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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)