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Nitrogenase enzyme complex

Nitrogenase

Electron transport Fe protein MoFe protein Assembling β α γ γ α β Fe-Mo-Cofactor Regulator J
Electron transport
Fe protein
MoFe protein
Assembling
β
α
γ γ
α
β
Fe-Mo-Cofactor
Regulator
J
H
D
K
T Y
E NX
U SVWZM F
L
A
B Q

Physical association of nif genes in Klebsiella pneumoniae

Nitrogenase enzyme complex Nitrogenase Electron transport Fe protein MoFe protein Assembling β α γ γ α

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

Regulation of nitrogen fixation ( K. pneumoniae)

NtrC
NtrC

P

in absence of N-compounds NtrC NtrB ADP ATP
in absence of
N-compounds
NtrC
NtrB
ADP
ATP
nitrC binding site σ54 = nitrA binding site nifLA operon
nitrC binding site
σ54 = nitrA
binding site
nifLA operon
Regulation of nitrogen fixation ( K. pneumoniae ) NtrC P in absence of N-compounds NtrC NtrB

σ54 = nitrA binding site

nif structural genes

nifA binding site

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

Function of NtrA, σ54 , the nitrogen σ factor

Nitrogen present, no transcription

Function of NtrA , σ 54 , the nitrogen σ factor Nitrogen present, no transcription

Function of NtrA, σ54 , the nitrogen σ factor

P
P

Nitrogen absent, NtrB phosphorylates NtrC, which activates RNA polymerase

Regulation of nitrogen fixation ( K. pneumoniae)

NtrC
NtrC

P

in absence of N-compounds NtrC NtrB ADP ATP
in absence of
N-compounds
NtrC
NtrB
ADP
ATP
nitrC binding site σ54 = nitrA binding site nifLA operon
nitrC binding site
σ54 = nitrA
binding site
nifLA operon
Regulation of nitrogen fixation ( K. pneumoniae ) NtrC P in absence of N-compounds NtrC NtrB

σ54 = nitrA binding site

nif structural genes

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 NtrC NtrB ADP ATP
in absence of
N-compounds
NtrC
NtrB
ADP
ATP
NtrC P transcription σ54 = nitrA binding site nifLA operon NifL NifA NifA
NtrC
P
transcription
σ54 = nitrA
binding site
nifLA operon
NifL
NifA
NifA
N-compound regulation of NifLA operon in absence of N-compounds NtrC NtrB ADP ATP NtrC P transcription

σ54 = nitrA binding site

nif structural genes

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 NtrC NtrB ADP ATP
in absence of
N-compounds
NtrC
NtrB
ADP
ATP
NtrC P transcription σ54 = nitrA binding site nifLA operon
NtrC
P
transcription
σ54 = nitrA
binding site
nifLA operon
NifL
NifL
NifA NifA transcription nif structural genes σ54 = nitrA binding site
NifA
NifA
transcription
nif structural genes
σ54 = 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 NtrC NtrB ADP ATP
in absence of
N-compounds
NtrC
NtrB
ADP
ATP
NtrC
NtrC

P

transcription

Oxygen and N-compound regulation of nif structural genes via nifL in absence of N-compounds NtrC NtrB
  • NifL

NifA

NifA

nifLA operon

NifL NifA NifA
NifL
NifA
NifA

σ54 = nitrA binding site

Oxygen and N-compound regulation of nif structural genes via nifL in absence of N-compounds NtrC NtrB

in presence of O 2 or N-compounds

Oxygen and N-compound regulation of nif structural genes via nifL in absence of N-compounds NtrC NtrB

σ54 = nitrA binding site

nif structural genes

nifA binding site

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

Measuring N 2 fixation rates

Acetylene reduction assay

Acetylene reduction assay • Football has been filled with acetylene • Glass jars contain the plant

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

Acetylene reduction assay A typical trace following gas chromatography The greatest peaks are of residual acetylene

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

Hydrogen evolution assay • Reduction of dinitrogen to ammonia N + 8H + 8e → 2NH

Reduction of dinitrogen to ammonia N 2 + 8H + + 8e - 2NH 3 + H 2

H 2 is evolved at ratio of 1 molecule per 2 molecules of N 2 reduced

– So, can use hydrogen sensor to

measure H 2 evolution to quantify N 2

fixation

Hydrogen evolution assay

Hydrogen evolution assay • Reduction of dinitrogen to ammonia N + 8H + 8e → 2NH

Reduction of dinitrogen to ammonia N 2 + 8H + + 8e - 2NH 3 + H 2

H 2 is evolved at ratio of 1 molecule per 2 molecules of N 2 reduced

– So, can use hydrogen sensor to

measure H 2 evolution to quantify N 2

fixation

Hydrogen evolution assay

Hydrogen evolution assay
The operation of nitrogenase. The iron protein (Fe) takes electrons from central metabolism electron carriers and
The operation of nitrogenase. The iron protein (Fe) takes electrons from central metabolism electron carriers and

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. N 2 is converted to ammonia and the electrons in H 2 are recycled by hydrogenase. (D. Benson)

Stable isotope assays

Combustion GC Mass separation detector magnet ion source
Combustion
GC
Mass separation
detector
magnet
ion source

Mass Separation

magnet
magnet
Mass Separation magnet ( mass 29 ( mass 28 14 15 N N ) 14 2
Mass Separation magnet ( mass 29 ( mass 28 14 15 N N ) 14 2

(

mass 29 (

mass 28

14

15

N

N

)

14

2

N)

mass 30 ( 15 N 2 )

ion source

Stable isotope lab

Stable isotope lab
Stable isotope lab

Table 15-3 (pg. 380 of text)

Crop

Common name

δ 15 N

Agropyron

Pubescent

5.13

trichophorum

wheatgrass

A.elongatum

Tall wheatgrass

3.04

A.dasystachyum

Northern

3.00

Wheatgrass

Elymus angustus

Wild ryegrass

2.31

Melilotus officinales

Yellow sweet

-0.72

clover

Medicago sativa

Alfalfa

0.82

Astragalus cicer

Milk vetch

1.90

Atmospheric N 2

 

0.00

Soil N

 

7.00

Lifestyles of N 2 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

Diazotrophic bacteria in consortia Living stromatolites Middle Proterozoic formations of the Hakatai Shale in Grand Canyon
Diazotrophic bacteria in consortia Living stromatolites Middle Proterozoic formations of the Hakatai Shale in Grand Canyon

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

Diazotrophic bacteria in consortia soil crusts
Diazotrophic
bacteria
in consortia
soil crusts
Cyanobacteria in soil crusts
Cyanobacteria in
soil crusts

Diazotrophic bacteria in consortia

Diazotrophic bacteria in consortia
Nostoc
Nostoc

Cyanobacteria

Photosynthetic and dinitrogen fixing

– heterocysts separate the two functions

Microcystis Anabaena Free-living
Microcystis
Anabaena
Free-living

Cyanobacteria

Oldest known fossils

– 3.5 bybp (oldest rocks are 3.8 bypb)

colonial chroococcalean

filamentous Palaeolyngbya
filamentous Palaeolyngbya

Cyanobacteria heterocysts

Cyanobacteria heterocysts Heterocyst

Heterocyst

Cyanobacteria heterocysts Heterocyst

Symbiotic N 2 -fixation: Azolla - Anabena

Symbiotic N -fixation: Azolla - Anabena S. Navie
S. Navie
S. Navie

Symbiotic N 2 -fixation: Azolla - Anabena

Symbiotic N -fixation: Azolla - Anabena
Symbiotic N -fixation: Azolla - Anabena
Symbiotic N -fixation: Azolla - Anabena
Symbiotic N -fixation: Azolla - Anabena
Symbiotic N -fixation: Azolla - Anabena

Symbiotic N 2 -fixation: Azolla - Anabena

Takao Furuno
Takao Furuno

Rice-Azolla-Ducks, Korea

Rice-Azolla-Fish, China

Symbiotic N -fixation: Azolla - Anabena Takao Furuno Rice-Azolla-Ducks, Korea Rice-Azolla-Fish, China Azolla to feed cows,
Symbiotic N -fixation: Azolla - Anabena Takao Furuno Rice-Azolla-Ducks, Korea Rice-Azolla-Fish, China Azolla to feed cows,

Azolla to feed cows, Thailand

Cycas micronesica © Paul Cox
Cycas micronesica © Paul Cox
Cycas micronesica
© Paul Cox
Cycad root nodules

Cycad root nodules

Cycad root nodules
Cycad root nodules
β-N-methylamino-L-alanine (BMAA)
β-N-methylamino-L-alanine
(BMAA)
Cycas micronesica © Paul Cox
Cycas micronesica
© Paul Cox
(BMAA)
(BMAA)
(BMAA) β -N-methylamino-L-alanine Guam flying fox ( Pteropus mariannus ) bio-magnification

β-N-methylamino-L-alanine

(BMAA) β -N-methylamino-L-alanine Guam flying fox ( Pteropus mariannus ) bio-magnification

Guam flying fox (Pteropus mariannus) bio-magnification

Soak the prunes overnight in 1 cup of the wine, then heat for about ten minutes
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.
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.
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
The preparation of this recipe requires an ingredient which is now a
protected species.
Flying Fox with Prunes and Cream Sauce

β-N-methylamino-L-alanine

Amyotrophic Lateral Sclerosis

β -N-methylamino-L-alanine Amyotrophic Lateral Sclerosis Mimics glutamate and acts as agonist at glutamate receptor Spinal cord
β -N-methylamino-L-alanine Amyotrophic Lateral Sclerosis Mimics glutamate and acts as agonist at glutamate receptor Spinal cord

Mimics glutamate

and acts as agonist

at glutamate

receptor

β -N-methylamino-L-alanine Amyotrophic Lateral Sclerosis Mimics glutamate and acts as agonist at glutamate receptor Spinal cord

Spinal cord

Actinorhizal symbioses

Frankia in alder root nodules

Actinorhizal symbioses Frankia in alder root nodules
Frankia root nodules Frankia vesicles Spores & hyphae

Frankia root nodules

Frankia vesicles

Frankia root nodules Frankia vesicles Spores & hyphae
Frankia root nodules Frankia vesicles Spores & hyphae
Frankia root nodules Frankia vesicles Spores & hyphae

Spores & hyphae

Ceanothus

Ceanothus Colletia Dicaria
Colletia
Colletia

Dicaria

Ceanothus Colletia Dicaria
Ceanothus
Ceanothus

Myrica faya

Actinorhizal root nodules

Myrica faya Actinorhizal root nodules Native to Canary Islands
Myrica faya Actinorhizal root nodules Native to Canary Islands

Native to Canary Islands

Myrica faya Invasive in Hawai’ i (no native N 2 - fixing pioneers)
Myrica faya
Invasive in Hawai’ i
(no native N 2 - fixing pioneers)
Legumes & N-fixing bacteria Soil-dwelling rhizobia infect legume roots © Simms
Legumes & N-fixing bacteria Soil-dwelling rhizobia infect legume roots © Simms
Legumes & N-fixing bacteria Soil-dwelling rhizobia infect legume roots © Simms

Legumes & N-fixing bacteria

Soil-dwelling rhizobia infect legume roots

© Simms
© Simms

Signals early in infection

– Complex handshaking between legume root and rhizobium

• Signals early in infection – Complex handshaking between legume root and rhizobium Correct signal Incorrect
Correct signal Incorrect signal
Correct
signal
Incorrect
signal

Legume & N-fixing bacteria

• Rhizobia engulfed into nodule cells

• Differentiate into bacteroids

Legume & N-fixing bacteria • Rhizobia engulfed into nodule cells • Differentiate into bacteroids © Simms
© Simms
© Simms
Legume & N-fixing bacteria • Rhizobia engulfed into nodule cells • Differentiate into bacteroids © Simms
Legume & N-fixing bacteria • Rhizobia engulfed into nodule cells • Differentiate into bacteroids © Simms

Illustration: M.S. Hargrove

Illustration: M.S. Hargrove Leghemoglobin Photo: D. Hume Photo and illustration: R. F. Denison

Leghemoglobin

Illustration: M.S. Hargrove Leghemoglobin Photo: D. Hume Photo and illustration: R. F. Denison

Photo: D. Hume

Illustration: M.S. Hargrove Leghemoglobin Photo: D. Hume Photo and illustration: R. F. Denison

Photo and illustration: R. F. Denison

Illustration: M.S. Hargrove Leghemoglobin Photo: D. Hume Photo and illustration: R. F. Denison
Ralstonia β Burkholderia Rickettsia Devosia Azorhizobium Sinorhizobium Methylobacterium Bradyrhizobium Proteobacteria Nitrobacter, Afipia α Mesorhizobium, Aminobacter, Phyllobacterium
Ralstonia
β
Burkholderia
Rickettsia
Devosia
Azorhizobium
Sinorhizobium
Methylobacterium
Bradyrhizobium
Proteobacteria
Nitrobacter, Afipia
α
Mesorhizobium,
Aminobacter, Phyllobacterium
Bartonella
Brucella
Sinorhizobium
Ensifer
Rhizobium
Agrobacterium
-Rhizobial symbioses have evolved ~10 times
-Nested parasites & non-symbionts
Sawada et al. 2003

Symbiotic plasmid of Rhizobium etli

Symbiotic plasmid of Rhizobium etli Víctor González et al. Genome Biology 2003 4(6):R36

plasmid 42d

  • M. loti MAFF303099

plasmid NGR234a

  • M. loti MAFF303099

  • B. japonicum

  • S. meliloti pSymA

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

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

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

Figure 3. Three-dimensional representation of the net of life Victor Kunin et al. Genome Res. 2005;

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

Figure 3. Three-dimensional representation of the net of life Victor Kunin et al. Genome Res. 2005;

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

Hazards of symbiotic life (or an animal dispersal agent?) Clover Root Curculio Sitona hispidula (Fabricus)
Hazards of symbiotic life (or an animal dispersal agent?) Clover Root Curculio Sitona hispidula (Fabricus)
Hazards of symbiotic life (or an animal dispersal agent?) Clover Root Curculio Sitona hispidula (Fabricus)

Clover Root Curculio Sitona hispidula (Fabricus)