Different phenotypic classes of Sinorhizobium meliloti mutants defective in synthesis of K antigen

Abstract

For Sinorhizobium meliloti (also known as Rhizobium meliloti) AK631 to establish effective symbiosis with alfalfa, it must be able to synthesize a symbiotically active form of its K antigen, a capsular polysaccharide containing a Kdo (3-deoxy-D-manno-octulosonic acid) derivative. Previously isolated mutants defective in the synthesis of K antigen are resistant to bacteriophage phi16-3. By screening ca. 100,000 Tn5-mutagenized R. meliloti bacteria for resistance to bacteriophage phi16-3, we isolated 119 mutants, 31 of which could not be complemented by genes previously identified as being required for K-antigen synthesis. Of these 31 new mutants, 13 were symbiotically defective and lacked the K antigen. Through genetic and phenotypic analyses, we have grouped these mutants into four distinct classes. Although all of these mutants lack the K antigen, many also have altered lipopolysaccharides (LPS), suggesting that the biochemical pathways for the synthesis of K antigen and LPS have common enzymatic steps. In addition, we have found that these and other classes of K-antigen-defective mutants of S. meliloti AK631 exhibit unique patterns of sensitivities to phage strains to which the parental strain was resistant. Our studies have identified new classes of genes required for both the synthesis of K antigen and the symbiotic proficiency of S. meliloti AK631. Some of these classes of genes also play a role in LPS synthesis.

FIG. 1

PAGE analysis of phenol-water-extracted material from AK631 and class I mutants (A) and class II, III, and IV mutants (B). K antigen and LPS were visualized via alcian blue-silver staining. The four classes of mutants can be readily distinguished from the parental strain, AK631, by their lack of K antigen. Classes can be distinguished through the properties of their R-LPSs. In class I and class III mutants, the R-LPS is indistinguishable from that of the wild type (note that the lower R-LPS band is absent in the rkp-180 lane due to underloading of the lane). In the class II mutants, the R-LPS separates into the phenol phase during the phenol-water extraction. In the case of the class IV mutants, the R-LPS runs similarly to that of the exoB⁺ parental strain of AK631. WT, wild type; LMW PS, lower-molecular-weight polysaccharide.