title: An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.
abstract: 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 mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous 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. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.
authors.affiliation: Laboratorium voor Genetica, Universiteit Gent, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Goethals
authors.forename: B
authors.initials: B
authors.lastname: Leyman
authors.forename: G
authors.initials: G
authors.lastname: Van Den Eede
authors.forename: M
authors.initials: M
authors.lastname: Van Montagu
authors.forename: M
authors.initials: M
authors.lastname: Holsters
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics; D007887:Fabaceae / Q000382:microbiology*; D005798:Genes, Bacterial / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics; D019081:O Antigens; D010641:Phenotype; D010946:Plants, Medicinal; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D012230:Rhizobiaceae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013559:Symbiosis; D002237:Carbohydrate Dehydrogenases
chemical_list: D001426:Bacterial Proteins; D002238:Carbohydrate Epimerases; D006836:Hydro-Lyases; D008070:Lipopolysaccharides; D008359:Mannose-6-Phosphate Isomerase; D009713:Nucleotidyltransferases; D019081:O Antigens; D011135:Polysaccharides, Bacterial; C085241:OAC1 protein, Azorhizobium caulinodans; C085243:dTDP-D-glucose synthase; C022771:dTDPglucose 4,6-dehydratase; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D002237:Carbohydrate Dehydrogenases