Redrawn from it6i-wtnb/BNF.html Nitrogenase enzyme complex Physical association of nif genes in...

Redrawn from www.asahi-net.or.jp/~it6i-wtnb/BNF.html

Nitrogenase enzyme complex

Physical association of nif genes in Klebsiella pneumoniae

NitrogenaseMoFe protein

Fe proteinElectron transport

AssemblingFe-Mo-Cofactor

Regulator

H D K T Y E NX U SVWZM F L A B QJ

Redrawn from http://www.science.siu.edu/microbiology/micr425/425Notes/12-NitrFix.html

NtrC NtrC

in absence of N-compounds

NtrB

ADP ATP

nifLA operonnitrC binding site 54 = nitrA binding site

nif structural genes 54 = nitrA binding site

nifA binding site

P

Regulation of nitrogen fixation (K. pneumoniae)

Nitrogen present, no transcription

Function of NtrA, 54 , the nitrogen factor

Nitrogen absent, NtrB phosphorylates NtrC, which activates RNA polymerase

Function of NtrA, 54 , the nitrogen factor

P

Redrawn from http://www.science.siu.edu/microbiology/micr425/425Notes/12-NitrFix.html

NtrC NtrC

in absence of N-compounds

NtrB

ADP ATP

nifLA operonnitrC binding site 54 = nitrA binding site

nif structural genes 54 = nitrA binding site

nifA binding site

P

Regulation of nitrogen fixation (K. pneumoniae)

Redrawn from http://www.science.siu.edu/microbiology/micr425/425Notes/12-NitrFix.html

NifANifANifL

transcription

NtrC

in absence of N-compounds

NtrB

ADP ATP

nifLA operon54 = nitrA binding site

NtrC P

N-compound regulation of NifLA operon

nif structural genes 54 = nitrA binding site

nifA binding site

Redrawn from http://www.science.siu.edu/microbiology/micr425/425Notes/12-NitrFix.html

NifANifA

NifL

transcription

transcription

NtrC

in absence of N-compounds

NtrB

ADP ATP

nifLA operon54 = nitrA binding site

nif structural genes 54 = nitrA binding site

N-compound regulation of NifLA operon

NtrC P

Redrawn from http://www.science.siu.edu/microbiology/micr425/425Notes/12-NitrFix.html

NifANifANifANifA

NifLNifLin presence

of O2 or N-compounds

transcription

NtrC

in absence of N-compounds

NtrB

ADP ATP

nifLA operon54 = nitrA binding site

nif structural genes 54 = nitrA binding site

nifA binding site

Oxygen and N-compound regulation of nif structural genes via nifL

NtrC P

Measuring N2 fixation rates

Acetylene reduction assay

• Football has been filled with acetylene

• Glass jars contain the plant samples being evaluated

• Sterile vacutainers (normally used to collect blood) are used to take the gas sample following incubation

• Several hundred samples can be taken each day

http://www.soils.umn.edu/academics/classes/soil3612/Nitrogen_Fixation/Measurement.htm

Acetylene reduction assay

• A typical trace following gas chromatography

• The greatest peaks are of residual acetylene

• Those next to them the ethylene peak

http://www.soils.umn.edu/academics/classes/soil3612/Nitrogen_Fixation/Measurement.htm

Hydrogen evolution assay

• Reduction of dinitrogen to ammonia

N2 + 8H+ + 8e- 2NH3 + H2

• H2 is evolved at ratio of 1 molecule

per 2 molecules of N2 reduced

– So, can use hydrogen sensor to measure H2 evolution to quantify N2

fixation

Hydrogen evolution assay

• Reduction of dinitrogen to ammonia

N2 + 8H+ + 8e- 2NH3 + H2

• H2 is evolved at ratio of 1 molecule

per 2 molecules of N2 reduced

– So, can use hydrogen sensor to measure H2 evolution to quantify N2

fixation

Hydrogen evolution assay

The operation of nitrogenase. The iron protein (Fe) takes electrons from central metabolism electron carriers and transfers them to the molybdenum iron protein (MoFe) expending a fair amount of ATP. N2 is converted to ammonia and the electrons in H2 are recycled by hydrogenase. (D. Benson)

Stable isotope assays

GC Mass separationCombustion

detector

ion source

magnet

mass 29 (15N14N)

ion source

magnet

mass 30 (15N2)

mass 28 (14N2)

Mass Separation

Stable isotope lab

Crop Common name 15N

Agropyrontrichophorum

Pubescentwheatgrass

5.13

A.elongatum Tall wheatgrass 3.04

A.dasystachyum NorthernWheatgrass

3.00

Elymus angustus Wild ryegrass 2.31

Melilotus officinales Yellow sweetclover

-0.72

Medicago sativa Alfalfa 0.82

Astragalus cicer Milk vetch 1.90

Atmospheric N2 0.00

Soil N 7.00

Table 15-3 (pg. 380 of text)

Lifestyles of N2 fixing bacteria (diazotrophs)

• Free living

• Living in consortia– e.g. stromatolites, soil crusts

• Plant associative (living in rhizosphere)

• Symbiotic

Middle Proterozoic formations of the Hakatai Shale in Grand

Canyon National Park. Lens cap is 55 mm.

Living stromatolites

Diazotrophic bacteria

in consortia

Diazotrophic bacteria

in consortiasoil crusts

Cyanobacteria in soil crusts

Diazotrophic bacteria

in consortia

Cyanobacteria• Photosynthetic and dinitrogen

fixing– heterocysts separate the two

functions

Anabaena

Microcystis

Nostoc

Free-living

Cyanobacteria• Oldest known fossils

– 3.5 bybp (oldest rocks are 3.8 bypb)

filamentous Palaeolyngbya

colonial chroococcalean

Cyanobacteria heterocysts

Heterocyst

Symbiotic N2-fixation: Azolla - Anabena

S. Navie

Symbiotic N2-fixation: Azolla - Anabena

Rice-Azolla-Fish, China

Azolla to feed cows, Thailand

Rice-Azolla-Ducks, KoreaTakao Furuno

Symbiotic N2-fixation: Azolla - Anabena

© Paul Cox

Cycas micronesica

Cycad root nodules

© Paul Cox

Cycas micronesica-N-methylamino-L-alanine (BMAA)

Guam flying fox(Pteropus mariannus)

bio-magnification

-N-methylamino-L-alanine (BMAA)

Flying Fox with Prunes and Cream Sauce

6 flying foxes (in case you are wondering, these are bats) 1 pound prunes 1 ¼ cup white wine salt, pepper 1/4 cup flour 2 oz. butter 1 tbsp red currant jelly 1 cup thick cream

Remove the flesh from the flying foxes. Either plunge the animals in boiling water for a while, then skin them and remove the flesh from the bones, or roast the animals for a little over an open fire, remove, and when cool, break open down the backbone and remove the flesh from the skin.

Soak the prunes overnight in 1 cup of the wine, then heat for about ten minutes in the wine before using. Season the flying fox meat with salt and pepper and roll in flour. Saute in butter over a low heat until brown. Add the rest of the wine, cover and cook another 20 minutes. Add the juice from the prunes, and transfer the prunes onto a serving dish. Cook the meat in the prune juice, uncovered, for another 10 minutes, then place on the serving dish with the prunes.

The preparation of this recipe requires an ingredient which is now a protected species.

Spinal cord

Amyotrophic Lateral Sclerosis-N-methylamino-L-alanine

Mimics glutamate and acts as agonist at glutamate receptor

Frankia in alder root nodules

Actinorhizal symbioses

Frankia vesicles

Frankia root nodulesSpores & hyphae

ColletiaDicaria

Ceanothus

Ceanothus

Myrica faya

Native to Canary Islands

Actinorhizal root nodules

Invasive in Hawai’i (no native N2 - fixing pioneers)

Myrica faya

Legumes & N-fixing bacteria

© Simms

Soil-dwelling rhizobia infect legume roots

• Signals early in infection– Complex handshaking between legume root

and rhizobium

Incorrect signal

Correctsignal

Legume & N-fixing bacteria• Rhizobia engulfed into

nodule cells• Differentiate into

bacteroids

© Simms

Illustration: M.S. Hargrove

Photo: D. Hume

Photo and illustration: R. F. Denison

Leghemoglobin

RhizobiumAgrobacterium

Sawada et al. 2003

SinorhizobiumEnsifer

Mesorhizobium,

BradyrhizobiumNitrobacter, Afipia

Methylobacterium

Sinorhizobium

DevosiaAzorhizobium

Ralstonia

Burkholderia

Rickettsia

BartonellaAminobacter, Phyllobacterium

-Rhizobial symbioses have evolved ~10 times-Nested parasites & non-symbionts

Brucella

Proteobacteria

Symbiotic plasmid of Rhizobium etli

Víctor González et al. Genome Biology 2003 4(6):R36

The nodulation genes nodABCDIJ are represented in blue

The nitrogen-fixation genes nifHDKNEXAB, fixABCX and fdxBN are represented in yellow

plasmid 42d

M. loti MAFF303099

plasmid NGR234a

M. loti MAFF303099

B. japonicum

S. meliloti pSymA

Víctor González et al. Genome Biology 2003 4(6):R36

Victor Kunin et al. Genome Res. 2005; 15: 954-959

Figure 3. Three-dimensional representation of the net of life

Hazards of symbiotic life (or an animal dispersal agent?)

Clover Root CurculioSitona hispidula (Fabricus)