Identification of Sinorhizobium meliloti Genes Influencing Synthesis of Surface Polysaccharides and Competitiveness

Abstract

A new approach to isolating mutants with altered composition of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) in nodule bacteria of alfalfa Sinorhizobium meliloti based on analysis of their respiratory activity was proposed. Random Tn5-mob mutants of symbiotically effective strain SKhM1-105 were tested for slime-production ability and coloration on diagnostic media containing the indicator of reducing equivalents, triphenyltetrasolium chloride (TTC), the inhibitor of respiratory activity, 2-methyl-4-chlorphenoxybutyric acid and Congo Red, the stain for LPS and exopolysaccharides (EPS). Electrophoretic analysis (SDS-PAGE) of polysaccharides in seven mutants, markedly differing from the parental strain with respect to their growth on diagnostic media demonstrated that (1) the production of CPS was either decreased (in mutants T64 and T835) or blocked (in T71, T94, T124, T134, and T170); (2) the R form of LPS had changed mobility (in T134); (3) the S form of LPS contained only one component instead of two (T71). In symbiosis with alfalfa Medicago sativa, all mutants exhibited a decreased competitive ability for adsorption on roots of host alfalfa plant, compared to the parental strain. At early stages of symbiosis, mutants had a tendency toward a decrease in the number of nodules, in comparison with the parental strain (a statistically significant decrease was observed in mutants T71 and T64). In mutants T64, T71, and T134, nodulation competitiveness was lower than in the parental strain. Analysis of Tn5-mob tagged sequences of the mutants revealed homologies with the genes encoding methionine synthase, luciferase-like monooxygenase, UDP-glucuronic acid epimerase, sensor hystidine kinase, acetoacetyl-CoA-reductase, oligopeptide uptake ABC transporter, and a transcription activator.