CHARACTERIZATION OF GENES INVOLVED IN BIOSYNTHESIS OF SOME AMINO ACIDS IN Rhizobium meliloti

Abstract

The present study was undertaken to isolate and characterize amino acid auxotrophic mutants of R meliloti. Atotal of thirty one amino acid auxotrophs of R. meliloti Rmd 201 were isolated The current investigation deals with the nature of the metabolic blocks and the role of these mutants in the symbiotic process. For obtaining auxotrophic mutants, R. meliloti ZB201 ( Cys", Rif) was subjected to random Tn5 mutagenesis. Transposon Tn5 carrying suicide vector pGS9 was used for Tn5 mutagenesis. Conjugation (patch mating) was earned out between R meliloti ZB201 and E^ coli WA803 (pGS9)to obtainTn5 derivatives. The mating time of sixteen hours wasfound sufficient for getting good frequency of Tn5 derivatives. Atotal of five thousands kanamycin resistant Tn5 exconjugants were isolated. All the Tn5 derivatives were found to be chloramphenicol sensitive, indicating positive suicide of pGS9 vector. Sixteen auxotrophic mutants were obtained when the growth of Tn5 derivatives was tested on minimal medium. These auxotrophs were found to be highly unstable and moreover their nutritional requirement could not be specified. Tn5 mutagenesis was done again with R meliloti Rmd201 (Strr derivative of R. meliloti AK631). Tn5 derivatives were obtained at a frequency of 0.4 x 10"4 ttansconjugants/recipient. Six thousand Kin' mutants were isolated and tested for nutritional auxotrophy. Seventeen auxotrophs were identified: proline" (three), isoleucine" (one), aspartic acid- (two), methionine' (one), tyrosine- (one), glutamic acid' (one), leucine' (one), cysteine' (one), phenylalanine" (two), valine" (one), threonine" (two) and histidine' (one). Auxott'ophic mutants of R. meliloti Rmd201 were also isolated by N-methyl- NT'-nitro-N-nitrosoguanidine (NTG) mutagenesis followed by penicillin enrichment. Aconcentration of lOOug/ml was used to yield six thousand NTGinduced mutants. Thirty auxotrophs were isolated and tested for nutritional requirements. Sixteen mutants were found to possess multiple auxotrophies. Fourteen auxotrophs for amino acids were obtained as follows : five for threonine, two for proline, three for (ii) lysine, one for asparag.ne and tyrosine and one for each valine, tryptophan and methionine. All the amino acid auxotrophs of R meliloti were subjected to reversion analysis. The revertants of seventeen Tn5 derived auxotrophs were found to be Km' indicating precise excision of Tn5 at a frequency of 1010 . NTG induced auxotrophs also reverted at the same frequency. All the purified auxotrophic mutants were tested for their symbiotic properties by inoculating them on Medicago sativa seedlings grown aspetically on nitrogen free agar slants in test tubes. After eight weeks, data on individual plants were recorded for nodule number, nodule colour, nodule size, nodule shape, plant height, dry plant weight and total nitrogen per plant. Out of thirty one auxotrophic mutants, one auxotroph (proline) was found to be Nod', seven auxotrophs (threonine', valine", histidine", aspartic acid", proline", leucine and phenylalnine") were found to have reduced nitrogen fixing ability and two auxotrophs (tryptophan' and lysine) were found to be more efficient in nitrogen fixation. When the revertants were similarly examined, they resembled the wild type strain Rmd201, indicating that changes in the symbiotic behaviour of the above strains could be attributed to their auxottophic mutations. In all the above plant experiments, the bacteria were isolated from the nodules to confirm that nodules were colonized by the same strain with which the plant was inoculated. The symbiotically altered auxotrophic mutants were tested for their symbiotic ability to fix nitrogen by inoculating them on nitrogen free agar supplemented with their auxotrophic requirement. The symbiotic properties ofall the auxotrophs except four (requiring aspartic acid, lysine, tryptophan and threonine) were restored on supplementation with their nuhitional requirement. The results with these four auxotrophs indicate either exogenous supply of amino acid is not accessible to the bacteroids in the nodules or the involvement of a particular intermediate of biosynthetic pathway in the symbiotic process. (iii) The plasmid constructs pGRl and pGR3 containing known symbiotic functions were transferred to different auxotrophs having altered symbiotic ability. Both pGRl andpGR3 could not restore the symbiotic nitrogen fixation ability indicating that the symbiotic genes on nod-nif region present on megaplasmid were not mutated in these auxotrophic mutants. Cross feeding and intermediate supplementation of amino acid auxotrophs were done to identify the metabolic blocks in the amino acid biosynthetic pathways. From the results, it appears that auxotrophs were having defects in the different steps of the biosynthetic pathways. The clones complementing the different auxodophic mutations were isolated from the gene bank of R. meliloti by doing uniparental crosses. For five auxotrophs, recombinant clones could not be obtained. The reason for this could be that these clones were not represented in the gene bank. The soluble proteins of whole cell extracts from the wild type and its auxotrophic mutants was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).No marked difference between protein patterns of auxotrophic mutants and the wild type was found. These auxotrophs were tested for their ability to utilise various sugars, response to high osmolality and high temperatures. The number of arabinose", xylose", lactose', galactose" and maltose" auxotrophs were three , two, one, one and one, respectively. Two auxotrophs were osmosensitive and one was temperature sensitive. The symbiohcally affected auxodophs were also tested for their surface properties. Two auxotrophs were Exo, two were j]( 1-3) glucan", two were Cel" andone was found unable to produce lipopolysaccharides. These results show the pleiotropic naftire of these auxotrophic mutations.