The objectives of this grant are to characterize the unique structure of the LPS from R. leguminosarum biovar phaseoli (recently reclassified Rhizobium etli) strain CE3 and to elucidate the role of this molecule in the nitrogen-fixing symbiotic infection of its legume host it is known that Rhizobium LPS are required for a successful infection of the legume host. Gross changes in the LPS, e.g. loss of the O-chain polysaccharide, result in aborted infections. Monoclonal antibodies have shown that during infection there are subtle structural changes that occur in the LPS during infection. By using defined mutants in the LPS biosynthetic pathway (provided by Dr. Dale Noel of Marquette University), the complete structure of the LPS will be determined, as well as those epitope changes that occur during symbiotic infection. Analysis of LPSs from mutants at various stages of the infection process will (a) facilitate the identification of genes in the LPS biosynthetic pathway, and (b) enable us to determine how the O-chain, core oligosaccharides and lipid A linked in the complete LPS molecule. The LPS from these mutants, as well as from the parent strain (CE3) will be used to isolate those LPS fragments that carry the epitopes important in symbiotic infection. These epitopes will be identified by ELISA inhibition assays. The monoclonal antibodies (i.e. JIM26, JIM27, JIM28 and JIM29) have been provided by Dr. Nick Brewin of the John Innes Institute in the UK. Additionally, a graduate student will visit Dr. Noel's laboratory to isolate mutants that are unable to undergo those epitope changes that occur during symbiotic infection. These mutants will be analyzed to determine their structural (by us) and symbiotic (by Dr. Noel) defects. Structural determination of the various LPSs and LPS fragments will be carried out by glycosyl composition and methylation analyses, NMR, and high resolution mass spectrometry. We currently know the structures of the O-chain repeating unit, two core oligosaccharides, and the lipid A from the R. leg. bv. phaseoli CE3 LPS. The lipid A of Rhizobium is unique in that it does not have phosphate, and its sugar backbone consists of galacturonic acid, glucosamine and 2-aminogluconic acid. We have shown that Rhizobium extracts have all the enzymes necessary for the synthesis of a lipid A precursor common to enteric lipid As, Kdo2IVa. Thus, in Rhizobium this common precursor is probably processed to form the unique rhizobial lipid A. One such enzyme, a specific Kdo2IVa 4'- phosphatase has already been identified. The last objective of this proposal is to characterize the other enzymes that process Kdo2lVa into the unique rhizobial lipid A. Dr. Chris Raetz (Duke University) has agreed to collaborate with us in this aspect of the project.
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