Nitrogen Cycle,

Nitrogenase, nodule
formation and nif
gene
N2 cycle
Nitrification
From ammonia to nitrate conversion depend on nitrifying bacteria and soil content,
the denitrification occurs on nitrate.
Denitrification
N2, NO and N2O is produce from
Merits and Demerits

Ammonification
Definition,
Its Merit and Demerits
It constitute only 15% of atmospheric nitrogen
Ammonia in fertilizers

Anammox
Anaerobically ammonia react with nitrite and
form N2
N2 fixation
From N2 ammonia is produce by prokaryotes
N2 cycle
Nitrification
Denitrification
Ammonification
Anammox
N2 fixation
N2 cycle
Biological N2 fixation
• Nitrogenase
•Alternate
nitrogenase
•Unique Nitrogenase
• biochemistry of
nitrogen fixation
•O2 inactivation
•Host dependence
nif regulon
•NifA, NifL, Ntrc
•Control by nitrogenous compound

nif regulon
 Physical process of nitrogen fixation

Haber Process
300
atm
N2(g) + 3H2(g) 2NH3(g) + 92 kJ

450oC
(Super heated Steam)
Symbiotic Nitrogen
fixation
•Rhizobia
•Leghaemoglobin
•Cross –inoculation group
•Attachment and infection
•Infection thread
•Nodule formation
Nitrogenase Complex (nif gene
cluster)
1. Dinitrogenase (Mo, Fe protein)
It has a cofactor, it is called FeMo-co. it the actual
centre for N2 reduction.

2. Dinitrogenase Reductase (Fe protein)

Nitrogenase inhibited by O2
1. it is possible in aerobic nitrogen fixer but not in purified enzymes
2. nitrogenase in search of organisms is protected from the O2 inactivation by
one of several different mechanisms
3. Including Rapid removal of O2 by respiration, the production of O2 retarding
slim layers or compartmentalization.
4. In case of Azotobacter it protects its nitrogenase from O2 inactivation by
conformational change by interacting with other proteins.

Electron flow in Nitrogen fixation

1. electron flow from electron donor to dinitrogenase reductase,
dinitrogenase to nitrogen
2. electron flow from dinitrogenase reductase to nitrogen ferredoxin or
flavodoxin (both are low potential iron sulphur protein)
3. oxidation of pyruvate to acetyl-coa and carbon dioxide gives electron to
ferredoxin
4. 8 electrons are used in nitrogen fixation but two of them are released as
a hydrogen and two ATP is used in this reaction.
Other Nitrogenase
1. many bacteria and archaea produce known molybdenum nitrogenase in limiting or
absence of molybdenum. It is call alternative nitrogenous
2. Vanadium or iron is present in the place of molybdenum in Alternative
nitrogenase , and with cofactor it is formed if FeVa-col instead FeMo-co.
3. it is the backup system of nitrifying bacteria or archaea in absence of molybdenum.
4. another different kind of nitrogenase is found in Streptomyces
thermoautotrophicus. Here str2 and str1 play the role for dinitrogenase reductase
and dinitrogenase.
5. the nitrogenous of Streptomyces thermoautotrophicus is insensitive to O2,
6. str1 is similar to dinitrogenous in having three different polypeptides, but str2 not
similar with dinitrogenase reductase and it is similar to Mn containing enzymes
called superoxide dismutase.
7. here are the electrons move from CO-O2-str2-str1, CO dehydrogenasae is the
enzyme which help to transfer of electron from CO and O2 to str2.
8. There is in advantage of this kind of nitrogenase because it use less than half of the
ATP.
• Assaying of nitrogenase by Acetylene reduction
• Genetics of nitrogen fixation
1. klebsiella pneumoniae is a good nitrogen fixer and it's gene for dinitrogenase
and dinitrogenase reductase is a part of a complex regulon or network of
operon called nif regulon. In addition to nitrogenase structural genes, the genes
for a FeMo-co synthesis, genes controlling the electron transport proteins and in
number of regulatory genes are also present in nif regulon.
2. dinitrogen is composed of α (product of nifD) and β (the product of nifK).
Dinitrogenase reductase is a protein dimer consisting of two identical subunit
(the product of nifH). FeMo-co enzyme is encoded by several genes including
nifN,V, Z, W, E and B, we as well as nifQ encoded molybdenum processing
enzyme.
3. nifA gene encodes a positive regulatory protein that activates the transcription
of nif genes.
4. Nitrogenase encoded by nifHDK, where as the alternative nitrogen is encoded
by vnfHDK for vanadium system and anfHDK for iron only system.
 Regulation of nitrogenase synthesis
1. Nitrogenase inhibited by O2 as well as by Ammonia, nitrate and certain
amino acid.
2. NifA protein is a positive regulator and NifL is a negative regulator of nif gene
expression. NifL contains a molecule of FAD, this molecules is involved in O2
sensing, in presence of sufficient O2 NifL block the synthesis of O2 liable
nitrogenase.
3. Ammonia represses the nitrogen fixation by NtrC protein which in Ammonia
limiting condition is active and promote the transcription of NifA. Is called
Ammonia switch off effect found in some Proteobacteria.

Regulation of nitrogenase activity

1. Ammonia inhibit the nitrogenase activity. Is called Ammonia switch off effect
found in some Proteobacteria.
2. Ammonia switch off has also been observed in nitrogen fixing archaea, in
Methanococcus species, Clostridium.
3. This Ammonia Sensing System function not by detecting Ammonia directly but by
detecting the carbon compound α-ketoglutarate.
4. A direct precursor of amino acid glutamate. A shortage of Alpha ketoglutarate
sensed excess of glutamine and thus nitrogenase activity would be unnecessary.
 Biological Reaction

N2 + 8H+ + 8e- + 16ATP + 16H2O 2NH3 + 16ADP + 16Pi + H2 + 16H+

Nitrogen assimilation
Non legume symbiosis
Free living nitrogen fixer

Anabaena
Molecular biology of root nodule formation
 Gene clusters on R. meliloti pSym plasmid

(nol) (nod) (nif) (fix)

F G H I N D1 A B C I J Q P G E F H D 3 E K D H A B C

NML REF D ABCIJT CBA HDK E N

Gene clusters on R. leguminosarum bv trifolii pSym plasmid

- - - D 2 D1 Y A B C S U I J - - -

Gene cluster on Bradyrhizobium japonicum chromosome

Host plant role in nodulation

1. Production and release of nod gene inducers

- flavonoids

2. Activation of plant genes specifically required for

successful nodule formation - nodulins

3. Suppression of genes normally involved in repelling

microbial invaders - host defense genes
 Nodulins

Nitrogen
Bacteroid
fixation
development Nodule
Root hair senescence
invasion
Bacterial late
attachment nodulins

early nodulins Nodulins?

Pre-infection Nodule
Infection function and Nodule
and nodule maintenance senescence
formation
Early nodulins

At least 20 nodule-specific or nodule-enhanced genes

are expressed in plant roots during nodule formation;
most of these appear after the initiation of the visible
nodule.

Five different nodulins are expressed only in cells

containing growing infection threads.
These may encode proteins that are part of the
plasmalemma surrounding the infection thread, or
enzymes needed to make or modify other molecules
Twelve nodulins are expressed in root hairs and in
cortical cells that contain growing infection threads.
They are also expressed in host cells a few layers ahead
of the growing infection thread.

Late nodulins
The best studied and most abundant late nodulin is
the protein component of leghaemoglobin.
The heme component of leghemoglobin appears to be
synthesized by the bacteroids.
Other late nodulins are enzymes or subunits of enzymes
that function in nitrogen metabolism (glutamine
synthetase; uricase) or carbon metabolism (sucrose
synthase). Others are associated with the peribacteroid
membrane, and probably are involved in transport
functions.

These late nodulin gene products are usually

not unique to nodule function, but are found in other
parts of the plant as well. This is consistent with the
hypothesis that nodule formation evolved as a
specialized form of root differentiation.
There must be many other host gene functions
that are needed for successful nodule formation.

Example: what is the receptor for the nod factor?

These are being sought through genomic and proteomic

analyses, and through generation of plant mutants
that fail to nodulate properly

The full genome sequencing of Medicago truncatula

and Lotus japonicus , both currently underway, will
greatly speed up this discovery process.