STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

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Nitrogen fixing symbioss between a Rhizobium and its legume host is a complex process involving the recognition of signal molecules from both the symbiont and the host legume which results in a highly coordinated expression of genes in both the plant and symbiont. The result of this process is a root nodule which contains nitrogen-fixing bacteria, called bacteroids. Many recent reports have shown that the major outer membrane component of rhizobia, the lipopolysaccharide (LPS), is essential in this symbiotic infection process. Mutants which lack a portion of the LPS known as the O-antigen polysaccharide (O-chain) are defective either infection thread formation or in the release of the bacteria into the cortical root cells. Additionally it has been shown, using monoclonal antibodies, that subtle structural changes occur in the LPS during symbiotic infection and that these changes are likely to be crucial in forming a nitrogen-fixing nodule. These structural alterations are also observed when the bacteria are grown under conditions which, in part, mimic those inside the root nodule, e.g. low pH (4.8). The main objective of this proposal is to characterize these important LPS structural alterations that are required for symbiotic infection. Specifically, with regard to the LPSs R. leguminosarum bv. phaseoli CE3 (symbiont of bean) and bv. victae (symbiont of pea), the objectives are to: determine the structure of the O-antigen polysaccharides; determine how the LPS core oligosaccharide, lipid A and O- antigen structural regions are joined to form a complete LPS molecule; and determine the LPS structural changes that occur during symbiosis. The O- antigen structures will be determined using methylation, GC-MS, FAB-MS and NMR analytical techniques. The LPSs with and without the O-chain will be characterized to determine how the lipid A, core oligosaccharide and O- chain are joined together. The LPSs from bacteria grown at pH 4.8 will be characterized to determine the structural alterations that are occuring during nodulation. Based on the results of this proposed work, it should (in the future) be able to identify the genes required for these LPS alterations and to determined how their expression is regulated by the host plant.