AN AZORHIZOBIUM-CAULINODANS ORS571 LOCUS INVOLVED IN LIPOPOLYSACCHARIDE PRODUCTION AND NODULE FORMATION ON SESBANIA-ROSTRATA STEMS AND ROOTS

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant , strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic muta nt was inoculated on roots or stems of the host, no nodules developed (Nod(-)). Compared with the wild type, strain ORS571-X15 produced lipo polysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a diff erent O-antigen structure with a lower degree of polymerization. A cos mid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was re lieved and nodulation was almost completely restored, but the strain w as still nonmotile. LPS preparations from these complemented strains r esembled the wild-type LPS, although minor quantitative and qualitativ e differences were evident. The sequence of the locus hit by the Tn5 i n ORS571-X15 (the oac locus) revealed a striking homology,vith the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosy nthesis. The Tn5 insertion position was mapped to the oac3 gene, homol ogous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. The refore, it was proposed that the O antigen of the mutant strain is dev oid of such sugars.