Ba cterial si ngle- c ell approaches to the relationship between diversity and function

BaBacterial cterial sisingle-ngle-ccell ell approaches to the approaches to the relationship between relationship between diversity and function diversity and function in the in the SSeaea

Marine Biodiversity Cluster Meeting - Brussels-July’03Marine Biodiversity Cluster Meeting - Brussels-July’03

EVK3-CT2002-00078EVK3-CT2002-00078November 2002 - October 2005November 2002 - October 2005

1 µm1 µm

Image:

K. Jü

rgens

The project’s ultimate goalThe project’s ultimate goal

Roundicoccus Roundicoccus southamptiisouthamptii

TinymonasTinymonas bremenensisbremenensis

Dalibacter Dalibacter banyuleusbanyuleus

(all names are fiction)(all names are fiction)

SpirovibrioSpirovibrio kalmariensiskalmariensis

75% of BCD75% of BCD

dominates dominates DMSP uptakeDMSP uptake

preferentiallypreferentiallygrazed by HNFgrazed by HNF

very sensitivevery sensitiveto viral attackto viral attack

C) Single-cell approachesC) Single-cell approaches

linkagelinkageB) Bacterial diversityB) Bacterial diversity

A) Bacterial biogeochemical A) Bacterial biogeochemical functionfunction


By developing new methodologies, sampling By developing new methodologies, sampling different European seas and through laboratory and different European seas and through laboratory and mesocosm experiments, we will address the main mesocosm experiments, we will address the main objectives of BASICS:objectives of BASICS:

The identification of the most important prokaryotic organisms associated with the

biogeochemical functioning (in the carbon and sulfur cycling) of the sea, through the development of single-cell analysis techniques applied to pelagic microbes. BASICS will also study how resilient the

link is between the diversity and the C and S biogeochemical cycling by bacterioplankton, in the face of the most important global environmental changes expected in European coastal waters.

Objective 1: To describe bacterioplankton diversity in the coastal seas of Europe

Objective 2: To describe the seasonal changes in the cycling of carbon and sulfur mediated by planktonic bacteria in surface waters of European coastal seas

Objective 3: To design, test and fine-tune different methods and research strategies for the single-cell analysis of natural bacterioplankton organisms

Objective 4: To link bacterial diversity and biogeochemical function (in the cycles of C and S) and identify the bacterial phylotypes responsible for the crucial steps in oceanic biogeochemical cycling, and to refine recently developed conceptual frameworks for the links between species richness (number of dominant coexisting species) and biogeochemical cycling

Objective 5: To estimate the effect of environmental changes affecting the ocean’s bacterially-mediated biogeochemical function, global bacterial diversity and the link between bacterial diversity and C and S cycling

-) Why bacteria ?-) Why bacteria ?A) Why bacterial diversity ? A) Why bacterial diversity ? B) Why biogeochemical function ?B) Why biogeochemical function ?

• • why the C cyclewhy the C cycle• • why the S cyclewhy the S cycle

C) Why study the linkage ?C) Why study the linkage ?D) The key: Single-Cell methodsD) The key: Single-Cell methodsE) Project organizationE) Project organizationF) Project partnershipF) Project partnership

• • The sampling sitesThe sampling sites• • A few of the first resultsA few of the first results

• • Most of Earth’s living biomassMost of Earth’s living biomass• • Most abundant living particles in the seaMost abundant living particles in the sea• • Only significant DOM transformers Only significant DOM transformers • • Responsible for most of ocean’s respirationResponsible for most of ocean’s respiration• • Largest living surface in the oceanLargest living surface in the ocean

• • The largest “unknown pool” of genomic and The largest “unknown pool” of genomic and metabolic (i.e. functional) diversitymetabolic (i.e. functional) diversity

Bacteria are...Bacteria are...

• • bacteria play far more important ecological roles in naturalbacteria play far more important ecological roles in natural environments than their small sizes would suggest (Brock environments than their small sizes would suggest (Brock et al.’88) • “L’essentiel est invisible pour les yeux” (Antoine et al.’88) • “L’essentiel est invisible pour les yeux” (Antoine de Saint-Exupéry)de Saint-Exupéry)• “• “small is beautiful !”small is beautiful !”

Chis

holm

20

00





Trying to make VISIBLE what is invisible

BacteriaBacteria

ArchaeaArchaea

Stéphan Daigle National Geographic

Pure culture

GenesProteinsActivity

Molecular biology

Hey, it’s me !

SYSTEM CULTURABILITY (%)

Marine 0.001 - 0.1

Freshwater 0.25

Mesotrophic lake 0.1 - 1

Estuary 0.1 - 3

Activated sludge 1 - 15

Sediments 0.25

Soil 0.3

Culturing native prokaryotesCulturing native prokaryotes

Are these few isolated bacteria relevant in plankton biogeochemistry ?Are these few isolated bacteria relevant in plankton biogeochemistry ?

Overview of techniques in molecular Overview of techniques in molecular ecologyecology

Species compositionFingerprinting

DGGE ARDRA T-RFLP

Cloning and sequencing

Bulk nucleic acid extract

Quantitative rRNA hybridization

Microbial biomass

Sea water microbes

Biomass collection

In situ hybridization Detection

single cells

Nucleic acid extraction

DNA reassociation kineticsDNA cross-hybridization Abundance

some phylotypes

Comparison among

communities

PCR amplification

PCR productComparison

among communities

Fingerprinting: lmw RNA

BASICS partners will follow the BASICS partners will follow the seasonality of bacterial diversity in seasonality of bacterial diversity in several sites in the North, several sites in the North, Mediterranean and Baltic Seas and the Mediterranean and Baltic Seas and the English ChannelEnglish Channel

We will use a variety of techniques:We will use a variety of techniques:

- fingerprinting techniques (DGGE, T-RFLP, SSCP...)- fingerprinting techniques (DGGE, T-RFLP, SSCP...)- detection of single phylotypes/groups (FISH)- detection of single phylotypes/groups (FISH)- cloning and sequencing- cloning and sequencing- culture isolation- culture isolation

• • Describe seasonality in “diversity” (what is there, who’s Describe seasonality in “diversity” (what is there, who’s the most abundant)the most abundant)

• • Usage of different techniques (fingerprinting &clon Usage of different techniques (fingerprinting &clon libraries & isolation...)libraries & isolation...)

• • Common frameworkCommon framework• • Characterization of isolatesCharacterization of isolates• • Biotechnological exploitation of the isolatesBiotechnological exploitation of the isolates

Objective 1: To describe bacterioplankton diversity in the coastal seas of Europe

The BactLibThe BactLib(Bacterial (Bacterial Culture database)Culture database)

The ecologically-The ecologically-referencedreferencedphylotype databasephylotype database





To know the main To know the main routes of C circulationroutes of C circulationis a prerequisite... is a prerequisite...

... for understanding... for understandingthe fluxes of C in the oceanthe fluxes of C in the ocean

JGO

FS

It is the belief of BASICS that too muchIt is the belief of BASICS that too mucheffort has been put in the past in describingeffort has been put in the past in describingbacterial diversity in the ocean...bacterial diversity in the ocean...

... barely telling what the position and... barely telling what the position anddepth of the sample was...depth of the sample was...

How can we understand the role that specificHow can we understand the role that specificbacteria will play in nature then ???bacteria will play in nature then ???

Fig. 2.2. The feedback linking oceanic plankton and climate through the production of atmospheric sulfur. The original hypothesis postulated that production of dimethylsulfide (DMS) by phytoplankton, and its subsequent ventilation and oxidation in the atmosphere feeds cloud condensation nuclei in marine stratus, thereby increasing cloud albedo. If the consequent reduction in solar irradiance forced phytoplankton toproduce less DMS, then a negative feedback would operate, thus stabilizing climate. Recent advances suggest that it is not only phytoplankton but the whole food web (with bacteria playing a crucial role) that releases DMS.

- DMSP is a labile organic molecule which can represent 15% of - DMSP is a labile organic molecule which can represent 15% of BCD BCD and close to 100% of S demandand close to 100% of S demand- DMS participates in climate feedback- DMS participates in climate feedback

Kie

ne e

t al. 2

000

Kie

ne e

t al. 2

000

BASICS partners will follow the BASICS partners will follow the seasonality of microbial seasonality of microbial biogeochemical cycling in several sites biogeochemical cycling in several sites in the North, Mediterranean and Baltic in the North, Mediterranean and Baltic Seas and the English ChannelSeas and the English Channel

We will measure a large We will measure a large amount of stocks and rates:amount of stocks and rates:

- Bulk DOC and nutrients- Bulk DOC and nutrients- Algal activity and biomass- Algal activity and biomass- Viral and protozoan stocks and activity- Viral and protozoan stocks and activity- DMS and DMSP stocks and rates- DMS and DMSP stocks and rates- etc.- etc.

• • Seasonal studies in C and S cyclingSeasonal studies in C and S cycling• • Key biogeochemical steps little studiedKey biogeochemical steps little studied

Objective 2: To describe the seasonal changes in the cycling of carbon and sulfur mediated by planktonic bacteria in surface waters of European coastal seas

Ocean Projects in IGBP II

BASICSBASICS





Nitrate

Phyto

Zoop

P-Z-N Dynamics: Populations Change through TimeP-Z-N Dynamics: Populations Change through Time

PhytoplanktonPhytoplankton

ZooplanktonZooplankton

T. M

ichaels

Does it matter what biology is hidden within each box?Does it matter what biology is hidden within each box?

Phyto

DiatomsPrasinophytesPrymnesiophytes

ProchlorococcusSynechococcus

Phyto

T. M

ichaels

PhytoPhyto

ZooZoo

Dynamic Green Ocean Model

Buitenhuis et al. 2003

N2 fixersDMS

producers

coccolith.Nano

phytoplankton

Fe NO3Si

CaCO3

PO4 NH4

DOM

diatoms

Biogeochemical fluxes are a function of community structureBiogeochemical fluxes are a function of community structure

Bacteria & Nutrients

Stays Suspended

Sinking Particle

s

Pico-Phyto

Nano-Phyto

Micro-Phyto

Micro-Zoo

Meso-Zoo

Bacteria & Nutrients

Sinking Particles

SALPS

T. M

ichaels

1 mm

salp

euphausiid

copepod

Fecal PelletsD

. S

tein

berg

Bacteria are abundant and importantBacteria are abundant and important

ButBut

We are unable of grouping them in We are unable of grouping them in biogeochemically relevant and biogeochemically relevant and distinctdistinct““boxes”boxes”

we don’t know whether they all do the same, or we don’t know whether they all do the same, or not...not...

BecauseBecause

• • Multistep strategyMultistep strategy• • Coocurrence analysis (Synthesis workshop !)Coocurrence analysis (Synthesis workshop !)• • Design of oligonucleotide probesDesign of oligonucleotide probes• • Test of BGQ function in isolatesTest of BGQ function in isolates• • Test during an experimental algal bloomTest during an experimental algal bloom

• • Each approach is partial and has some risk of failureEach approach is partial and has some risk of failure

Objective 4: To link bacterial diversity and biogeochemical function (in the cycles of C and S) and identify the bacterial phylotypes responsible for the crucial steps in oceanic biogeochemical cycling, and to refine recently developed conceptual frameworks for the links between species richness (number of dominant coexisting species) and biogeochemical cycling





• • Flow cytometry sorting: standarization & controlsFlow cytometry sorting: standarization & controls• • FISH improvementsFISH improvements• • Combination of techniques: MicroFISH, MicroACT, etc...Combination of techniques: MicroFISH, MicroACT, etc...• • Capillary electrophoresisCapillary electrophoresis• • X-Ray microanalysisX-Ray microanalysis• • .... ???.... ???

Objective 3: To design, test and fine-tune different methods and research strategies for the single-cell analysis of natural bacterioplankton organisms

The power is in the combination of methodsThe power is in the combination of methods

Hybridized

DAPI-stained

Fluorescence in situ Hybridization:("phylogenetic staining")

Environmental sample

Extracted nucleic acidsDNA rRNA

rDNA clonesNucleic acid probe

rDNA Sequences

rDNAdatabase

Comparative Analysis

Hybridization Sequencing

MPIMMMPIMM

DAPI + AU

Roseobacter + AU

MicroFISH

35S DMSP

ICMICM

Cottrell & Kirchman 2000Cottrell & Kirchman 2000

HMW-DOMHMW-DOM

LMW-DOMLMW-DOM

CytophagaCytophaga

-Proteobacteria-Proteobacteria

-Proteobacteria-Proteobacteria

% a

ctiv

e c

ells

% a

ctiv

e c

ells

% cells in sample

0

20

40

60

0 20 40 60

Protein

C C

0

20

40

60

0 20 40 60

Aminoacids

C CAtlanticAtlanticOceanOcean

Cell sorting by FCM

Further analyses of sorted fractions

* Activity (radioactivity)* Identification* Isolation* Chemical analyses (C; N; P,….)

PrelabelingRadioactive substratesNucleic acid probesPhysiological probes

Laser(488 nm)

Trash

FACSCalibur FACSVantageHigh speed cell sorter

OOBOOB

PML/SOC/MPIMMPML/SOC/MPIMM

Blu

e fl

uore

scence

(D

NA

)

103

102

Threshold101

100 101 102 103

Red fluorescence (protein)

Flow Citometry

DMSP producing phytoplankton bloom in the North SeaEmiliania huxleyi y Prorocentrum minimum

FISH

Roseobacter

Cytophaga/Flavovacterium

SAR86

Zubkov et al. 2001Zubkov et al. 2001

PML/SOC/MPIMMPML/SOC/MPIMM

Abundance highly correlated with

DMSP consumption

Capillary electrophoresis

Sample

Data recording

+ -Power Supply

Inlet buffer reservoir Outlet buffer reservoir

Absorbance detector

Capillary

Basic scheme

NIOZNIOZ

Protein separation

Sample

Injection

Separation

Detection

Detection window

UV absorbance

SDS+

---

+++

++ +

+

+-

-

- --

- -

-

- -

-

-

-

-

+

+

+

+

NIOZNIOZ

• • Functional redundancy, ecosystem stability...Functional redundancy, ecosystem stability... ... effects of env. change on ... effects of env. change on

- diversitydiversity- BGQ function- BGQ function- their linkage- their linkage

• • Environmental perturbations in microcosmsEnvironmental perturbations in microcosms

Objective 5: To estimate the effect of environmental changes affecting the ocean’s bacterially-mediated biogeochemical function, global bacterial diversity and the link between bacterial diversity and C and S cycling





WP 4: SCA method developmentWP1: Seasonal studies of bacterial diversity

WP3: Seasonal studies of biogeochemical C & S cycling

WP7: Functional stability of the link facing global change

WP6: Experimental determination of the factors that regulate the link between bacterial diversity and function

WP5: Linking bacterial diversity with biogeochemical function

WP2: Explotaition of phylotypes and isolate information

WS0: Coordination

WS1: Synthesis WS on bacterial diversity in coastal european seas and C & S cycling

WS3: Synthesis WS on SCA

WS5: Summary

Phase 1

Phase 2

Phase 3

WS2: Mesocosm experiment

WS4: Microcosm experiments

WP2:WP2:ExploitationExploitation

WP6:WP6:Factors regulateFactors regulate

WP5: WP5: LinkingLinking

WP7:WP7: Funct. stabil.Funct. stabil.

WP3:WP3:C/S-CyclingC/S-Cycling

WP1:WP1:DiversityDiversity

WP4:WP4: SCA devSCA dev





BarcelonaBarcelonaBanyulsBanyuls

TexelTexel

KalmarKalmar

ToulouseToulouseVillefrancheVillefranche

SouthamptonSouthampton

BergenBergen

BremenBremen

PlymouthPlymouth

BarcelonaBarcelona Pep Gasol / Rafel SimóPep Gasol / Rafel Simó

BanyulsBanyuls Philippe LebaronPhilippe Lebaron

TexelTexel Gerhard HerndlGerhard Herndl

KalmarKalmar Åke HagströmÅke Hagström

ToulouseToulouse Pascal BordatPascal Bordat

VillefrancheVillefranche Markus WeinbauerMarkus Weinbauer

SouthamptonSouthampton Mike Zubkov / Peter BurkillMike Zubkov / Peter Burkill

BergenBergen Frede Thingstad / Mikal HeldalFrede Thingstad / Mikal Heldal

BremenBremen J. Pernthaler / B. FuchsJ. Pernthaler / B. Fuchs

PlymouthPlymouth Steve ArcherSteve Archer





• Shelf = 20% of Ocean NPP; supports 90% of Marine Fisheries Production

• Coastal population = 2.2 billion (40% of total)

(Jackson et al. 2001)

Before fishingAfter fishing

• Nutrient inputs microbialization of food chains

Sampling sitesSampling sites

• • Blanes Bay (ICM)Blanes Bay (ICM)• • Banyuls - MOLA & MILA station (OOB)Banyuls - MOLA & MILA station (OOB)• • Baltic proper landsort (UNIK)Baltic proper landsort (UNIK)• • North Sea Texel site (NIOZ)North Sea Texel site (NIOZ)• • Villefranche point “B” (LOV)Villefranche point “B” (LOV)• • L4 English Channel station (PML & SOC)L4 English Channel station (PML & SOC)• • Helgoland site G (MPIMM)Helgoland site G (MPIMM)

General characteristics of Blanes BayGeneral characteristics of Blanes Bay

• Typical Mediterranean waters: warm, salty and nutrient-poor• Typical Mediterranean waters: warm, salty and nutrient-poor

• Oligotrophic coastal system (annual average chlorophyll of 0.5 µg l• Oligotrophic coastal system (annual average chlorophyll of 0.5 µg l -1-1) )

• Relatively unaffected by human or freshwater influence• Relatively unaffected by human or freshwater influence

• Separated from oceanic waters by a southwest current associated with • Separated from oceanic waters by a southwest current associated with a front in the continental slope (10-20 miles offshore)a front in the continental slope (10-20 miles offshore)

• Episodic intrusions of oceanic waters caused by the Blanes canyon• Episodic intrusions of oceanic waters caused by the Blanes canyon

Half mile from

harbour

Depth of 20 m

ICMICM

Seasonality of Seasonality of phytoplanktonphytoplankton

Main peak of chlorophyll Main peak of chlorophyll aa during late winter, during late winter, driven by high driven by high atmospheric pressures atmospheric pressures together with irradiances together with irradiances and temperatures higher and temperatures higher than similar latitudes in than similar latitudes in the Atlanticthe Atlantic

ICMICM

5•10•12•14•17•23•27•35•40•34•33•48•54•13•28•36•31•38•18•22•41•43•50•24•49•2227512192531118262932293941Dec97Jan Feb AprJun Jul SepOctNovDec Mar

3 September

2July

29 July

9 October

4 November

1 Desember

26 March

29 April

3 June

27 January

22 Desember 97

25 February

5 February

12 February

19 February

3 March

18 March

11 March

1.0

SummerFall

Spring

Winter

Seasonal succession of Seasonal succession of

bacterioplanktonbacterioplankton

ICMICM

Temporal dynamics of bacterial Temporal dynamics of bacterial groups and populationsgroups and populations

ICMICM

Four sites 70 km appartFour sites 70 km appart

One sample for seasonOne sample for season

Comparison of bacterial composition Comparison of bacterial composition

by DGGEby DGGE

How representative is the Blanes site?How representative is the Blanes site?

CO

Blanes-Jul

Blanes-Nov

Masnou-Jul

Barcelona-Jul

Masnou-Nov

Blanes-Jan

Blanes-Apr

Masnou-Apr

Barcelona-Apr

Harbour-Jan

Masnou-Jan

Barcelona-Jan

Barcelona-Nov

CC

Harbour-Apr

Harbour-Jul

Harbour-Nov

1.0

Summer

Winter

Spring

ICMICM

PML/SOC seasonal sampling site

Time series station since 1988(Roger Harris and PML Zooplankton group)

Measurements: (weekly throughout the year)

•Environmental: Chl a, Temp, Salinity, Optics, POC, PON

•Biological: Phytoplankton, Zooplankton, Bacteria, Viruses

•Processes: Primary production (FRRF), Calanus egg production, L4 location:

Western English ChannelPML/SOCPML/SOC





MPIMMMPIMM

Horseradish-peroxidase-labeled FISH probes and catalyzed reporter deposition (CARD)

(tyramide signal amplification, TSA)

HRP

protein

fluorescently labeled tyramide

Signal AmplificationPermeabilization

HRP

HRP HRP

Hybridization

MPIMMMPIMM

Sediment

Surface

water

Exposure time:

10 seconds 1 second

10 seconds 1 second

conventional FISH

FISH & signal amplification

MPIMMMPIMM

G2 EuryarchaeaDAPI-staining

Euryarchaeota in coastal North Sea surface waters

Coastal North Sea, % detection by FISH

Month

03 05 07 09 11 01

Probe detection rate [% of total cells]

0

10

20

30

40

50

G2-Euryarchaeota

MPIMMMPIMM

And still, other unplanned BASICS returns...And still, other unplanned BASICS returns...

• • Development and optimization of molecular tools Development and optimization of molecular tools (useful with other microorganisms and in other (useful with other microorganisms and in other ecosystemsecosystems

• • Better knowledge of the spatial and temporal scales of Better knowledge of the spatial and temporal scales of bacterial biodiversity changesbacterial biodiversity changes

• • Applicability of the “key species” concept to bacteriaApplicability of the “key species” concept to bacteria• • Autoecology of bacteriaAutoecology of bacteria• • Indicator bacterial species of ecosystem perturbationsIndicator bacterial species of ecosystem perturbations• • Biotechnological exploitation of effort made at the Biotechnological exploitation of effort made at the

“pure science” (knowledge for its own sake) level“pure science” (knowledge for its own sake) level•• ......

Single-cell approachesSingle-cell approaches

linkagelinkageBacterial diversityBacterial diversity

Bacterial biogeochemical Bacterial biogeochemical functionfunction


www.icm.csic.es/bio/projects/basicswww.icm.csic.es/bio/projects/basics

Documents

Ba cterial si ngle- c ell approaches to the relationship between diversity and function