Genetics of Nitrogen Fixation

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Genetics of Nitrogen Fixation
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Nitrogen Fixation

• The growth of all organisms depend on the
  availability of Nitrogen (e.g. amino acids)
• Nitrogen in the form of Dinitrogen (N2) makes up
  80 of the air we breathe but is essentially
  inert due to the triple bond (N?N)
• In order for nitrogen to be used for growth it
  must be "fixed" (combined) in the form of
  ammonium (NH4) or nitrate (NO3) ions.

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Nitrogen Fixation

• To break N2 apart so that its atoms can combine
  with other atoms requires the input of
  substantial amounts of energy.
• Three processes are responsible for most of the
  nitrogen fixation in the biosphere
• 1. atmospheric fixation
• 2. industrial fixation
• 3. biological fixation

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ATMOSPHERIC N2 FIXATION
Lightning H OH- O2 N2
HNO3
HNO3 comes to surface of earth through
precipitation. Amount of N fixed by lightning is
relatively small but important in natural
ecosystems.
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Industrial Fixation

• Under great pressure, at a temperature of 600 oC,
  and with the use of a catalyst, atmospheric
  nitrogen and hydrogen (usually derived from
  natural gas or petroleum) can be combined to form
  ammonia (NH3).
• Ammonia can be used directly as fertilizer, but
  most of its is further processed to urea and
  ammonium nitrate (NH4NO3).
• Haber-Bosch
• 3CH4 6H2O --gt 3CO2 12H2
• 4N212H2 --gt 8NH3 (high T,press)
• (100-200 atm, 400-500C, 8,000 kcal kg-1 N)

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Biological Fixation

• The ability to fix nitrogen is found only in
  certain bacteria.
• Some live in a symbiotic relationship with plants
  of the legume family (e.g., soybeans, alfalfa).
• Some establish symbiotic relationships with
  plants other than legumes (e.g., alders).
• Some nitrogen-fixing bacteria live free in the
  soil.
• Nitrogen-fixing cyanobacteria are essential to
  maintaining the fertility of semi-aquatic
  environments like rice paddies.

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Biological Fixation cont.

• Biological nitrogen fixation requires a complex
  set of enzymes , Nitrogenase, and a huge
  expenditure of ATP (4,000 kcal kg-1 N).
• Although the first stable product of the process
  is ammonia, this is quickly incorporated into
  protein and other organic nitrogen compounds.
• Scientist estimate that biological fixation
  globally adds approximately 140 million metric
  tons of nitrogen to ecosystems every year.

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Nitrogenase

• All nitrogen fixing bacteria use highly conserved
  enzyme complex called Nitrogenase
• Nitrogenase is composed of of two subunits an
  iron-sulfur protein and a molybdenum-iron-sulfur
  protein
• Aerobic organisms face special challenges to
  nitrogen fixation because nitrogenase is
  inactivated when oxygen reacts with the iron
  component of the proteins

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Nitrogenase
FeMo Cofactor
Fd(ox)
N2 8H
Fd(red)
8e-
2NH3 H2
nMgATP
nMgADP nPi
4C2H2 8H 4C2H2
Dinitrogenase reductase
Dinitrogenase
N2 8H 8e- 16 MgATP ? 2NH3 H2 16MgADP
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Nitrogenase
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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 B Q
J
Physical association of nif genes in Klebsiella
pneumoniae
Redrawn from www.asahi-net.or.jp/it6i-wtnb/BNF.ht
ml
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Regulation of nitrogen fixation (K. pneumoniae)
in absence of N-compounds
P
NtrB
ADP
ATP
nifLA operon
?54 nitrA binding site
nitrC binding site
nif structural genes
nifA binding site
?54 nitrA binding site
Redrawn from http//www.science.siu.edu/microbiolo
gy/micr425/425Notes/12-NitrFix.html
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Function of NtrA, ?54 , the nitrogen ? factor
Nitrogen present, no transcription
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Function of NtrA, ?54 , the nitrogen ? factor
Nitrogen absent, NtrB phosphorylates NtrC, which
activates RNA polymerase
P
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Regulation of nitrogen fixation (K. pneumoniae)
in absence of N-compounds
P
NtrB
ADP
ATP
nifLA operon
?54 nitrA binding site
nitrC binding site
nif structural genes
nifA binding site
?54 nitrA binding site
Redrawn from http//www.science.siu.edu/microbiolo
gy/micr425/425Notes/12-NitrFix.html
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N-compound regulation of NifLA operon
in absence of N-compounds
NtrB
ADP
ATP
transcription
nifLA operon
?54 nitrA binding site
nif structural genes
nifA binding site
?54 nitrA binding site
Redrawn from http//www.science.siu.edu/microbiolo
gy/micr425/425Notes/12-NitrFix.html
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N-compound regulation of NifLA operon
in absence of N-compounds
NtrB
ADP
ATP
transcription
nifLA operon
?54 nitrA binding site
transcription
nif structural genes
?54 nitrA binding site
Redrawn from http//www.science.siu.edu/microbiolo
gy/micr425/425Notes/12-NitrFix.html
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Oxygen and N-compound regulation of nif
structural genes via nifL
in absence of N-compounds
NtrB
ADP
ATP
transcription
nifLA operon
?54 nitrA binding site
in presence of O2 or N-compounds
nif structural genes
nifA binding site
?54 nitrA binding site
Redrawn from http//www.science.siu.edu/microbiolo
gy/micr425/425Notes/12-NitrFix.html
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Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
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Activation of NifA
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
NtrC-RNA polymerase
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Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
ATP
Ferredoxin
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Electron Transport
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
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Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
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Electrons Donated to N2
Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
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Electrons Donated to N2
Formation of NH3
Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
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Genetics of Nitrogenase
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Genetic Regulation of Nitrogen Fixation in
Rhizobia

• Symbiotic nitrogen fixation genes in the broadest
  sense can be divided into nod, nif, and fix
  genes.
• The nod gene products are required for the early
  steps in nodule formation
• Rhizobial nif genes are structurally homologous
  to the 20 K pneumoniae nif genes, and it is
  inferred that a conserved nif gene plays a
  similar role in rhizobia as in K pneumoniae.
• The term "fix gene" is used for genes that are
  essential for nitrogen fixation but do not have a
  homologous counterpart in K pneumoniae, e.g.,
  genes involved in development and metabolism of
  bacteroids.

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Regulation of nif/fix Gene Expression in S.
meliloti




nifA
fixABCX
syrA
nifHDKE
other nif/fix
nifBQ
-
fixN


FixK
-

fixK
Nitrogen Starvation

FixL
Symbiosis
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Both cluster I and cluster II are located on
megaplasmid 1 (1,700 kb).
B. japonicum and apparently alsoA. caulinodans do
not have plasmids. Hence, the nif and fix (and
nod) genes are located on the chromosome in these
organisms,
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Symbiotic plasmid of Rhizobium etli
Víctor González et al. Genome Biology 2003
4(6)R36
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plasmid 42d M. loti MAFF303099plasmid NGR234aM.
loti MAFF303099 B. japonicumS. meliloti pSymA
The nodulation genes nodABCDIJ are represented in
blue The nitrogen-fixation genes nifHDKNEXAB,
fixABCX and fdxBN are represented in yellow
Víctor González et al. Genome Biology 2003
4(6)R36
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Formation of a Root Nodule
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Genetics of Nodulation

• Legume plants secrete specific flavonoids, which
  are detected by interaction with bacterial NodD
  proteins.
• When NodD binds a flavonoid it activates other
  nodulation genes. Some of the nod genes code for
  enzymes that make Nod factors, which are
  recognized by the plant.
• There are many different flavonoids and Nod
  factors and lots of variety in the host
  specificity between plants and Rhizobia

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Genetics of Nodulation

• Legume plants secrete specific flavonoids, which
  are detected by interaction with bacterial NodD
  proteins.

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Genetics of Nodulation

• When NodD binds a flavonoid it activates other
  nodulation genes. Some of the nod genes code for
  enzymes that make Nod factors, which are
  recognized by the plant.

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Nod factor biosynthesis
NodM
NodC
Nod factor R-group decorations determine host
specificity
NodB
Nod Factor a lipooligosaccharide
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• Signals early in infection
• Complex handshaking between legume root and
  rhizobium

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• Other interactions also occur. For example, a
  carbohydrate binding lectin on the surface of
  root cells of clover (Trifolium) specifically
  binds to lipopolysaccharide of Rhizobium trifolii
  which contains 2-deoxyglucose. This stimulates
  the bacteria to move to the root. The bacteria
  enter the outer root tissue and produce
  cytokinins (plant hormones) which cause division
  of plant cells to form nodules. The bacteria
  lose their outer membranes and become irregular
  in shape - "bacteroids".

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Infection Process

• Attachment
• Root hair curling
• Localized cell wall degradation
• Infection thread
• Cortical cell differentiation
• Rhizobia released into cytoplasm
• Bacterioid differentiation (symbiosome formation)
• Induction of nodulins

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How is specificity controlled?
Specific modifications to the Nod factor
structure alter the specificity of purified Nod
factors change the host range of the bacterium

• S. meliloti
• - nodH,P,Q add sulfate to the reducing end of
  the Nod factor molecule.
• - nodH mutants fail to nodulate alfalfa, but
  gain the capacity to nodulate vetch.
• R. leguminosarum
• nod X adds acetate to the non-reducing end,
  and restricts host range in pea genotypes
• carrying sym2.

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The Biology of Symbiotic Development
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Attachment and infection
Rhizobium
Nod factor (specificity)
Invasion through infection tube
Flavonoids (specificity)
Nitrogen fixation
Bacteroid differentiation
Formation of nodule primordia
From Hirsch, 1992. New Phyto. 122, 211-237
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Some nitrogen fixing organisms

• Free living aerobic bacteria
• Azotobacter
• Beijerinckia
• Klebsiella
• Cyanobacteria (lichens)
• Free living anaerobic bacteria
• Clostridium
• Desulfovibrio
• Purple sulphur bacteria
• Purple non-sulphur bacteria
• Green sulphur bacteria

• Free living associative bacteria
• Azospirillum
• Symbionts
• Rhizobium (legumes)
• Frankia (alden trees)

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Rank of Biological Nitrogen Fixation
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END