.�Nitrogenase�activity,�with�C2H2



as�substrate,�and H2 production�were�quantitated�by�a�combination�ofgas�chromatography�with,�respectively,�a�flame�ionization�and�athermal�conductivity�detector,.�

Results:�Compared�to�phototrophic�growth,�photoheterotrophicgrowth�on�fructose�increased�A. variabilis‘�specific�H2 production�rate30-fold,�and�nitrogenase�activity�two-fold.�Cultures�for�microarraystudies�were�harvested�four�days�after�inoculation.�Cell�purificationand�cell-specific�RNA�extraction�were�confirmed�by�microscopicexamination�and�RT-qPCR�with�cell-specific�genes,�respectively.�Weare�using�cDNA�microarray�experiments�to�study�cell-specific�generegulation�in�response�to�changes�in�growth�conditions,�fromphototrophic�to�photoheterotrophic�and�to�fully�heterotrophic.�Togroup�genes�accurately�that�show�similar�expression�patterns�in�eachgrowth�condition�or�in�each�cell�type,�expression�data�will�be�clusteredusing�a�self-organizing�tree�algorithm�and�visualized�as�heat�maps.�Weexpect�to�be�able�to�categorize�types�of�responses�as�connecteddirectly�or�indirectly�to�H2 production.

Conclusions: We�will�report�microarray�studies�of�cell�type-specificgene�expression�in�a�filamentous�cyanobacterium.�We�expect�that�ourresults�will�answer�previously�unanswered�questions�on�cell-specificgene�regulation�and�H2 metabolism.�

P.124

FACTORS INFLUENCING PRODUCTION AND RELEASE OF THECYANOBACTERIAL TOXIN CYLINDROSPERMOPSIN.

Karina�Preußel,�Jutta�Fastner,�Ingrid�Chorus.

Umweltbundesamt�(Federal�Environmental�Agency),�Berlin,�Germany.

Introduction: The�cyanobacterial�secondary�metabolitecylindrospermopsin�(CYN)�is�an�inhibitor�of�eukaryotic�proteinsynthesis.�Unlike the�well�examined�cyanobacterial�peptide�toxins(microcystins),�CYN�often�occurs�with�a�high�proportion�of�extracellulardissolved�toxin�in�water�bodies�affected.�The�dissolved�CYN�fractionhas�important�implications�both�for�aquatic�ecosystems and�for�humanhealth:�as�CYN�inhibits�an�ubiquitous�process�in�growth�andmaintenance�metabolism�of�organisms,�the�dissolved�toxin�mayimpact�a�wide�range�of�aquatic�organisms�(including�plants)independently�of�their�trophic�relationships�to�cyanobacteria.

It�is�so�far�unknown�if�the�high�amounts�of�dissolved�CYN�in�persistingcyanobacterial�blooms�and�in�stationary�phases�of�batch�cultureexperiments�arise�from�passive�leakage�and�simultaneous�enrichmentdue�to�its�poor�biodegradability,�or�if�specific�environmental�factorsmay�enhance�an�active�CYN�release�from�cells.

Our�investigations�aim�at�improving�the�understanding�of�the�factorswhich�influence�CYN�production�and�release�especially�towardshuman�health�risk�assessment.

Methods: Semicontinous�as�well�as�batch�culture�experiments�withdifferent�CYN�producing�cyanobacterial�strains�were�performed�tomeasure�CYN�production�and�release�under�various�cultivationconditions.

Nine�different�combined�light-temperature�conditions�as�well�as�theinfluence�of�nitrogen�and�phosphorus�limitation were�tested.

Intracellular�and�extracellular�CYN�contents�were�analysed�by�LC-MS/MS�and�HPLC-PDA.

Results: Biovolume�related�CYN�contents�varied�at�maximum�by�afactor�of�2.7�in�Aphanizomenon strains�at�the�conditions�tested,�withtemperature�as�the�factor�causing�the�strongest�effects.�ExtracellularCYN�concentrations�amount�to�11�–�26�%�of�total�CYN�but�mayincrease�up�to�58�%�under�conditions�which�were�postulated�to�act�as

physiological�stress.

During�the�adaptation�phase�to�nitrogen�limitation�when�formation�ofadditional�heterocysts�is�induced�for�assimilation�of�atmosphericnitrogen,�the�intracellular�CYN�content�of�Aphanizomenon strainsincreases�up�to�twice�the�amount�found�in�cells�adapted�to�nitrogensaturation.�In�contrast�in�batch�cultures�with�limiting�factors�other�thannitrogen,�intracellular�CYN�decreases�continuously�and�CYN�release�issubstantially�higher�than�in�nitrogen�limited�cultures.�

Conclusions: The�variation�of�the�total�CYN�concentration�in�strainsfrom�temperate�lakes�responds�to�environmental�variables�in�a�rangesimilar�to�the�range�described�previously�for�the�well�investigatedcyanobacterial�peptide�toxins�(microcystins).

We�confirmed�that�CYN�release�could�not�only�be�related�to�cell�lysisand�propose�a�further�active�process�which�seems�to�be�regulated�byvarious�environmental�factors.�Incipient�nitrogen�limitation�reducedCYN��release�from�cells,�while�in�contrast�other�physiological�stressconditions,�particularly�photoinhibition�enhanced�release.

Risk�assessment�thus�needs�to�take�into�account�that�large�amounts�ofdissolved�CYN�may�occur�in�water�bodies�even�while�cyanobacterialblooms�are�intact.

P.125

VITAMIN-E DEFICIENCY RESULTS IN OVER-ACCUMULATION OFGLYCOGEN IN THE CYANOBACTERIUM SYNCHOCYSTIS SP.PCC6803.

Lisa�Rosgaard, Yumiko�Sakuragi.

Department�of�Plant�Biology�and�Biotechnology, Faculty�of�LifeSciences,�University�of�Copenhagen;��VKR�Research�Centre�“ProactivePlant”;�Strategic�Research�Consortium�“Fuel-for-Life”,�Faculty�of�LifeSciences,�University�of�Copenhagen;�Denmark.

Introduction: Photoautrotrophic�cyanobacteria�are�one�of�the�mostimportant�primary�producers�on�Earth.�They�are�responsible�for�nearly70%�of�CO2 assimilation�in�the�aquatic�environment�andunderstanding�of�their�carbon�metabolism�is�therefore�very�important(1).This�project�is�focussed�on�investigating�the�role�of�vitamin�E�and�itsrole�in�carbon�metabolism�in�the�model�cyanobacterium�Synechocystissp. PCC 6803.�The�previous�work�has�demonstrated�that�theSynechocystis mutant�slr1736- which�is�deficient�in�vitamin�E,�exhibitca.�20%�increased�photosynthetic�capacities�and�increased�total�sugarcontent (2).�We�hypothesize�that�vitamin�E�functions�as�regulator�ofphotosynthesis�and�carbon�metabolism,�in�addition�to�its�antioxidantactivity (2). We�want�to�understand�the�mechanism�by�which�vitamin�Eregulates�the�carbon�metabolism�and�in�this�work�we�examined�thecarbohydrate�content�of�the�slr1736- mutant.�

Methods:�The�total�sugar�content�was�determined�by�the�phenol-sulfuric�acid�method�as�described�in�(3)�and�the�total�hexose�contentwas�determined�using�anthrone�in�sulfuric�acid�according�to�(4).Glycogen�was�extracted�by�ethanol�precipitation�from�liquid�culturesof�wildtype�PCC6803�and�the�slr1736- mutant.�The�glycogen�extractwas�hydrolyzed�by�amyloglucosidase�and�the�amount�of�glucosereleased�was�quantified�by�colorimetric�determination�using�a�glucoseoxidase�and�peroxidase�coupled�assay.

Results: Analysis�of�the�slr1736- mutant�showed�that�amount�of�totalsugars�was�40%�higher�compared�to�the�wildtype.�The�content�of�totalhexoses and�glycogen was�also�determined;�the�slr1736- containedapproximately�30%�more�hexoses�and�ca.�70%�more�glycogen�ascompared�to�the�wildtype.�The�increase�in�total�hexose�content�waslargely�attributed�to�the�increase�in�glycogen.



Conclusion: The�results�confirm�the�previous�results�showingincreased�total�sugar�content�in�the�slr1736- strain.�The�increase�inhexose�content�found�in�the�slr1736- mutant�could�be�attributed�to�anincrease�in�glycogen�compared�to�the�wildtype.�This�suggests�aconnection�between�vitamin�E�and�glycogen�metabolism�and�furtherwork�to�elucidate�the�mechanism�of�glycogen�accumulation�in�slr1736-

is�in�progress�and�will�be�presented.

Bryant�DA�(2003).�Proc.�Natl.�Acad.�Sci.�USA�17:�9647-9640.

Sakuragi�Y,�Maeda�H,�DellaPenna�D,�Bryant�DA�(2006).�Plant�Physiol.141:�508-52.

Masuko,�T.,�Minami,�A.,�Iwasaki,�N.,�Majima,�T.,�Nishimura,�S-I.,�lee,�Y.(2005).�Analytic�Biochem.�39:69-72.

Fry,�S.�C.�(1988).�In:�The�Growing�Plant�Cell�Wall.�Wilkins,�M�(Ed).�pp.64-101.�Longman�Scientific�&�Technical,�Harlow,�UK.

P.126

OVEREXPRESSION OF PKNE (ALR3732) INHIBITS HETEROCYSTDEVELOPMENT IN ANABAENA SP. STRAIN PCC 7120.

Sushanta�Kumar�Saha,�James�W.�Golden.

Division�of�Biological�Sciences,�Molecular�Biology�Section,�Universityof�California,�San�Diego,�CA,�USA.

Introduction: Protein�kinases�play�a�key�role�in�signaling�pathways�bymodifying�the�activity�of�their�target�proteins.�The�nitrogen-fixingfilamentous�cyanobacterium Anabaena PCC�7120�contains�severaleukaryotic-type�serine/threonine�protein�kinases.�Reported�increasesof�intracellular�Ca2+ concentration�during�heterocyst�differentiationprompted�us�to�determine�the�spatiotemporal�expression�of�fourputative�Ca2+ dependent�serine/threonine�kinases,�and�then�furthercharacterize�the�role�of�pknE in�heterocyst�development.

Methods: Anabaena strains�were�constructed�to�contain�GFPreporters,�and�to�knockout�and�overexpress�pknE.�Strains�werecharacterized�by�standard�and�fluorescence�microscopy,�determinationof�growth�characteristics,�and�western�immunoassay�of�proteins.

Results:We�constructed�reporter�strains�containing�gfp reporterfusions�with�the�upstream�intergenic�regions�of�the�four�kinase�genesall2334,�alr3732 (pknE),�all4668,�and�all4838.�All�of�these�kinase�geneshad�low�levels�of�GFP�reporter�expression�when�grown�on�BG-11medium.�Upon�nitrogen�step-down,�the�GFP�fluorescence�wasincreased�in�differentiating�cells�from�the�promoters�PpknE and�Pall4668,not�changed�from�Pall2334,�and�very�low�throughout�the�filament�fromPall4838.�We�further�characterized�pknE because�it�showed�strong�up-regulation�of�GFP�reporter�expression in�differentiating�cells�afternitrogen�step-down.�The�pknE gene�was�inactivated�by�singlerecombination.�The�resulting�strain�was�nearly�normal,�but�had�shorterfilaments�with�slightly�higher�heterocyst�frequency�compared�to�thewild type�after�nitrogen�depletion.�Overexpression�of�pknE from�itsnative�promoter�on�a�multicopy�shuttle�plasmid�completely�inhibitedheterocyst�formation,�and�the�strain�was�unable�to�grow�on�nitrogen-depleted�medium.�However, overexpression�from�thecopper-inducible�petE promoter,�which�is�expressed�in�vegetativecells, did�not�inhibit�heterocyst�development�but�caused a�24�h�delayin�heterocyst�differentiation.�The�PpetE-pknE strain�also�formed�granularpleomorphic�vegetative�cells�after�4�days�of�growth�and�had�defectivecell�division�resulting�4�to�10�cell-lengths�long�cells on�BG-11�medium.We�examined�expression�of�the�hetR gene�in�pknE mutant�andoverexpression�backgrounds.�In�the�pknE mutant�background,�normalpatterned�expression�of�the�PhetR-gfp reporter�was�observed�duringheterocyst�development.�In�the strain�overexpressing�pknE from�itsown�promoter,�PhetR-gfp reporter�expression�was�inhibited;�and�western

blot�immunoassay�showed no�detectable�HetR�protein�in�this strain.Because�of�hetR positive�autoregulation,�the�pknE overexpressionphenotype�could�be�caused�by�a�block�either�upstream�ordownstream�of�HetR.�In�genetic�epistasis�experiments,�we�found�thatthe�introduction�of�pknE on�a�multicopy�plasmid�into�three�strains�thatproduce a�higher�frequency�of�heterocysts,�AMC451�(∆patS),AMC1289�(PpetE-hetRR223W), and�AMC1537�(∆hetR containing�hetR-His6), caused�complete�inhibition�of�new�heterocyst�formation�afternitrogen�step-down.�Therefore,�it�appears�that�overexpression�of�pknEblocks�heterocyst�differentiation�downstream�of�HetR.

Conclusions: The�pknE gene�is�developmentally�regulated,�and�itsproper�expression�is�required�for�both�normal�vegetative�cell�growthand�cell�division,�and�for�normal�heterocyst�development.

P.127

BIOCHEMICAL STUDIES OF THE PROTEINS ENCODED BY SOXGENES INVOLVED IN THIOSULFATE AND SULFIDE OXIDATIONFROM THE GREEN SULFUR BACTERIUM CHLOROBACULUMTEPIDUM.

Hidehiro�Sakurai,�Takuro�Ogawa,�Toshinari�Furusawa,�Michiko�Shiga,Ryohei�Nomura,�Daisuke�Seo,�Naomi�Hosoya-Matsuda,�KazuhitoInoue.

Res.�Inst.�Photobiol.�H2�Production,�Kanagawa�Univ.,�Hiratsuka,Kanagawa,�Japan.

Introduction:�The�green�sulfur�bacterium�Chlorobaculum tepidum,utilizes�various�sulphur compounds�such�as�sulfide,�thiosulfate�andelemental�sulphur as�electron�donors�for�phototrophic�growth.�The�sox(sulfur oxidizing�system)�gene�cluster�of�this�bacterium�consists�ofsoxF2XYZAKBW genes.�We�have�purified�and�characterized�threeproteins�(the�core�TOMES,�thiosulfate�oxidizing�multi-enzyme�system)indispensable�for�in�vitro�thiosulfate�oxidation.��We�have�also�purifiedSoxF2,�and�studied�its�effects�on�thiosulfate�oxidizing�activity�of�thecore�TOMES�as�well�as�its�sulfide oxidation��activities.

Methods: C. tepidum cells�were�photoautotrophically�grown�withsulfide and�thiosulfate�as�electron�donors,�and�proteins�were�purifiedto�apparent�homogeneity�as�described�elsewhere�(Ogawa�et�al.,�2008,Ogawa�et�al.,�in�preparation).�

Results:�From�C. tepidum cells,�we�have�purified�three�factorsindispensable�for�thiosulfate-dependent�reduction�of�small,�mono-heme�cytochrome�(cyt)�c-554:�SoxYZ, SoxB�and�SoxAXK�(the�coreTOMES).�The�last�component�is�trimeric�protein�(also�called�cyt�c-551)composed�of�mono-heme�cyt�SoxA,�mono-heme�cyt�SoxX,�and�thecolour-less�product�of�the�hypothetical�ORF�CT1020 (soxK).�The�SoxA,X�and�K�were�separately�expressed�in�Escherichia coli.�In�the�presenceof�other�two�core�TOMES�factors,�the�recombinant�SoxA�and�SoxXshowed�a�low�but�discernible�activity�of�thiosulfate-dependent�cyt c-554�reduction.�Further�addition�of�the�recombinant�SoxK�greatlyincreased�the�activity,�and�the�total�activity�was�as�high�as�that�of�thenative�SoxAXK�complex.�The�recombinant�SoxK�participated�information�of�a�tight�complex�with�SoxA�and�SoxX,�and�is�referred�to�asSAXB�(SoxAX binding�protein).�The�homologues�of�SAXB gene�(soxK)are�found�in�roughly�about�one�third�of�thiosulfate�oxidizing�bacteriawhose�sox gene�cluster�sequences�have�been�deposited�so�far,ranging�over�Chlorobiciae, Chromatiaceae, Hydrogenophilaceae, etc.Each�of�the�deduced�SoxA�and�SoxX�proteins�of�various�bacteriaconstitute�groups�that�are�distinct�from�those�found�in�bacteria�thatapparently�lack�soxK gene�homologues.

We�have�purified�SoxF2�as�a�monomeric�flavoprotein.�The�coreTOMES�catalysed�oxidation�of�both�thiosulfate�and�sulfite�with�various



cytochromes�as�electron�acceptors,�and��SoxF2�stimulated�the�formerand�inhibited�the�latter�reaction.�SoxF2�alone�catalysed�oxidation�ofsulfide�with�a�Km value�of�about�2�μM�with�various�cytochromes�aselectron�acceptors.�

Conclusions: The�components�of�the�core�TOMES�of�C. tepidum arebasically�similar�to�those�found�in�various�thiosulfate�oxidizing�bacteriasuch�as�the�purple�bacterium�Allochromation vinosum and�thefacultative�organotrophic�bacterium�Paracoccus pantotrophus.However,�we�have�found�that�SoxAX�protein�additionally�binds�a�newsubunit�SoxK�which�is�absent�in�some�of�them.

Although�soxF�gene�is�found�in�the�sox gene�cluster�of�variousbacteria,�its�biochemical�function�in�thiosulfate�oxidation�has�not�beendemonstrated�by�in�vitro�experiments.�We�have�shown�for�the�firsttime�that�it�stimulates�thiosulfate�oxidizing�activity�of�the�core�TOMESby�reconstituted�system.�

References:

Ogawa,�T.�et�al.,�SoxAX�binding�protein,�a�novel�component�of�thethiosulfate-oxidizing�multienzyme�system�in�the�green�sulphurbacterium�Chlorobaculum tepidum.�J.�Bacteriol.�190:�6097-6110(2008).

Ogawa�et�al.,�(in�preparation)�.��

P.128

SUCROSE METABOLISM IN MICROCYSTIS AERUGINOSA PCC7806.

María�A.�Kolman,�Laura�E.�Giarrocco,�Corina�M.�Berón,�Graciela�L.Salerno.

CEBB-MDP,�CIB,�FIBA,�Mar�del�Plata,�Prov.�Buenos�Aires,�Argentina.

Introduction: Sucrose�is�one�of�the�most�common�non-reducingdisaccharides�in�Nature.�It�has�been�extensively�studied�in�plants,where�it�is�the�main�transport�photosynthetic�product,�a�source�ofcarbon�and�energy,�and�a�molecule�associated�with�environmentalstress�and�signal�transduction.�During�the�last�decade,�Suc�metabolismwas�demonstrated�in�cyanobacteria.�Suc�is�synthesized�through�a�two-step�pathway�involving�SPS (U/ADP-glucose:D-fructose-6-phosphate2-α-D-glucosyltransferase)�and SPP�(sucrose-6F-phosphate-phospho-hydrolase),�either�in�unicellular�or�filamentous�cyanobacterium�strains.However,�Suc�cleavage�by�SuS�(A/UDP-glucose:D-fructose�2-α-D-glucosyl�transferase),�has�only�been�reported�in�filamentousheterocyst-forming�cyanobacteria,�where�Suc�was�demonstrated�toplay�a�crucial�role�in�N2 fixation�and�in�polysaccharide�production.�

Objective: The�aim�of�this�study�was�to�investigate�the�role�of�Suc�inMycrocystis aeruginosa,�a unicellular�non-N2 fixing�strain, well�knownas�one�of�the�most�common�bloom-forming�cyanobacteria�in�freshwater�environments.

Methods: M. aeruginosa PCC�7806�was�cultured�in�BG11�medium.DNA�fragments�corresponding�to�Suc�metabolism�candidate�geneswere�PCR�amplified.�The�open�reading�frames�(orfs)�were�ligated�intothe�pRSET�vector.�The�recombinant�His–tagged�proteins�wereexpressed�in�Escherichia coli BL21�(DE3)pLysS.�Proteins�were�purifiedby�Ni2+ affinity�chromatography,�while�protein�extracts�and�purification,enzyme�activities�and�expression�analyses�were�performed�as�reportedpreviously�for�Anabaena [Cumino�et al.�2007;�Curatti�et al.�2008].

Results:We�searched�in�the�M. aeruginosa genome�for�sequenceshomologous�to�those�of�functionally�characterized�genes�coding�forSPS,�SPP�and�SuS.�We�retrieved�three�contiguous�orfs in�the�contig328: IPF_1566, IPF_1564,�and�IPF_1565�whose�deduced�amino-acidsequences�are�about 55%,�53%,�and�72%�identical�to�the�protein

sequences�of�Anabaena sp.�PCC�7120�SPP�SPS, and�SuS-A,respectively.�The�resulting�His-tagged�fusion�proteins�showed�SPP,SPS,�and�SuS�activity.�In�addition,�cell�free�extracts�from�M. aeruginosawere�chormatographed�through�an�ion�exchange�column�and�theenzyme�activities�were�assayed�in�the�eluted�fractions.�When�westudied�the�biochemical�properties�of�the�enzymes,�we�found�that�SPSspecificity�was�similar�to�that�of�other�cyanobacterial SPSs,�andinterestingly,�SuS�activity�could�only�be�measured�in�the�Suc�cleavagedirection.�Expression�analyses�by�RT-PCR�and�Northern�blottingindicated�that�the�three�genes�are�transcribed�during�standard�cultureconditions�of�the�cyanobacterium.

Conclusions: This�is�the�first�report�showing�that�both�Suc�synthesisand�cleavage�take�place�in�a�potential�toxic�cyanobacterium.Remarkably,�we�showed�that�SuS�is�also�present�in�a�unicellular,�non-N2-fixing�strain.�The�fact�that�this�enzyme�acts�only�in�the�Suc�cleavagedirection�suggests�that�it�might�correspond�to�a�novel�class�of�SuS.Further�studies�on�this�enzyme�may�shed�some�light�into�its�reactionmechanism�and�into�the�role�of�Suc�in�Microcystis cells.

References:

Cumino�AC,�Marcozzi�C,�Barreiro�R,�Salerno�GL.�Plant�Physiol.�143,1385-1397�(2007)

Curatti�L,�Giarrocco�LE,�Cumino�AC,�Salerno�GL.�Planta�228,�617-625(2008)

P.129

REGULATION OF AUTOTROPHIC AND HETEROTROPHICMETABOLISM IN SYNECHOCYSTIS SP. STRAIN PCC 6803 BY TWO-COMPONENT SYSTEM CLUSTERS ON BOTH THE CHROMOSOMEAND A PLASMID.

Louis.�A.�Sherman,�Sowmya�Nagarajan.

Deptartment�of�Biological�Sciences,�Purdue�University,�West�Lafayette,IN,�USA.

Introduction: Synechocystis sp.�strain�PCC�6803�(Synechocystis)�is�afreshwater�cyanobacterium�with�a�3.57�Mb�chromosome�and�7plasmids,�including�3�over�100�kb.��The�chromosome�contains�80ORFs�(>2.5%)�that�code�for�Two�Component�System�(TCS)�proteinsthat�help�the�cell�adapt�to�environmental�changes.�We�are�interestedin the�TCS�in�a�three-gene�cluster�that�contains�a�histidine�kinase(Hik31),�a�DNA-�binding�response�regulator�(Rre34),�and�an�upstreamhypothetical�protein�(Uhp).��These�genes�were�strongly�up�or�down-regulated�under�several�growth�and�stress�conditions�in�the�wild�type(WT)�and�various�mutants�in�microarray�studies.�Importantly,�there�areduplicate�(>95%�identical)�operons�of�this�TCS�on�the�chromosome(sll0788-sll0790)�and�on�the plasmid�pSYSX�(slr6039-slr6041).�Nodetailed�study�of�plasmid-encoded�genes�has�been�performed�inSynechocystis�and�the�presence�of�these�two�clusters�(with�differentpromoters)�raises�important�questions�as�to�the�function�anddifferential�regulation�of�the�paralogs.�This�is�the�only�duplicated�TCSin�Synechocystis, and�the�only�cluster�present�on�both�thechromosome�and�a�plasmid,�among�16�sequenced�cyanobacteria.�

Methods:We�have�constructed�deletion�mutants�that�lack�all�threegenes�in�the�putative�operon�or�hik31 alone,�on�either�thechromosome or�the�plasmid,�and�on�both�the�chromosome�and�theplasmid.�Methodology�included�growth�under�defined�physiologicalconditions,�careful�measurements�of�cell�doubling�and�morphology,spectral�analysis�and�microarrays.

Results:�We�report�differences�in�morphology,�pigment�content�andgrowth�between�the�cultures.�Initial�phenotype�analysis�suggests�thatthe�chromosomal�operon�is�involved�in�negative�control�of�autotrophic



events,�whereas�the�plasmid�operon�is�involved�in�positive�control�ofheterotrophic�events.�First,�the�deletion�of�the�entire�plasmid�operonand�the�deletion�of�both�operons�resulted�in�strains�that�were�not�ableto�grow�in�the�presence�of�5�mM�glucose�under�12h�L-12h�Dconditions.��Secondly,�the�plasmid�operon�mutant�grows�better�oncontinuous�light�than�under�12h�LD�conditions.��Thirdly,�the�doublehik31 mutant�demonstrated�significant�alterations�in�pigmentcomposition�when�grown�in�the�presence�of�glucose�undermoderately�high�light�(150-200�µmoles�photons�m-2s-1).��Fourth,�thedouble�operon�mutant�demonstrated�the�poorest�growth�under�allconditions�studied�to�date.��Finally,�the�chromosome�operon�mutantgrows�best�under�many�conditions,�including�photoautotrophically�inhigh�light�and�mixotrophically�in�low�light.

Conclusions:��These�results�suggest�the�importance�of�genes�on�boththe�chromosome�and�the�plasmid�on�the�regulation�of�majormetabolic�processes.��In�addition,�all�three�genes�in�the�operonappear�to�be�functionally�important.��The�unique�nature�of�theseoperons�and�the�intriguing�phenotypes�of�the�mutants�indicate�thatthis�system�is�a�valuable�model�for�the�study�of�regulation.�Supportedby�a�grant�from�the�DOE.

P.131

PROTEOME DYNAMICS IN NOSTOC SP. PCC 7120 AND NOSTOCPUNCTIFORME ATCC 29133.

Karin�Stensjö1, Saw�Yen�Ow2,�Tanai�Cardona1,�Arnaud�Taton3,�AnnMagnuson1,�Peter�Lindblad1,�Phillip�C.�Wright2.1Department�of�Photochemistry�and�Molecular�Science,�The�ÅngströmLaboratories,�Uppsala�University,�Uppsala,�Sweden; 2Biological�&Environmental�Systems�Group,�Department�of�Chemical�and�ProcessEngineering,�The�University�of�Sheffield,�Sheffield,�United�Kingdom;3Center�for�the�Study�of�Biological�Complexity,�VirginiaCommonwealth�University,�Richmond,�VA,�USA.�

Introduction Nostoc sp.�PCC�7120�and�Nostoc punctiforme ATCC29133�are�multicellular�cyanobacteria capable�of�fixing�atmosphericnitrogen.�The�nitrogen�fixing�process�takes�place�in�heterocysts;specialized�cells�with�altered�metabolism�to�mediate�the�N2-fixingprocess.�One�of�our�aims�is�to�further�the�understanding�of�themetabolic�balance�between�the�heterocysts�and�the�photosyntheticCO2 fixing�vegetative�cells�through�the�use�of�quantitative�shotgunproteomics.�

Methods Relative�comparisons�between�the�proteomes�of�enrichedheterocysts,�vegetative�cells�and�complete�filaments�were�achievedusing�a�quantitative�proteomic�approach.�A�4-plex�or�8-plex�isobaricpeptide�tagging�technology�(iTRAQ) workflow�coupled�withchromatographic�pre-fractionation�and�tandem�mass�spectrometrywas�used.�Reliability�in�our�datasets is�exemplified�by�biologicalreplicates�for�all�samples.

Results More�than�500�proteins�from�each�strain�were�identified�withconfident�quantifications.�Observations�provided�by�purifiedheterocyst�analysis�enabled�the�elucidation�of�the�dominant�metabolicprocesses�between�the�respective�cell�types.�Approximately�20%�ofthe�identified�proteome�showed�significant�differential�regulation. Keyindicators�like�the�nitrogenase�enzyme�complex�and proteins�of�theoxidative�pentose�phosphate�cycle�were�as�expected more�abundantin�heterocysts�of�both�strains.�

More�interesting�are�the�findings�of�proteins,�such�as�thioredoxinsinvolved�in�redox�regulation�and�superoxide�dismutases�involved�incell�protective�processes�with�a�different�abundance�in�heterocysts�ascompared�to�vegetative�cells.�

Conclusions By�using�purified�heterocysts�we�are�able�to�present�aquantitative�investigation�with�much�higher�detail�relating�Nostoc sp.PCC�7120�and�N. punctiforme heterocyst�species�compared�to�in�ourearlier�investigation,�providing�resolutions�unachievable�by�wholefilament�proteomics.�Complementary�data�from�the�twocyanobacterial�strains�exhibit�similar�metabolic�trends.�Findingsobtained�from�this�study�contribute�to�the�understanding�of�heterocystspecific�metabolism�important�for�downstream�research�for�bio-fuelproduction�from�N2 fixing�heterocystous�cyanbacteria.

P.132

USING 23NA AND 31P NMR SPECTROSCOPY TO STUDY PH ANDNA+ HOMEOSTASIS IN SYNECHOCYSTIS SP. STRAIN PCC 6803.

Jessica�Li�Tse,�Hatice�Gizem�Yayla,�Mary�M�Allen,�Nancy�H�Kolodny.

Wellesley�College,�Wellesley,�MA,�USA.

Cyanobacteria�live�in�many�extreme�conditions,�including�hypersaline,hyperosmotic,�and�of�recent�interest,�increasingly�acidic�environmentscaused�by�pollution�and�industrial�waste.�Cells�have�been�shown�tosurvive�in,�and�adapt�to,�acidic�conditions�by�raising�the�pH�of�theirenvironment�[1],�but�the�mechanisms�that�they�use�to�regulate�theirinternal�(pHin)�and�external�(pHex)�pH�are�speculative.�We�hypothesizethat�the�cyanobacteria�use�Na+/H+ antiporters�to�regulate�their�internalpH.�The�model�organism�Synechocystis sp.�strain�PCC�6803�containsfive�putative�antiporter�genes,�which�code�for�transmembrane�proteinsthat�actively�transport�Na+ into�the�cell�cytoplasm�and�H+ out�of�thecell,�or�vice-versa,�depending�on�the�pH�of�the�environment [2].�ABruker�Avance�400�MHz�NMR�spectrometer�was�used�to�detectphosphorus�signals�in�solution,�such�as�internal�and�external�inorganicphosphate�to�determine�pHin and�pHex,�respectively,�sugar�phosphate,and��-,��-,�and��-ATP.�Sodium�signals�in�solution,�both�inside�(Na+in)and�outside�(Na+ex)�the�cyanobacteria,�were�detected�using�

23Na�NMRspectroscopy.�Since�the�Na+in and�Na

+ex peaks�resonate�at�the�same

frequency,�a�shift�reagent,�Na4HTmDOTP,�was�used�to�resolve�theNa+ex from�the�Na

+in peak�by�shifting�the�Na

+ex peak�downfield.�

23Naand�31P�NMR�spectra�were�acquired�for�control�cells�buffered�inminimal�BG-11�+�Na2CO3 medium�(pH�≈�10)�and�acid-stressed�cellsbuffered�in�60�mM�PIPES�medium�(pH�≈�6.5).�An�initial�23Na�NMRspectrum�was�acquired�after�addition�of�Na4HTmDOTP,�followed�by�atimed�series�of�31P�NMR�spectra�to�determine�the�pHin of�the�cells�anda�final�23Na�NMR�spectrum.�23Na�NMR�spectra�were�analyzed�todetermine�the�ratio�of�the�integral�of�Na+ex to�the�integral�of�Na

+in.�The

chemical�shifts�of�the�internal�inorganic�phosphate�peak�show�thatalthough�the�cell�samples�were�suspended�in�different�pHenvironments,�the�cells�were�able�to�maintain�a�pHin of�about�7.2.�ThepHex of�control�cells�decreased�over�time,�while�the�pHex of�the�acid-stressed�cells�increased,�suggesting�that�pH�regulation�does�occur.Addition�of�external�inorganic�phosphate�in�control�cells�producedspectra�in�which�the�internal�and�external�inorganic�phosphate�peakswere�completely�resolved,�which�was�not�seen�in�spectra�of�acid-stressed�cells.�In�both�the�control�and�acid-stressed�cell�samples,�using2.2�mM�Na4HTmDOTP,�the�integral�of�Na

+ex to�the�integral�of�Na

+in

decreased�over�time�between�initial�and�final�23Na�NMR�spectraacquisition,�suggesting�that�the�amount�of�Na+in increased�over�time.�

[1]�Huang�J.J. et al.�(2002)�Arch.�Microbiol. 177,�486�–�493.�

[2]�Wang�H-L et al.�(2002)�Mol.�Microbiol. 44,�1493�–�1506.�



P.133

WHICH IS THE INTRINSIC FORM OF PHOTOSYSTEM II, AMONOMER OR A DIMER?

Mai�Watanabe1,�Masako�Iwai2,�Rei�Narikawa1,�Masahiko�Ikeuchi1.1University�of�Tokyo,�Department�of�Life�Sciences�Biology;�2TokyoUniversity�Science,�Faculty�Science�and�Technology,�Department�ofApplied�Biological�Science;�Japan.

Introduction: Photosystem�II�(PSII)�is�a�membrane�protein�complexconsisting of�at�least�20�protein�subunits,�many�pigments�and�lipids.To�date,�both�dimeric�and�monomeric�PSII�complexes�have�beenprepared�from�a�wide�range�of�oxygenic�phototrophs.�However,�theoxygen�evolution�activity�of�PSII�was�often�higher�in�the�dimer�than�inthe�monomer.�Thus,�it�is�widely�believed�that�the�PSII�complex�mayfunction�as�a�dimer�in�the�thylakoid�membrane.�The�crystal�structuresof�the�dimeric�PSII�from�thermophilic�cyanobacteria�have�beendetermined�up�to�the�resolution�of�2.9�Å.�Here,�we�report�conversionbetween�the�dimeric�and�monomeric�complexes�of�PSII�by�treatmentof�a�detergent�and�gene�disruption.

Methods: The�thylakoid�membranes�from�Thermosynechococcuselongatus were�solubilized�with�various�concentrations�of�n-dodecyl-b-D-maltoside�(DM)�and�separated�by�blue-native�PAGE�(BN-PAGE)�andsubsequent�SDS-urea-PAGE�(16-22%�acrylamide).�Small�membranepolypeptide�gene�psbTc was�inactivated�by�replacement�with�achloramphenicol�cassette�and�complete�segregation�was�confirmed.

Results: When�the�thylakoid�membranes�were�solubilized�with�lowconcentrations�of�DM,�the�recovery�of�the�PSII�monomer�was�higherthan�the�dimer�in�BN-PAGE,�while�solubilization�with�highconcentrations�of�DM�gave�rise�to�lower�recovery�of�the�PSII�monomerthan�the�dimer.�The�chlorophyll�content�in�BN-PAGE�estimated�bymonochromatic�scanning�at�670�nm�confirmed�the�inverse�correlationbetween�the�dimeric�and�monomeric�bands�depending�on�the�DMconcentrations,�despite�that�maximal�recovery�of�the�total�PSII�wasachieved�above�0.6%�DM.�This�correlation�was�verified�as�spotintensity�in�the�two-dimensional�PAGE.�Namely,�spots�for�the�PSIIintegral�subunits�reflected�the�chlorophyll�distribution�in�BN-PAGE,being�exclusively�found�in�the�monomer�and�the�dimer�bands.�Theseresults�suggest�that�solubilization�with�high�concentration�of�DMartificially�facilitates�the�dimerization�of�the�monomer.�We�also�studiedsequential�treatment�of�PSII�with�0.5%�and�4.5%�DM.�Aftersolubilization�of�the�thylakoids�with�0.5%�DM,�the�solubilized�PSII�wasrecovered�by�centrifugation�and�further�treated�with�or�withoutaddition�of�4.5%�DM�(final�5.0%).�Results�thus�obtained�were�verysimilar�to�those�in�the�one-step�solubilization.�This�indicates�that�amajor�part�of�the�dimer�is�generated�from�the�monomer�aftersolubilization�by�action�of�the�additional�DM�treatment.�In�the�crystalstructure,�there�are�four�DM�molecules�at�the�monomer-monomerinterface�of�PSII.�This�may�suggest�that�the�dimeric�PSII�in�the�crystal�isartificially�stabilized�by�insertion�of�the�DM�molecules.�There�are�alsoPsbTc�polypeptides�at�the�monomer-monomer�interface�in�the�PSIIcrystal.�We�studied�the�PsbTc-depleted�PSII�by�BN-PAGE�aftersolubilization�with�DM.�At�low�concentrations�of�DM,�very�little�amountof�the�PSII�dimer�was�recovered,�while�the�recovery�of�the�dimer�wasincreased�at�high�concentrations.�This�result�suggests�that�PsbTc�isessential�for�the�stability�of�the�dimer�in�low�concentrations�of�DM,�butis�not�essential�for�the�dimerization�driven�by�high�concentrations�ofDM.

Conclusion: We�conclude�that�the�PSII�complex�exists�in�the�thylakoidas�a�monomer�or�a�loosely�associated�dimer�which�is�surrounded�bylipids.�PsbTc�is�essential�for�stabilization�of�the�dimer�but�not�essentialfor�the�dimerization�driven�by�high�concentration�of�DM.

P.134

THREE INDIVIDUAL POINT MUTATIONS IN SYNECHOCOCCUSPCC6301 RUBISCO CONFER INCREASED OXYGEN TOLERANCEIN VIVO UNDER AEROBIC CHEMOAUTOTROPHIC GROWTHCONDITIONS IN RHODOBACTER CAPSULATUS SBI/II-.

Brian�Witte,�Stephanie�Scott,�Sriram�Satagopan,�F.�Robert�Tabita.

Ohio�State�University,�Columbus,�OH,�USA.

Introduction: Ribulose�bisphosphate�carboxylase/oxygenase(RubisCO)�is�the�key�enzyme�of�the�Calvin-Benson-Bassham�(CBB)pathway,�and�is�directly�or�indirectly�responsible�for�most�of�theorganic�carbon�on�Earth.��Despite�its�importance,�the�enzymepossesses�confused�substrate�specificity�and�often�wastes�cellularcarbon�in�incorporating�O2 instead�of�CO2.�This�study�uses�randommutagenesis�of�the�RubisCO�gene�from�Synechococcus PCC6301�anda�powerful�biological�selection�system�that�was�previously�developedin�Rhodobacter capsulatus to�select�for�mutant�enzymes�that�conferincreased�oxygen�tolerance�in vivo.��Further,�the�mutant�RubisCOshave�been�purified�and�characterized�in vitro.��We�hypothesize�that�aspecific�region�of�the�RubisCO�large�subunit�is�especially�important�forinteractions�with�O2.

Methods: The�genes�encoding�the�large�(rbcL)�and�small�(rbcS)subunits�of�RubisCO�from�Synechococcus PCC�6301�were�cloned�intandem�into�pUC19.��The�vector�with�insert�was�used�as�the�templatein�repeated�rounds�of�mutagenic�pcr.��The�pool�of�mutagenized�rbcLSwas�subcloned�into�the�broad�host�range�plasmid�pRPS-MCS3�andsubsequently�transferred�into�the�RubisCO-deletion�strain�of�R.capsulatus SBI/II-.�Individual�colonies�were�used�for�creating�replicaplates�for�incubation�under�photoheterotrophic�(minimal�salts�plusmalate,CO2/H2 atmosphere),�photoautotrophic�(minimal�salts,�CO2/H2

atmosphere)�and�chemoautotrophic�(minimal�salts�plates,�CO2/H2/O2

atmosphere).��Levels�of�intracellular�RubisCO�were�monitored�usingWestern�blots�to�ascertain�the�approximate�fraction�of�total�proteincomprised�of�RubisCO.�Mutanted�6301�genes�that�complementedgrowth�under�all�autotrophic�conditions�were�subcloned�intoEscherichia coli expression�vector�pSKB3�and�the�products�purifiedand�characterized�in vitro.

Results: Using�the�6301�numbering�scheme,�mutations�at�residues259,�269�and�375�enabled�6301�RubisCO�to�complement�growth�inSBI/II- under�aerobic�chemoautotrophic�conditions.��Growth�curves�ofSBI/II- complemented�with�each�mutant,�as�well�as�WT�6301,�areprovided.��The�kinetic�data�with�recombinant�proteins�indicated�thateach�mutation�confers�a�subtly�different�effect�on�RubisCOmechanism,�although�Km�(CO2), Km(O2) and�kcat were�affected�todifferent�degrees.

Conclusions: It�is�difficult�to�say�that�any�one�of�the�mutant�RubisCOsis�truly�“better”�although�they�do�definitely�confer�an�increasedtolerance�for�oxygen�under�chemoautotrophic�growth�conditions�andenable�growth�under�conditions�where�the�wild–type�enzyme�doesnot.�Interestingly,�none�of�the�residues�identified�in�this�study�arelocated�directly�within�the�active�site�of�the�enzyme.��Previous�studieshave�shown�that�alterations�to�active�site�residues�always�results�in�aloss�of�enzyme�function.��Rather,�this�study�seems�to�indicate�theimportance�of�residues�neighboring�the�active�site�that�could�affectthe�access�of�gaseous�substrates�to�the�enediol�of�ribulosebisphosphate�in�the�reaction�center.��It�is�also�possible�that�one�or



more�of�these�residues�alter�interactions�with�R. capsulatus intracellularchaperones�such�that the�observed�growth�under�aerobic�conditionsmay�be�due�in�part�to�greater�stability�of�the�mutant�enzymes.Nevertheless,�the�combination�of�in�vivo�complementation�and�growthunder�selective�conditions�combined�with�in vitro enzymologicalstudies,�projects�an�intriguing�new�direction�for�research�into�the�basisfor�RubisCO’s�interaction�with�oxygen.�

P.135

THE REDOX-SENSING MECHANISM OF REGB IN RHODOBACTERCAPSULATUS.

Jiang�Wu,�Carl�Bauer.

Indiana�University,�Bloomington,�Indiana,�USA.

RegB/RegA�is�a�global�redox-responding�two-component�system�thatregulates�many�energy-generating�and�energy-utilizing�processes�inRhodobacter capsulatus. Previous�studies�have�shown�that the�sensorkinase�RegB�modulates�its�kinase�activity�and�sense�redox�signalsthrough�two�different�signal-input�sites:�a�ubiquinone-binding�sitelocated�in�a�periplasmic�loop�which�can�monitors�the�oxidation�statesof�ubiquinone�pool�and�a�redox-active�cysteine�in�the�cytosolicdomain�that�responds�to�cellular�redox�state.�The�ubiquinone-bindingsite�and�redox-active�cysteine�appear�to�work�independently�and�bothcontrol�a�part�of�RegB�activity.�

To�further�elucidate�the�mechanism�by�which�RegB�senses�the�redoxsignal,�the�roles�of�two�highly�conserved�residues in�thetransmembrane�domain,�R31�and�Q38,�have�been�analyzed�bymutagenesis�study.�Mutations�at�both�of�these�residues�compromisedthe�inhibitory�effect�of�oxidizing�condition�on�RegB�activity,�indicatingthat�R31�and�Q38�are�involved�in�redox�sensing.�To�investigate�howexactly�RegB�interacts�with�its�redox�signal�ubiquinone,�the�bindingaffinities�of�oxidized�ubiquinone�analog�Q0�and�reduced�Q0 to�RegBhave�been�determined�by�Isothermal�Titration�Calorimetry�(ITC).�ITCdetermined�the�Ka�of�oxidized�Q0 to�RegB�while�there�is�nodetectable�binding�affinity�for�reduced�Q0.�Consistent�with�ITC�data,�invitro phosphorylation�assay�showed�that�two�mutations�in�theubiquinone-binding�site,�N110Q�and�F112Y,�will�give�RegB�higheractivity�in�the�presence�of�oxidized�Q0.�In vivo, both�mutants�hadelevated�photosystem�expression�levels�under�aerobic�conditions.Thus,�we�conclude�that�oxidized�ubiquinone�is�the�form�that�bind�toRegB�and�inhibit�RegB�activity,�when�ubiquinone�is�converted�toreduced�form,�it�will�be�dissociated�from�RegB�and�RegB�activity�willbe�released.��

P.136

DISCOVERY OF NOVEL O2-TOLERANT NIFE-HYDROGENASESFROM ENVIRONMENTAL MICROBES TO CONSTRUCT ACYANOBACTERIAL RECOMBINANT FOR SOLAR H2 PRODUCTION.

Qing�Xu,�Gergely�Maroti,�Walter�Vargas,�Yingkai�Tong,�Shibu�Yooseph.

J.�Craig�Venter�Institute,�Rockville,�MD,�USA.

Hydrogen�is�a�clean�alternative�to�gasoline�and�other�fossil�fuels.Photosynthetic�cyanobacteria�have�attracted�considerable�attention�inrecent�years�since�they�can�photolytically�split�water�to�support�H2

production�through�hydrogenase�catalysis.��However,�one�majordrawback�of�the�biophotolysis�process�is�that�their�H2-evolvinghydrogenases�are�extremely�sensitive�to�O2.��To�develop�an�O2-tolerant�biophotolytic�system,�we�are�searching�for�O2-tolerantNiFe-hydrogenases�that�can�be�transferred�into�cyanobacteria.��Theoceans�harbor�an�abundance�of�microorganisms�with�H2-productioncapability�and�they�are�good�resources�for�identifying�novel�O2-

tolerant�hydrogenases.�The�Venter�Institute�recently�conducted�ametagenomic�study�of�the�marine�planktonic�microbes�in�the�globalocean.��We�searched�the�metagenomic�data�of�this�global�oceansampling�project�for�putative�novel�NiFe-hydrogenases�by�usingprobabilistic�modeling�methods.��One�of�the�novel�hydrogenasesidentified�from�the�global�ocean�was�cloned�from�environmental�DNAsamples,�which�shows�strong�homology�to�a�known�O2-toleranthydrogenase�from�a�phototropic�bacterium�Thiocapsa roseopersicina.The�cloned�environmental�DNA�contains�the�structural�genes�(hynSand�hynL)�of�this�novel�hydrogenase.��Located�immediately�upstreamof�hynS/hynL,�two�accessory�genes�(hupH and�hynD)�were�alsoidentified.��Through�heterologous�expression�in�T. roseopersicina,�weconverted�the�environmental�DNA�into�a�functional�NiFe-hydrogenase.��Transferring�the�novel�NiFe-hydrogenase�intocyanobacterium�Synechococcus elongates PCC7942�is�in�progress.��

P.137

RHODOBACTER CAPSULATUS HBRL AND IRR: TWO MORETRANSCRIPTION FACTORS INVOLVED IN THE REGULATION OFTHE HEME BIOSYNTHESIS.

Sébastien�Zappa,�Carl�E.�Bauer.

Department�of�Biology,�Indiana�University,�Bloomington,�IN,�USA.

The�purple�photosynthetic�bacterium�Rhodobacter capsulatuspresents�a�versatile�metabolism,�capable�of�growing�from�dark�aerobicto�light�anaerobic�conditions.�In�order�to�achieve�such�metabolicadaptations,�the�synthesis�of�pigments�has�to�be�tightly�regulated�andthe�balance�between�the�major�tetrapyrroles�heme,bacteriochlorophyll�and�cobalamin�maintained�at�adequate�levels.�Ithas�been�shown�in�the�lab�that�several�transcription�factors�areinvolved�in�the�regulation�of�tetrapyrrole�synthesis, i.e. RegA,�CrtJ,AerR.�More�recently,�studies�were�undertaken�about�two�newtranscription�factors,�namely�HbrL�and�Irr.�While�the�first�one�is�amember�of�the�LysR�family,�the�second�is�a�Fur�representative.�Bothwere�studied�with�regard�to�their�effect�on�heme�biosynthesis,�as�afunction�of�the�exogenous�heme�content�or�the�availability�of�iron�inthe�growth�medium.�Their�respective�roles�were�investigated�bygenetic�and�biochemical�approaches.�Indeed,�gene�disrupted�strainswere�produced�and�their�phenotypes,�regarding�the�expression�ofheme�synthesis�genes,�were�analyzed.�In�addition,�both�protein�wereheterologously�expressed�in�Escherichia coli TUNER(DE3),�using�thepSUMO�vector.�Purification�of�the�recombinant�protein�by�affinitychromatography�enabled�to�undertake�studies�regarding�DNA�and/orco-factor�binding.�It�appeared�that�the�HbrL�deleted�strain�is�verysensitive�to�iron-chelated�media,�while�the�Irr�mutant�shows�a�cellularheme�content�approximately�25%�higher�than�the�wild-type�and�theHbrL-deleted�strains.�Regarding�gene�expression�of�the�hemebiosynthesis�pathway,�while�HbrL�shows�a�control�on�hemA,�hemB�andhemZ,�it�looks�like�Irr�has�an�effect�on�hemC�and�hemE.�Finally,�thebiochemical�characterization�gave�some�insights�of�the�DNA�bindingmechanism,�such�the�as�the�binding�of�HbrL�to�promoters�such�ashemA�and�puf,�as�shown�by�gel�mobility�shift�analysis,�and�preliminaryDNAseI�footprinting�assay.�As�a�conclusion,�this�work�confirms�thatHbrL�and�Irr�are�involved�in�the�regulation�of�the�complex�tetrapyrrolesynthesis�pathway,�in�addition�to�previously�characterized�transcriptionfactors.



P.138

METABOLIC ENGINEERING OF SYNECHOCYSTIS SP. PCC 6803 - A SYSTEMS BIOLOGY APPROACH.

Yvonne�Zilliges,�Jan-Christoph�Kehr,��Wolfgang�Lockau,�ThomasBörner.

Humboldt-Universität�zu�Berlin,�Institut�für�Biologie, Berlin,�Germany.

Introduction: Cyanobacteria,�also�known�as�blue-green�algae, are�theonly�known�oxygenic�photoautotrophic�prokaryotes.�They�play�a�majorrole�in�global�ecology�and�the�assimilation�of�inorganic�carbon�fromthe�atmosphere.�The�genome�of�the�unicellular�cyanobacteriumSynechocystis sp.�Strain�PCC�6803�was�the�first�to�be�fully�sequencedof�any�phototrophic�organism.�This�strain�thus�represents�an�idealcandidate�for�a�systems-level�description�of�primary�metabolism�andits�regulation�–�ultimately�aiming�at�an�iterative�construction�of�acomputational�model�of�the�core�metabolic�processes.�Thecombination�of�a�metabolic�network�model�with biochemical�andphysiological�data�allow�for�a�deep�understanding�of�photosyntheticand�metabolic�processes�and�their�regulation�in�a�systems�biologyapproach.

Methods and Results: In�our�studies,�we�analyzed�several�mutantswhere�enzymes�of�glycolysis�and�especially�of�pyruvate�metabolism,�ofmetabolism�of�storage�compounds�and�of�the�Calvin�cycle�weremanipulated.�These�mutants�were�characterized�with�respect�togrowth�parameters,�pigmentation�and�intra-�as�well�as�extracellularmetabolites�by�ion�chromatography�and�optic�enzymatic�tests�formetabolic�fingerprinting.�These�data�are�used�for�a�systems-leveldescription�of�primary�metabolism�and�its�regulation.�This�approach�toa�model�is�also�assisted�by�transcriptomic�data�and�will�be�furtherrefined�by�the�incorporation�of�experimentally�determined�kineticparameters�of�selected�enzymes.

Conclusion: The�establishment�of�an�accurate�model�combinesdifferent�data�from�transcriptomics,�metabolomics�and�kinetics.�Usingthe�established�methodology�of�flux-balance�analysis,�we�evaluate�themain�modes�of�metabolism,�identify�metabolic�bottlenecks�andcorrelated�reaction�sets.�

Based�on�the�computational�model,�mutations�could�be�introducedand�the�resulting�phenotypes�compared�to�the�theoretical�predictions.The�final�model�is�expected�to�permit�the�selection�of�candidategenes�for�the�optimization�of�biotechnological�processes.



ENVIRONMENTAL PROTEOMICS22�Bickerton�Ave.�Sackville,�NB��E4L�3M7Canada

Phone:� 506-536-7426Fax:� 506-536-1421Web:� www.environmentalproteomics.ca�

QUBIT SYSTEMS INC.700�Gardiners�Road,�Unit�105Kingston,�ON,�K7M�3X9Canada

Phone:� 613-384-1977�(888-262-2219)Fax:� 613-384-9118Web:� www.qubitsystems.com

Qubit’s�Photobioreactors�are�used�for�precise�phototrophiccultivation�of�algae�and�cyanobacteria.�They�feature�a�uniquecombination�of�cultivator�and�monitoring�device.�Temperature,aeration�gas�composition,�Light�intensity�and�spectral�compositioncan�be�set�with�a�high�accuracy.�In�addition,�cultivation�conditionscan�be�dynamically�varied�according�to�a�user�defined�protocol.�

SATLANTIC INC.Richmond�Terminal�Pier�93481�North�Marginal�RdHalifax,�NS�B3K�5X8Canada

Phone:� 902-492-4780Fax:� 902-492-4781Web:� www.satlantic.com�

Satlantic�develops�active�fluorometers�for�the�study�of�algal�andbacterial�photosynthesis.�We�also�offer�real-time�in-situ�nutrientsensors�and�sophisticated�instrument�integration�and�water�qualitysystems�that�enable�real-time�operational�decision-making.�Ourcustom�design�capabilities�enable�us�to�provide�our�clients�withthe�ideal�solution�package.�

Exhibitors





Notes

Abed,�Raeid P.090

Adams,�David OC-11.6,�P.035

Ahn,�Chi-Yong P.066

Akinwole,�Philips P.057

Al-Shehri,�Abdulrahman P.036

Alexova,�Ralitza OC-12.5

Allison,�L.F. P.037

Alvey,�Richard OC-10.5

Appel,�Jens OC-18.3

Attia,�Magdy P.038

Autenrieth,�Caroline P.001

Azai,�Chihiro P.002

Bagheri,�Homayoun P.078

Ballot,�Andreas OC-1.6

Batyrova,�Horcheska P.003

Bauer,�Carl PL-6.1

Beatty,�J.�Thomas PL-7.2,�P.004

Bennette,�Nicholas P.091

Bergman,�Brigitta PL-2.1

Bibby�,�Thomas�S. OC-1.2

Bird,�David P.058

Biswas,�Avijit P.092

Blankenship,�Robert PL-1.1

Bonsu,�Ernest�O. P.093

Boulay,�Clémence P.005

Brown,�Igor�I. P.059

Bryan,�Samantha P.094

Bryant,�Donald�A. OC-9.2

Burnap,�Robert P.006,�P.007

Burroughs,�Nigel P.039

Campbell,�Douglas P.095

Cardona,�Tanai P.032

Chen,�Min P.008,�P.096

Christman,�Harry OC-5.1

Cox,�Raymond�P. P.060

Csotonyi,�Julius�T. P.040

Daldal,�Fevzi PL-5.1

Dangel,�Andrew OC-7.1

De�Philippis,�Roberto OC-17.3,�P.041

Dey,�Swati P.097

Doná,�Clelia OC-14.2

Dorador,�Cristina P.079

Dragnea,�Vladimira P.009

Dragomani,�Tierna P.098

Druga,�Bogdan P.042

Duggan,�Paula P.099

Eaton-Rye,�Julian�J OC-6.1

Eddie,�Brian�J. P.100

Eisenhut,�Marion OC-6.6

Elhai,�Jeff OC-14.1

Espie,�George P.101

Ferreira,�Daniela P.043

Fewer,�David OC-13.2

Florencio,�Francisco�J. OC-10.4

Flores,�Enrique PL-3.1

Forchhammer,�Karl PL-3.3

Fortin,�Nathalie OC-15.5

Frigaard,�Niels-Ulrik OC-13.1

Fuhrmann,�Eva P.077

Gaertner,�Katrin P.010

Galmozzi,�Carla�V. P.102

Gao,�Qunjie P.103

Garcia�Costas,�Amaya P.061

Garcia-Pichel,�Ferran OC-16.5,�P.104

Ghosh,�Robin P.105

Giani,�Alessandra OC-3.3,�P.044

Golden,�Susan PL-4.1

Gomolla,�Dorothea P.011

Gorlenko,�Vladimir PL-1.3,�P.062

Grammel,�Hartmut OC-15.4

Grossman,�Arthur PL-2.2

Gugger,�Muriel P.080

Haande,�Sigrid P.063

Hagemann,�Martin PL-3.2

Hahn,�Alexander P.106

Haimovich,�Maya OC-11.5

Hanson,�Thomas�E. OC-4.1

Harwood,�Caroline PL-9.1

Hervás,�Manuel P.012

Hess,�Wolfgang OC-11.3

Hirose,�Yuu P.107

Hosokawa,�Norimune P.108

Inoue-Sakamoto,�Kaori P.045

Ishizuka,�Takami P.109

Iwamori,�Shunsuke P.110

Jackson,�Owen OC-5.2

Jacobsen,�Jacob P.046

Johansen,�Henning�Kristian P.064

Johnson,�Zackary OC-3.2

Joshi,�Gauri P.111

Jungblut,�Anne�D. OC-1.5

Kaplan,�Aaron OC-6.5,�OC-10.1

Karp,�Peter OC-2.4

Katayama,�Mitsunori P.112

Kawabata,�Tadashi P.013

Keren,�Nir OC-16.6

Kerfeld,�Cheryl OC-9.4

Khalfaoui�Hassani,�Bahia P.014

Kirilovsky,�Diana PL-6.2

Kitashima,�Masaharu P.113

Knaff,�David OC-8.1

Knoop,�Henning P.114

Kobayashi,�Masayuki P.015

Komada,�Jun P.070

Korn,�Anja P.016

Kurmayer,�Rainer OC-15.6

Kushige,�Hiroko P.115

Kyndt,�John OC-11.1,�P.017

Laguna,�Rick P.047

Le,�Thi�Anh�Tuyet P.048

Lechno-Yossef,�Sigal P.018

Lee,�Young-Ki P.071

Lindblad,�Peter OC-18.1

Liu,�Jinjie OC-5.4

Liu,�Zhenfeng OC-4.3

Ludwig,�Frank P.081

Ludwig,�Marcus P.019

Luther,�Amanda P.116

Maeda,�Isamu OC-17.1,�P.117

Magnuson,�Ann OC-12.3

Maness,�Pin-Ching OC-18.2

Masepohl,�Bernd PL-6.3

Masukawa,�Hajime P.049

Mazard,�Sophie P.065

McNeely,�Kelsey OC-12.2

Meeks,�Jack OC-13.3



Index of Presenters



Index of Presenters and Chairs

Mella-Herrera,�Rodrigo P.118

Meyer,�Terry P.020,�P.021

Midorikawa,�Takafumi P.119

Miller,�Mette OC-14.3

Mulkidjanian,�Armen�Y. PL-1.2,�P.022

Mullineaux,�Conrad OC-5.3

Mundt,�Sabine P.050

Narikawa,�Rei OC-16.3

Nenninger,�Anja P.082

Nicolaisen,�Kerstin P.120

Núñez-Cardona,�M.T. P.072,�P.083

Oh-oka,�Hirozo P.121

Ohki,�Kaori OC-3.1

Ohta,�Hisataka P.122

Otaki,�Hiroyo OC-2.6

Ouchane,�Soufian OC-7.3

Overmann,�Jörg OC-2.1

Owttrim,�George�W. P.023

Palinska,�Katarzyna�A. OC-1.4

Park,�Jeong-Jin P.123

Partensky,�Frederic PL-5.3

Pedersen,�Marie�Ø. OC-9.1

Pereira,�Sara P.051

Pitt,�Frances�D. OC-10.3

Preuflel,�Karina P.124

Razi,�Saiqa P.052

Redding,�Kevin OC-6.4

Rosgaard,�Lisa P.125

Saha,�Sushanta�Kumar P.126

Sakamoto,�Toshio OC-10.6

Sakurai,�Hidehiro OC-15.1,�P.127

Salerno,�Graciela�L. P.128

Sandmann,�Gerhard OC-8.3

Schaefer,�Steffani OC-6.3

Schluchter,�Wendy OC-16.2

Schmetterer,�Georg OC-16.1

Schumann,�Susanne P.084

Schwarz,�Christoph OC-2.5

Schwarz,�Rakefet OC-16.4

Setterdahl,�Aaron P.025

Severin,�Ina OC-2.3

Shabeb,�Mohamed P.085

Shastik,�Evgeny P.073

Shen,�Gaozhong OC-9.6

Sherman,�Louis PL-5.2,�P.129

Shimura,�Yohei P.074

Sicora,�Cosmin P.033

Sivonen,�Kaarina PL-9.3

Song,�Hong-Gyu P.053

Spence,�Edward P.067

Stadnichuk,�Igor OC-11.2

Stal,�Lucas OC-10.2

Steglich,�Claudia OC-11.4

Stensjö,�Karin P.131

Summerfield,�Tina OC-12.1

Summers,�Michael OC-5.5

Suzuki,�Eiji OC-12.4

Suzuki,�Iwane PL-7.1

Tabita,�F.�Robert KN-2.1

Takaichi,�Shinichi OC-8.2,�P.054

Tamagnini,�Paula�M. PL-9.2

Tang,�Kuo-Hsiang OC-4.2,�P.026

Tank,�Marcus OC-1.1

Tekucheva,�Darya P.055

Thiel,�Teresa OC-5.6

Thiel,�Vera P.086

Tomitani,�Akiko P.087

Tse,�Jessica�Li P.132

Tsukatani,�Yusuke P.027

Tsygankov,�Anatoly OC-15.3

Vasquez,�Yasmin P.034

Ventura,�Stefano OC-1.3,�P.068

Vermaas,�Wim OC-6.2,�OC-9.3

Vioque,�Agustin P.075

Vogl,�Kajetan P.076

Walsby,�A.E. KN-1.1

Watanabe,�Mai P.133

Watson,�Susan�B. OC-12.6

Wegener,�Kimberly�M. P.028

Wilmotte,�Annick P.069,�P.088,�P.089

Wilson,�Adjélé P.029

Witte,�Brian P.134

Wolk,�Peter�C. PL-4.2

Woronowicz,�Kamil OC-9.5

Wu,�Jiang P.135

Xu,�Qing P.136

Xu,�Yu P.056

Yamamoto,�Haruki P.030

Yeager,�Chris OC-15.2

Yin,�Liang OC-7.2,�P.031

Yurkov,�Vladimir OC-2.2

Zannoni,�Davide OC-17.2

Zappa,�Sébastien P.137

Zilliges,�Yvonne P.138

Documents

ISPP2009 Final Program