NOVEL RKP GENE CLUSTERS OF SINORHIZOBIUM-MELILOTI INVOLVED IN CAPSULAR POLYSACCHARIDE PRODUCTION AND INVASION OF THE SYMBIOTIC NODULE - THERKPK GENE ENCODES A UDP-GLUCOSE DEHYDROGENASE

The production of exopolysaccharide (EPS) was shown to be required for the infection process by rhizobia that induce the formation of indete rminate nodules on the roots of leguminous host plants. In Sinorhizobi um meliloti (also known as Rhizobium meliloti) Rm41, a capsular polysa ccharide (KPS) analogous to the group II K antigens of Escherichia col i can replace EPS during symbiotic nodule development and serve as an attachment site for the strain-specific bacteriophage phi 16-3. The rk pA to -J genes in the chromosomal rkp-1 region code for proteins that are involved in the synthesis, modification, and transfer of an as-yet -unknown lipophilic molecule which might function as a specific lipid carrier during KPS biosynthesis. Here we report that with a phage phi 16-3-resistant population obtained after random Tn5 mutagenesis, we ha ve identified novel mutants impaired in KPS production by genetic comp lementation and biochemical studies. The mutations represent two novel loci, designated the rkp-1 and rkp-3 regions, which are required for the synthesis of rhizobial KPS. The rkp-2 region harbors two open read ing frames (ORFs) organized in monocistronic transcription units. Alth ough both genes are required for normal lipopolysaccharide production, only the second one, designated rkpK, is involved in the synthesis of KPS. We have demonstrated that RkpK possesses UDP-glucose dehydrogena se activity, while the protein product of ORF1 might function as a UDP -glucuronic acid epimerase.