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

α βγ γαβ

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

NtrC NtrC

in absence ofN-compounds

NtrB

ADP ATP

nifLA operonnitrC binding site σ54 = nitrAbinding site

nif structural genes σ54 = nitrAbinding site

nifA binding site

P

Regulation of nitrogen fixation (K. pneumoniae)

Nitrogen present, no transcription

Function of NtrA, σ54 , thenitrogen σ factor

Nitrogen absent, NtrB phosphorylates NtrC,which activates RNA polymerase

Function of NtrA, σ54 , thenitrogen σ factor

P

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

NtrC NtrC

in absence ofN-compounds

NtrB

ADP ATP

nifLA operonnitrC binding site σ54 = nitrAbinding site

nif structural genes σ54 = nitrAbinding 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 ofN-compounds

NtrB

ADP ATP

nifLA operonσ54 = nitrAbinding site

NtrC P

N-compound regulation of NifLA operon

nif structural genes σ54 = nitrAbinding site

nifA binding site

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

NifANifA

NifL

transcription

transcription

NtrC

in absence ofN-compounds

NtrB

ADP ATP

nifLA operonσ54 = nitrAbinding site

nif structural genes σ54 = nitrAbinding 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 orN-compounds

transcription

NtrC

in absence ofN-compounds

NtrB

ADP ATP

nifLA operonσ54 = nitrAbinding site

nif structural genes σ54 = nitrAbinding 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 withacetylene

• Glass jars contain the plantsamples being evaluated

• Sterile vacutainers (normallyused to collect blood) areused to take the gas samplefollowing incubation

• Several hundred samplescan be taken each day

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

Acetylene reduction assay

• A typical trace followinggas chromatography

• The greatest peaks areof residual acetylene

• Those next to them theethylene 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 moleculeper 2 molecules of N2 reduced

– So, can use hydrogen sensor tomeasure 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 moleculeper 2 molecules of N2 reduced

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

fixation

Hydrogen evolution assay

The operation of nitrogenase. The iron protein (Fe) takes electronsfrom central metabolism electron carriers and transfers them to themolybdenum iron protein (MoFe) expending a fair amount of ATP.N2 is converted to ammonia and the electrons in H2 are recycled byhydrogenase. (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 δ15N

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 ofthe Hakatai Shale in Grand

Canyon National Park.Lens cap is 55 mm.

Livingstromatolites

Diazotrophicbacteria

in consortia

Diazotrophicbacteria

in consortiasoil crusts

Cyanobacteria insoil crusts

Diazotrophicbacteria

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 micronesicaβ-N-methylamino-L-alanine (BMAA)

Guam flying fox(Pteropus mariannus)

bio-magnification

β-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_ cup white wine salt, pepper 1/4 cup flour 2 oz. butter 1 tbsp red currantjelly 1 cup thick cream

Remove the flesh from the flying foxes. Either plunge the animals in boilingwater for a while, then skin them and remove the flesh from the bones, orroast the animals for a little over an open fire, remove, and when cool, breakopen down the backbone and remove the flesh from the skin.

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

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

Spinal cord

Amyotrophic Lateral Sclerosisβ-N-methylamino-L-alanine

Mimics glutamateand acts as agonistat glutamatereceptor

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-fixingbacteria

© Simms

Soil-dwelling rhizobia infectlegume 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 intobacteroids

© 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, Phyllobacteriumα

β

-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)