STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

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The lipopolysaccharides (LPSs) from Gram-negative bacteria are important molecules in determining whether or not a strain of bacteria will be a successful pathogen or will be recognized and destroyed by the defense mechanisms of the host cell. Lipopolysaccharides from the nitrogen-fixing Rhizobium bacteria are also thought to play a crucial role in the symbiotic infection of their host legumes. If this proposal is approved the funds will be used to determine the structures of the LPSs from one strain each of R. trifolii (symbiont of clover), R. leguminosarum (the symbiont of pea) and R. phaseoli (the symbiont of bean). In order to determine if genes required for infection and nodulation (nod genes) affect the structure of the LPSs, these molecules from one or two well defined symbiotic mutants will be characterized. In the case of R. trifolii the nod genes of interest reside on a plasmid which is required for nodulation (the pSym). These genes require induction by a legume flavone so the LPSs from induced bacteria will also be characterized. Analysis of the LPS from an R. leguminosarum strain which lacks its own pSym but contains an R. trifolii pSym and nodulates clover will also determine if the pSym nod genes affect LPS structure. The LPS from a mutant of R. phaseoli will be characterized since it has already been shown that this mutation produces a defective LPS and that this defect is linked to a symbiotic defect in which infection threads are aborted. The LPS will be isolated by phenol/water extraction and gel-filtration chromatography. The polysaccharide portions of the LPSs will be isolated and their structures determined by composition, methylation and GC/MS analyses and by 1H- and 13C-n.m.r analyses.