Study Of Molecular Mechanism Involved In Salt Tolerance Of Sinorhizobium Meliloti

Sinorhizobium meliloti 042BM was isolated from root nodules of alfalfa (Medicago sativa) from Xinjiang Region, it can survive in the presence of 0.6 mol/L NaCl in the FY minimal medium. Eight mutants sensitive to salt tolerance were obtained after screening about 20,000 mutants by mutagenesis with Tn5-1063, they were W2,W4, W8, W10, W11, W15, W19 and E4. Osmotolerance experiment showed that they could not grow in the presence of 0.4 mol/L NaCl in FY medium. Under the same osmotic stress, the mutants were only sensitive to salt but not to glucose except for W4 then they could be identified as salt-sensitive instead of osmosensitive. Their tolerance for temperature and pH was not different from wild type 042BM. A Southern analysis using luxAB of pRL1063a as probe revealed that 8 salt-sensitive mutants each contained a singled Tn5-1063 fusion, and that the location of each fusion was distinct. It suggested the salt-sensitive phenotype were made by Tn5 insertion.The DNA sequences flanking the Tn5-1063 were determined and seven different genes were identified. These genes include omp10 encoding a cell outer membrane protein, relA encoding (p)ppGpp synthetase, greA encoding a transcription cleavage factor, nuoL encoding NADH dehydrogenase I chain L transmembrane protein, a putative nuclease/helicase gene and two unknown genes.DNA sequences of the greA gene containing promoter were obtained by PCR, then they were cloned into shuttle plasmid pBBR1MCS-5, and introduced into sensitive mutants W2 through three-parental mating experiment. The salt-tolerance of W2 was recovered and it was demonstrated that the greA gene was a transcription factor involved in salt tolerance.The ORF of the relA were obtained by PCR and were cloned into the expression vector pET-28a. Significant growth was observed when relA was transformed into E. coli relA mutant. In contrast, the growth of the empty vector transformant was strongly inhibited on the SMG plate. Thus, it can be postulated that the ORF can encode the product which has the function of (p)ppGpp synthetase activity. Nodulation tests showed relA mutation leads to the symbiosis defect of 042BM. DNA sequences of the relA gene containing promoter were obtained by PCR, and they were cloned into shuttle plasmid pBBRlMCS-5, introduced into sensitive mutants W11 through three-parental mating experiment. The salt-tolerance of Wll was recovered and it was indicated that the relA gene was an important gene involved in salt tolerance.Based on our findings, we suggest that the regulation of salt tolerance of S. meliloti 042BM is on several levels in which many different genes can be involved:l) global regulation;2) macromolecule metabolism;3) stress products;4) energy metabolism/ion transportion;5) cell structure components.