Biotechnology of Nitrogen Fixation: Understanding

Nitrogen Fixation, Genetic Engineering in

Nitrogenase gene.
 BIOTECHNOLOGY OF NITROGEN
FIXATION
Nitrogen is a critical limiting element for plant growth and
production.
It is a major component of chlorophyll, proteins, ATP and
nucleic acids.

Plants acquire these forms of “combined” nitrogen by:

1) The addition of ammonia and/or nitrate fertilizer (from the
Haber-Bosch process) or manure to soil.
2) The release of these compounds during organic matter
decomposition.
3) The conversion of atmospheric nitrogen into the
compounds by natural processes, such as lightning, and
 Biological Nitrogen Fixation
Biological nitrogen fixation (BNF), discovered by Beijerinck
in 1901.

These organisms utilize the enzyme nitrogenase to catalyze

the conversion of atmospheric nitrogen (N2) to ammonia
(NH3).

Plants can readily assimilate NH3 to produce the

aforementioned nitrogenous biomolecules.

The prokaryotes include such as cyanobacteria, Azotobacter,

Azospirillum, Rhizobium and Bradyrhizobium.
 Process for Formation of Ammonia
Nitrogenase catalyzes the breaking of three bonds of N2
and the addition of three hydrogen atoms to each nitrogen
atom.

Microorganisms that fix nitrogen, require 16 moles of

adenosine triphosphate (ATP) to reduce each mole of
nitrogen.

Photosynthetic microorganisms, such as cyanobacteria, use

sugars produced by photosynthesis.

Associative and symbiotic nitrogen-fixing microorganisms

obtain these compounds from their host plants’
 Nitrogen Fixation by Free-Living Heterotrophs
Many heterotrophic bacteria live in the soil and fix
significant levels of nitrogen without the direct interaction
with other organisms.

Examples of this type of nitrogen-fixing bacteria include

species of Azotobacter, Bacillus, Clostridium,
and Klebsiella.

Because nitrogenase can be inhibited by oxygen, free-

living organisms behave as anaerobes or microaerophiles
while fixing nitrogen.

Wheat rotation farming system demonstrated that free-

 Associative Nitrogen Fixation
Species of Azospirillum are able to form close
associations with several members of
the Poaceae (grasses) & crops, such as rice, wheat, corn,
oats, and barley.

These bacteria fix appreciable amounts of nitrogen

within the rhizosphere of the host plants.

Symbiotic Nitrogen Fixation

The plant provides sugars for nitrogen-fixing
microorganism for the energy it needs for nitrogen
fixation.
 Legume Nodule Formation
The Rhizobium or Bradyrhizobium bacteria colonize the
host plant’s root system and cause the roots to form
nodules.

Legumes not being nodulated plants are typically

chlorotic, low in nitrogen content, and yield very little
seed.

The process begins when the rhizobia are attracted to

flavonoids released by the host legume’s roots.

For legumes like alfalfa, clover, and soybeans (others

like lupines and peanuts form nodules in other ways) the
Crop Nodulating Bacteria
Alfalfa Sinorhizobium melilotii
Rhizobium legumninosarum biovar
Beans
phaseoli and Rhizobium tropici

Clover Rhizobium leguminosarum biovar trifolii

Lotus Mesorhizobium loti
Peas Rhizobium leguminosarum biovar viceae
Bradyrhizobium japonicum, Bradyrhizobium
Soybean
elkanii, Rhizobium fredii
Sesbania Azorhizobium caulinodans
Table 1. Major cross inoculation groups of legumes.
 Structure and Operation of Nitrogenase
Active nitrogenase can be reconstituted by the addition of
purified Mo-Fe and Fe proteins of different
microorganisms.

For examples, proteins of Klebsiella pneumoniae and

Bacillus polymyxa and cyanobacteria

Nitrogenase contains the two proteins molybdoferredoxin

and azoferredoxin.

This MoFe cofactor is unique to nitrogen fixation and

distinct from the Mo-pterin cofactor of other Mo proteins
(e.g., nitrate reductase, xanthine oxidase).
 Organization of Nif Genes
Nitrogen fixation is carried out by three groups of genes.
These are;
1. Nod gene (responsible for nodule formation),
2. Nif gene (responsible for nitrogen fixation) and
3. Hup gene (responsible for nitrogen uptake).

All these three types of genes are present in a group on a

single chromosome.

This makes their copying and transfer mechanism simple for

genetic engineering purposes.
 Nod gene:
Most of the biological nitrogen fixing bacteria contains a
large plasmid called mega-plasmid.

Several special genes are present along with nod genes.

Such plasmids are absent in non-symbiotic bacteria.

A nod gene is a group of genes containing Nod A, B, C, D

genes having 8.5 kb length.

These genes have different lengths (196, 197, 402, 211

amino acid).

Nod genes of different rhizobium species have almost 70%

 Nif Genes
This gene is responsible for nitrogen fixation and present in
the genome of symbiotic and non symbiotic nitrogen fixing
bacteria.

 In symbiotic bacteria Rhizobium, it is present near nod

genes on the megaplasmid, while in non-symbiotic
cyanobacteria it is present on the main DNA.

Initially Nif gene has been transferred in E. coli.

Most of the cereal plants are monocots and any such effort
to transfer such Nif gene will revolutionize the yield,
economics and environmental pollution.
 Hup gene
Gene responsible for nitrogen uptake is Hup gene.

In symbiotic bacteria this gene recycles the hydrogen

produced during nitrogen fixation.

If this hydrogen can be recycled by nitrogenase enzyme we

can save a lot of energy, and this can be carried out by
improved Hup gene.

Klebsiella genome is quite similar to that of E. coli and

Salmonella typhimurium.

Therefore most of the techniques of genetic engineering can

 Nif Gene Organization in Klebsiella:
Several mutants of Klebsiella were developed by growing
the bacteria on medium containing a mutagen, methyl-nitro
nitro-so-guanidine.

Different mutants obtained were used in transformation,

transduction to map the Nif gene.

 This provided the information that Nif gene is downstream

to histidine operator.

 On the basis of this Nif gene of Klebsiella was transferred

in E. coli on the basis of homology in the plasmid and not in
genomic DNA.
 Transgenes with Nif Genes:
It is important to develop transgenic plants containing Nif
genes to solve the problem of nitrogen fertiliser supplement
to crop plants.

Ti (tumor inducing) plasmid and Cauliflower mosaic virus

based promoter was used to transfer Nif genes in to non-
legume plants.

To test the efficacy of this system, phaseolin gene from

legume (pulses) has been transferred to sunflower where it
was expressed and produced phaseolin.

Protoplasts isolation from root nodules and preparations of