Nitrogenase enzyme complex

Nitrogenase Electron transport MoFe protein Fe protein Assembling Fe-Mo-Cofactor Regulator

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

BQ

Physical association of nif genes in Klebsiella pneumoniae

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

Regulation of nitrogen fixation (K. pneumoniae)

in absence of N-compounds NtrB ADP ATP NtrC

NtrC

nitrC binding site

54 = nitrA binding site

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

Function of NtrA, 54 , the nitrogen factor

Nitrogen present, no transcription

Function of NtrA, 54 , the nitrogen factor

Nitrogen absent, NtrB phosphorylates NtrC, which activates RNA polymerase
P

Regulation of nitrogen fixation (K. pneumoniae)

in absence of N-compounds NtrB ADP ATP NtrC

NtrC

nitrC binding site

54 = nitrA binding site

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

N-compound regulation of NifLA operon

in absence of N-compounds NtrB ADP NtrC ATP NtrC

Ptranscription
nifLA operon

54 = nitrA binding site

NifL

NifA

nif structural genes

54 = nitrA binding site

nifA binding site

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

N-compound regulation of NifLA operon

in absence of N-compounds NtrB ADP NtrC ATP NtrC

Ptranscription
nifLA operon

54 = nitrA binding site

NifL
transcription nif structural genes

NifA

54 = nitrA binding site

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

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

in absence of N-compounds NtrB ADP NtrC ATP NtrC

Ptranscription
nifLA operon

54 = nitrA binding site

NifL NifA
in presence of O2 or N-compounds

NifL

NifA

nif structural genes

54 = nitrA binding site

nifA binding site

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

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

Combustion GC Mass separation magnet detector

ion source

Mass Separation

magnet

mass 30 (15N2) ion source

mass 28 (14N2) mass 29 (15N14N)

Stable isotope lab

Table 15-3 (pg. 380 of text)

Crop Agropyron trichophorum A.elongatum A.dasystachyum Elymus angustus Melilotus officinales Medicago sativa Astragalus cicer Atmospheric N2 Soil N Common name Pubescent wheatgrass Tall wheatgrass Northern Wheatgrass Wild ryegrass Yellow sweet clover Alfalfa Milk vetch 15N 5.13 3.04 3.00 2.31 -0.72 0.82 1.90 0.00 7.00

Lifestyles of N2 fixing bacteria (diazotrophs)

Free living Living in consortia
e.g. stromatolites, soil crusts

Plant associative (living in rhizosphere) Symbiotic

Diazotrophic bacteria in consortia Living stromatolites

Middle Proterozoic formations of the Hakatai Shale in Grand Canyon National Park. Lens cap is 55 mm.

Diazotrophic bacteria in consortia soil crusts

Cyanobacteria in soil crusts

Diazotrophic bacteria in consortia

Cyanobacteria
Photosynthetic and dinitrogen fixing
heterocysts separate the two functions
Nostoc

Microcystis Anabaena Free-living

Cyanobacteria
Oldest known fossils
3.5 bybp (oldest rocks are 3.8 bypb)
colonial chroococcalean filamentous Palaeolyngbya

Cyanobacteria heterocysts

Heterocyst

Symbiotic N2-fixation: Azolla - Anabena

S. Navie

Symbiotic N2-fixation: Azolla - Anabena

Symbiotic N2-fixation: Azolla - Anabena

Rice-Azolla-Fish, China

Takao Furuno

Rice-Azolla-Ducks, Korea

Azolla to feed cows, Thailand

Cycas micronesica

Paul Cox

Cycad root nodules

-N-methylamino-L-alanine (BMAA)

Cycas micronesica

Paul Cox

-N-methylamino-L-alanine (BMAA)

Guam flying fox (Pteropus mariannus) bio-magnification

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 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.

-N-methylamino-L-alanine

Amyotrophic Lateral Sclerosis

Mimics glutamate and acts as agonist at glutamate receptor

Spinal cord

Actinorhizal symbioses
Frankia in alder root nodules

Frankia vesicles

Frankia root nodules Spores & hyphae

Ceanothus

Colletia

Dicaria

Ceanothus

Myrica faya Actinorhizal root nodules

Native to Canary Islands

Myrica faya

Invasive in Hawaii (no native N2 - fixing pioneers)

Legumes & N-fixing bacteria

Soil-dwelling rhizobia infect legume roots

Simms

 Signals early in infection

Complex handshaking between legume root and rhizobium
Correct signal

Incorrect signal

Legume & N-fixing bacteria

Rhizobia engulfed into nodule cells Differentiate into bacteroids

Simms

Illustration: M.S. Hargrove

Leghemoglobin
Photo and illustration: R. F. Denison

Photo: D. Hume


Rickettsia

Ralstonia Burkholderia

Devosia Azorhizobium Sinorhizobium Methylobacterium Proteobacteria Bradyrhizobium Nitrobacter, Afipia Mesorhizobium, Aminobacter, Phyllobacterium

Bartonella Brucella

Sinorhizobium Ensifer Rhizobium Agrobacterium

-Rhizobial symbioses have evolved ~10 times Sawada et al. 2003 -Nested parasites & non-symbionts

Symbiotic plasmid of Rhizobium etli

Vctor Gonzlez et al. Genome Biology 2003 4(6):R36

plasmid 42d M. loti MAFF303099 plasmid NGR234a M. loti MAFF303099 B. japonicum S. meliloti pSymA

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

Vctor Gonzlez et al. Genome Biology 2003 4(6):R36

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

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

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

Clover Root Curculio Sitona hispidula (Fabricus)