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.
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).
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.
© 2018 Biocyclopedia | All rights reserved.