An integrated genetic and biochemical approach will be used to study Rhizobium meliloti extracellular polysaccharides, and the role of these polysaccharides in the formation of nitrogen fixing root nodules on alfalfa. The main focus will be on the acidic heteropolysaccharides, since genetic evidence indicates that these polysaccharides are important in nodule invasion by the bacteria. Mutants will be isolated with powerful screens, including staining by the fluorescent stain calcofluor, and mucoid colony formation to detect the polysaccharides. Genes involved in polysaccharide synthesis will be mapped, subjected to high resolution genetic analysis, and cloned. The regulation of polysaccharide synthesis will be investigated using gene fusions and biochemical techniques for quantifying various forms of the polysaccharides. The biochemical functions of some of the gene products will be studied by assaying in the wild type and the mutants for specific steps and intermediates in polysaccharide synthesis. A variety of approaches will be employed to establish the function of one of the polysaccharides in nodulation. These include probing nodules with antibody to the polysaccharide, assaying attachment of polysaccharide deficient mutants and of the polysaccharide itself to plant roots or extracted components of plant roots, investigating the effect of isolated polysaccharide on nodulation or steps in nodulation, the use of temperature sensitive mutants, and coinoculation of plant roots with different mutant strains of R. meliloti. Root nodulation is a model system for the study of development, and in particular for the study of polysaccharide function, which is likely to be important in mammalian development, and also plays a role in the virulence of certain human pathogens. Nodulation is also important in the production of human dietary nitrogen. The system is particularly amenable to genetic analysis because of the involvement of a well- characterized bacterial symbiont.
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