Biological Nitrogen Fixation

Genetics of Diazotrophs
Nodule formation and nitrogen fixation are the two biological processes which are controlled by genes of diazotrophs. From rhizobial genome numerous symbiotic genes (nod genes) encoding for nodulation, and nitrogen fixing genes (nif genes) have been identified. In free living and symbiotic nitrogen fixers nodule-forming and non-nodule forming nif genes are present on genome or megaplasmid in their cells. Organization, structure and function of nod genes, nif genes and Hup genes (hydrogen uptake genes) are briefly described below :

Nod Genes
Nodule forming species of Rhizobium consists of an extremely, large plasmid known as 'megaplasmid' in cell which possesses numerous genes coding for nodulation (nod genes) and nitrogen fixation (nif genes) (Rosenberg et al 1981). Both nod genes and nif genes are closely located. However, a physiological map of 135 Kb segment of megaplasmid was established which contained nod-nif regions.

Kondorosi et al. (1982) successfully transferred the megaplasmid of Rhizobium meliloti into other Rhizobium species and Agrobacterium tumifacnies with the result that the transconjugants became able to form nodules or nodule-like structures on alfalfa. This indicates that early steps of nodulation are encoded by this megaplasmid i.e. pRme4 lb in R. meliloti. However, it has been confirmed that nodulation occurs only in certain stages. When early period is over nod genes do not express. 

» Non-Symbiotic N2 fixation

» Diazotrophy

» Ecology of diazotrophs

» Special features of diazotrophs

» Sites of N2 fixation

» Nitrogenase and reductants

» Presence of hydrogenase

» Self regulatory systems

» Mechanism of N2 fixation
» Symbiotic N2 fixation

» Establishment of symbiosis

» Host specificity and root hair curling

» Infection of root hairs

» Nodule development

» Nodule development and maintenance

» Factors affecting nodule development

» Mechanism of N2 fixation in root nodules
» Genetics of diazotrophs

» Nod genes

» Nif genes

» Nif gene cloning

» Hup genes
The identified nod gene region is of 11.5 Kb in length. Nucleotide sequence and proteins encoded by 8.5 Kb fragment in E. coli has been determined. The nod genes consisted of 4 genes designated as nod A, B, C and D; the 4 genes code for proteins of 196, 217, 402 and 311 amino acid residues, respectively. Based on comparisons of nucleotide and amino acid sequences of nod A, B and C genes and amino acid sequence of nod A, B and C genes between different species of Rhizobium, about 69-72% homologous region has been characterized which is known as "common nod genes'' (Kondorosi et al., 1982).

The structural and functional conservation of common nod genes has become a tool to identify common nod genes of other species of Rhizobium. Recently, about 25 Kb fragment of megaplasmid containing all essential nod genes has been indentified and a recombinant plasmid (pPP346) has been constructed. A. tumifaciens cells became able to develop nodules on alfalfa roots when this plasmid was incorporated.

Nif Genes
A region lies on genetic material of free living and symbiotic N2 fixers which consists of nitrogen fixing nif genes. In Rhizobium leguminosarum, R, meliloti and other species nif genes are located on a megaplasmid adjacent to nod genes, whereas in cyanobacteria e.g. Anabaena 7120 and most of free living bacteria nif genes are localized on the chromosome. Early studies on nif genes were carried out in Klebsiella pneumoniae and the function was confirmed by transferring into E. coli cells. Thereafter, nif genes in Rhizobium, cyanobacteria and other N2 fixers were discovered. 

Researches were done on physical mapping to know the product of nif gene cluster. It was found that there are several parts of nif genes forming a gene clusture of 24 Kb nucleotides which are located between the genes encoding for histidine (his) and shikimic acid (shi A).
  A diagram of nif gene cluster of Klebsiella pneumoniae (A) and Anabaena sp. (B). I-nif genes in vegetative cells; II-nif genes in heterocyst after rearrangement.

Fig. 11.8. A diagram of nif gene cluster of Klebsiella pneumoniae (A) and Anabaena sp. (B). I-nif genes in vegetative cells; II-nif genes in heterocyst after rearrangement.


This cluster is organized in 7 operons i.e. transcription units (e.g. QB AL FM VSUX NE YKDH J). On the basis of mutational studies performed in all m/genes, the nature of various products of nif genes was determined. Now it is confirmed that nif HDKY operon encodes nitrogenase, whereas nif LA has regulatory function. In nif HDKY genes H, D and K encode for subunit of Fe-protein, Mo-Fe-Protein.

Nitrogenase acts only in the absence of ammonia and other nitrogen compounds as they inhibit its expression. Glutamine synthatase (GS) also losses its function. Glutamine synthatase gene encodes GS enzymes which is quite apart from nif genes.
Some filamentous cyanobacteria are composed of entirely vegetative cells (Golden et al, 1987). In absence of combined nitrogen source some photosysthetic vegetative cells differentiate into heterocysts at regular intervals along the filaments, terminal or lateral singly or in chains. In heterocysts, during differentiation many morphological, biochemical and genetical changes occur. At this time induction of nitrogenase takes place.
In Anabaena 7120 nif H, D and K have been identified with DNA probe of K. pneumoniae through hybridization. However, during heterocyst differentiation two DNA rearrangements occur. In Anabaena, organization of nif H,D and K genes differs in vegetative cells from that of K. pneumoniae where three genes (S, H and D) are contiguous and form an operon. NifH is in close proximity to m/D, while nifK is 11 Kb apart from nifD (Fig. 11.8B).
Organization of m/genes of vegetative cells and heterocysts was compared. It was found that in heterocysts the nifK and nifW were closely attached; size of nif gene cluster reduced from 17 Kb (in vegetative cell) to 6 Kb (in heterocyst) (Fig. 11.8B). This was proved by hybridizing the m/gene fragment with some of the m/gene probes by using restriction enzymes (Golden et al 1987).
In recent years, Azospirillium has attracted attention of workers for being as a possible source of biofertilizer due to presence of m/HDK clusture like K. pneumoniae. Hybridization experiment between m/probe of K. pneumoniae and total DNA of many strains of Azospirillum has confirmed the presence of nif HDK and nif A genes. Like Rhizobium sp., Azospirillum sp. also contain a megaplasmid and the sequence homologous to nod genes originated from a common ancestors (Elmerich et. al, 1987).
Elmerich (1987) reviewed the N2 fixing organisms associated with nonleguminous plants and described the presence of plasmids of various molecular weight in Anabaena, Azotobacter, Frankia and Rhizobium.
Cloning of nif genes
In many countries researches on m/gene transfer into higher plants, especially in monocots, and gene expression in them are in progress. However, success has been made in m/gene cloning into E. coli. Since nif genes are prokaryotic in origin, the best strategy of their transfer into non-leguminous crops would be to transfer m/genes into chloroplast. The transcriptional and translational machinery of chloroplast bears several prokaryotic features. These attempts would be successful because the chloroplasts are geared to the production of ATP and reducing power, as both of which are required for nitrogen fixation (Merrick and Dixon, 1984). But the major problems for doing so are (i) lack of chloroplast transferring techniques, and (ii) protection of nitrogenase from O2 evolved during photosynthesis. Some more aspects of nif gene cloning have been discussed elsewhere (see Enzyme engineering and Trasnfer of nif genes to eukaryotes).

Hup Genes
In some species of Rhizobium, hydrogen uptake (or Hup) genes have been reported which displayed the ability to recycle H2 (produced as a result of conversion of N2 to NH3) back to nitrogenase complex. This mechanism helps the plant to harvest the energy as being lost by the plants (Fig. 11.2).

Most of legumes loss 30-50 per cent of their energy as H2 gas which in turn reduces the efficiency of N2 fixation. Recently, Indian Scientists at IARI (New Delhi) have produced a genetically engineered N2 strain by transferring the Hup genes of R. leguminosarum into Rhizobium strain (which did not contain Hup genes). This strain infected the roots of chick-pea and developed root nodules. Hup system recycled H2 and reduced energy losses by 8-13 per cent. This is the world's first case of interspecific transfer of Hup genes. The successful transfer and expression of Hup genes has increased the possibility of improving symbiotic energy efficiency of chick-pea - Rhizobium system.