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Salt-tolerance genes of Sinorhizobium 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, which encodes a cation efflux system protein, while mutations in genes encoding other cation effux system proteins were found in seven (phaD2), two (phaF2) and two (phaG2) mutants. A mutation in the metH gene, encoding 5′ methyltetrahydrofolate homocysteine methyltransferase, was found in two of the salt sensitive strains. 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 and that metH mutants were highly sensitive to any of Na+, Li+, K+ and sucrose. Na+ intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na+ efflux in S. fredii RT19. 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.