Index >> Nitrogen Fixation Free Living and Associative Symbiotic Bacteria >> Genetics

Electrons are carried fro the nif J protein having pyruvate flavodoxin oxydoreductase activity to the nif F protein which is the substrate of the Fe protein.

Ammonia (NH₄) totally represses nif gene product biosynthesis. The genes involved in glutamine synthetase, an enzyme which regulates NH₄ assimilation are referred to as Gin while ntr denotes genes whose products regulate nitrogen assimilation. The genes which determine uptake hydrogenase activity are known as hup genes. In K. pneumoniae, ntrBC are linked to glnA, the structural gene for glutamine synthetase whereas whereas ntrA is unlinked. GlnA and ntrBC are organized in one or two operons transcribed from two promoters in the order P1glyAP2ntr BC.P1 promotes transcription under conditions of nitrogen limitation whereas P2 acts under nitrogen enriched situations.

In this way, when nitrogen is the limiting factor, the biosynthesisi of glutamine synthetase is derepressed including those operons under the control of nif which includes the nif regulator also. The product of ntrC acts as a general of all these operons which is again dependent on ntrA product. The organization of the nif cluster of K. pneumoniae.

From the current information on the organization and functioning of the nif gene is some N2 fixing species, it can be reasonably concluded that a basic group of nif genes is perhaps common to all diazotrophs including nif HDK genes which are highly conserved. These are the genes concerned with the processing of the metal clusters of the nitrogenase plus the regulatory genes.

Genetic analysis of Azotobacter vinelandii and A. chroococcum have revealed that a major nif cluster comparable to that of one in K. pneumoniae and comprising of genes nif HDKTY, nif ENX, nif USV, nif WZM, nif F, also occur in Azotobacter, The nif ABQ genes are also linked to each in a separate cluster and have been sequenced in Azotobacter A chroococcum is capable of producing two been sequenced in Azotobacter. A. chroococcum is capable of producing two nitrogenases?a Mo containing one in N-free molybdenum added medium, a Va containing one in the presence of vanadium and absence of molybdenum.

A. vinelandii can produce three enzymes a Mo nitrogenase, a Va nitrogenase and one without either of these two metals in a medium devoid of those metals. Even though nif regulation in Azotobacter appears to be considerably similar to K. pneumoniae, the situation is complicated because of the unknown mechanisms through which the three types nitrogenases are synthesised by the bacteria. Nevertheless, the nif regulation in Azotobacter is believed to be similar with regard to nir/nif system of K. pneumoniae.

Azospirillum spp. contain plasmids ranging from 90 to 120 megadaltons (Mda). They are difficult to cure, non-conjugat8ive and not easy to purify. Different species of Azospirillum cannot be differentiated on the basis of plasmid content. There are no indications that plasmids of the same molecular with corresponds to the same molecular species. Spontaneous loss of plasmids has been reported in strain sp7 of A. brasilense. No phenotype has been associated with the plasmids. Nif genes are likely to be chromosomal in nature.

A small number .of mutants Unpaired in N, fixation and metabolism (ex: impaired in glutamine synthetase activity) have been obtained using chemical mutagens. Hybridization of DNA of Azospirillum with Klebsiella pneumoniae nif probes revealed homology with nif HDK and nif A. The transcriptional organization on of the nif HDK cluster of A. brasilense sp7 was examined by Tn5 site directed mutagenesis, genetic Complementation and analysis of products.

Similarly, the presence of nif loci in the 20 KB region nif HDK was also examined by carrying out over 50 Tn5-induced mutations. The results showed the presence of nif loci about 5 and 12 Kb downstream from nif K. Using heterologous nif Probes from K. pneumoniae and Azorhizobium caulinodans new nif regions were characterized as nif E proximal to nif K and nif US distal to nif K.

Eventhough the corresponding gene(s) were not identified, an additional locus was detected between nif E and nif US. Nitrogen-fixing genes (fix) have been identified in rhizobia but not in K. brasilense by hybridization experiment with fix ABC genes of Bradyrihizobium japonicum and fix A of Azorhizobium caulinodans. Many of these results have been obtained by Claudine Elmerich and her colleagues at the Institute of Pasteur, Paris and summarized in.

Enterobacter agglomerans, a nitrogen fixing bacterium isolated from the innermost rhizosphere of wheat contains all genes essential for nitrogen fixation on large (100-150 Kb) self-transmissible indigenous plasmids in the form of a continous package.



When 22 strains of Bacillus azotofixans, a new nitrogen-fixing species were tested for DNA homology with Klebsiella pneumoniae nif genes, it was found to contain only sequences which are homologous to structural nif genes of K. pneumoniae.

Nitrogen fixation by methanogenic bacteria such as Methanobacterium and Methanosarcina have been recently discovered. By using Klebsiella pneumoniae and Anabaena probes, 14 methanogenic species were analysed and found to contain homology to nif H, suggesting the very ancient origin of nif genes since archaebacteria to which methanogens belong occupy the lowest position in the evolution of microorganisms.

The availability of sophisticated DNA recombination techniques has made it possible to think in terms of transfer of nif genes to a larger variety bf prokaryotic organisms and even to eukaryotic cells. This kind of exercise may ultimately lead to the introduction of nif genes into other eukaryotic systems. The main emphasis behind all these recombinant DNA research in relation to biological nitrogen fixation is to render crop plants self-sufficient with regard to nitrogen, one of the key elements in crop growth and grain production.

The first step in the genetic engineering strategy has been to construct an appropriate vehicle for transferring nitrogen-fixing (nif) genes. The vehicles are plasmids (extra-chromosomal circular DNA molecules) which are small amplifiable self-replicating units. The most useful plasmid has been RDI which has been used to transfer K. pneumoniae nif genes to Agrobacterium tumefaciens, Rhizobium meliloti and Azotobacter vinelandii.

The second step is to construct nitrogen-fixing cereal plants by transferring nif genes into eukaryotic plants. In this approach, the success depends on the construction of plasmid vehicles which overcome the barriers of DNA uptake, DNA replication and gene expression in the higher plants. One of the promising methods which has been proposed is to introduce nif genes into 'Agrobacterium tumefaciens' which produces a tumour called crown gall in a large number of dicotyledonous plants upon wounding and subsequent infection.

The bacterium has a plasmid that causes crown gall tumour and induces the plant to synthesize opines, which are nitrogen-rich compounds. The underlying mechanism is the in¬sertion of T-DNA (transfer DNA, a segment of the plasmid) into a chromosome of the plant cells which are infected. (For more information.) Through this genetic modification, other daughter plant cells acquire the property to regenerate crown-gall tumours without the need for fresh A. tumefaciens infection. The altered plant cells synthesize their own hormones and opines necessary for tumour growth. The procedure envisaged is to cut open the plasmid at a site within the T-DNA region and the foreign gene (in this instance, nif genes) can be spliced into it.

This reconstituted DNA is replicated when the tumour cells of a crown gall are grown in tissue culture and the dividing cells continue to carry T -DNA. The transmission may continue through successive progeny. This approach provides a mod l for transferring genes from a prokaryote to a eukaryote. Other two possible approaches for molecular cloning of nif genes involve plant viruses like cauliflower mosaic virus and chloroplast DNA.

Yeast is an example of simple eukaryote. Genes for nitrogen fixation have been inserted in yeast (Saccharomyces cerevisiae). The first step is aimed at producing a hybrid plasmid from the yeast cell and E. coli and in the second step nif genes from K. pneumoniae are cleaved into another E. coli plasmid to produce another hybrid plasmid. The two types of yeast cell genomes are then integrated to form a novel yeast genome carrying nif genes. Although integration of nif genes into the yeast genome has been achieved, the expression of nif, i.e., the ability of altered yeast cells to fix N2 is yet to be achieved.