Selection and characterization of salt-tolerant strains of Rhizobium phaseoli

Abstract

Rhizobium phaseoli TAL 113 is one of the fast-growing type of Rhizobium which can nodulate Phaseolus vulgaris. By an application of a sibling selection, 4 strains of 0.3 M NaCl tolerant-mutants were isolated and named as P1, P5, P19 and P21 respectively. It was found that all isolated mutants still possessed a modulated property as seen in their effectiveness of symbiotic nitrogen fixation. Multiplication of cells in the medium supplemented with 0.3 M NaCl was observed, only in mutants and the generation time was markedly increased about 4-6 times compared to that without salt. The maximal growth yield under1 salted-condition was also decreased about 1.5 times compared to that under unsalted condition. Every isolates of mutants possessed a similar surface antigen comparing to that of the WT as revealed in the certification of cell-surface antigen based on the fluorescent antibody technique. In addition, an identical value of % GC content, determined from the chromosomal DNA of mutant P19 and WT was equal to 61.2%, whereas R. japonlcum, a slow-growing rhizobium, possessed a higher value of 64.9%. It was concluded that a deviation in growth character from a fast to a slow-growing type, found in all mutants, was in situ property hindered as a nature of a salt-tolerant mutant of Rhizobium. The cellular morphology of the mutants when visualized by SEM was similar to that of the WT. But visualization of the cellular morphology of the mutants by TEM revealed a rough inclusion in the cell envelope including a more dense and compact character at the chromosomal center of the periplasm, especially when sample used was cells cultivated under salted condition. In addition, under salted condition the rate of oxygen consumption was two folds increased whereas a maintained level of ATPase activity was observed in both mutants and WT regardless of the presence of salt in the cultivating medium. Kinetic studies of rate of Na+ efflux revealed that cultivation of cells in the presence of salt caused an increased value in the initial rate of Na+ efflux in the mutant from 1.0 to 1.7 nmol per min per mg protein, whereas the initial rate of Na+ efflux in the WT was only 0.2 nmol per min per mg protein. Studying of the effect of oxidative inhibitors was resulted in that, only FCCP(p-trifluoromethoxy carbonyl cyanide phenylhydrazone)not DCCD ( N ,N'-dicyclohexylcarbodiimide) could inhibit the flux of Na^ out from the cells. The result from this study suggested that a mechanism to tolerate high salt concentration of the strains isolated was the enhancement in the rate of Na+ efflux driven by the respiratory energy which being inhibited by FCCP.