Cloning And Functional Study Of The Genes Related To Salt Tolerance Of Sinorhizobium Fredii RT19

Sinorhobium fredii RT19, isolated from saline soil in Tianjin of China, is a halotolerant soybean rhizobial strain which can tolerate up to 0.6mol/L NaCl. Salt-tolerance genes of S. fredii RT19 were identified by the construction and screening of a transposon Tn5-1063 library containing over 30,000 clones. Twenty-one salt-sensitive mutants were obtained and five different genes were identified by sequencing. Eight mutants were found with disruptions in the phaA2 gene, while mutations in other genes were found in seven (phaD2), two (phaF2) and two (phaG2) mutants. A mutation in the metH gene, encoding methionine synthase â…¡, was found in two of the salt sensitive strains. Based on the insertion locus of Tn5-1063, these salt-sensitive mutants were designated as phaA2 mutants: RTa-1 to 8; phaD2 mutants: RTd-1 to 7; phaF2 mutants: RTf-1, RTf-2; phaG2 mutants: RTg-1, RTg-2; metH mutants: RTh-1 and RTh-2, respectively.Further sequence analysis showed these four genes, phaA2, D2, F2 and G2, as well as other three genes phaB2, C2 and E2, form a single transcription unit, which share the highest identity with phaA2, B2, C2, D2, E2, F2 and G2 of 5. meliloti 1021 and also share very high similarity with the corresponding genes of putative multiunit Na~+/H~+ antiporter of Agrobacterium tumefaciens strain C58 and the corresponding genes of the putative monovalent cation/H~+ antiporters of Coxiella burnetii RSA 493.Growth experiments showed that phaA2, phaD2, phaF2 and phaG2 mutants were hypersensitive to Na~+/Li~+ and slightly sensitive to K~+ and not sensitive to sucrose, which suggested that pha2 gene cluster play a dominant role in the salt-tolerance of 5. fredii RT19. By contrast, metH mutants were highly sensitive to any of Na~+, Li~+, K~+ and sucrose. Moreover, recovery of growth of the metH mutants incubated with different concentrations of NaCl could be obtained by additions of methionine, choline and betaine, which showed that the metH gene is probably involved in osmoregulation in S. fredii RT19.The complete pha2 gene cluster was cloned into the vector pBBRl-MCS5 and the resulting vector is named pMCS5-ZEd-l-ZCa-3. Through the tri-parental conjugation, pMCS5-ZEd-l-ZCa-3 was transferred into the pha2 mutants. Complementary experiments showed that the pha2 gene cluster completely restored the growth of pha mutants even at the concentration of 0.6 mol/L NaCl. The gene metH was cloned by PCR into the vector pBBRl-MCS5 and the resulting vector is named pMCS5-metH. And pMCS5-metH was transferred into the metH mutants through the tri-parental conjugation, complementary experiments showed that metH gene also restored the growth of metH mutants on the plates of FY medium.Na~+ intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na~+ efflux in 5. fredii RT19. Monovalent cation/H~+ antiporter assay showed that wild type had very high Na~+(Li~+)/H~+ antiporter activity and the mutants lost this function, indicating that the proteins encoded by the pha2 gene cluster share Na~+(Li~+)/H~+ antiporter activity. And wild type showed higher K~+/H~+ antiporter activity than pha2 mutants, suggesting that the proteins encoded by this DNAregion also functioned as one or several K~+/H~+ antiporters. The four mutants with mutations in phaA2, phaD2, phaF2 and phaG2, respectively, showed the same physiological results, and these seven proteins share a very high identity with the "subunits" of a putative multisubunit Na~+/H~+ antiporter. Therefore, we consider that the pha2 gene cluster encodes a multisubunit monovalent cation/H~+ antiporter.