We study the interaction of bacterial cells with specific eukaryotic hosts, in a system which relates both to developmental events in multicellular organisms, and to bacterial pathogenesis. Rhizobium meliloti invades and induces nodule formation in specific host plants including alfalfa (Medicago sativa). As they invade, the bacteria alter the host's cell wall synthesis, induce normally non-dividing cells to resume cell division, and stimulate new host gene expression. The bacteria also differentiate structurally and express new genes. We have identified and cloned the bacterial genes used in invasion and nodule induction. Free living bacteria have little or no RNA homologous to the nod gene fragments, suggesting they may not be transcribed in free-living cells. No protein differences were observed between wild type bacteria and transposon Tn5 induced Nod- mutants. An in-frame translational gene fusion of nodC and lacZ has been constructed and this is being used to analyze nod gene expression in various growth conditions and during infection. The DNA sequence has been determined for most of this region, and in vitro, mini-cell and maxi-cell expression has been achieved for open reading frames in the nod gene segment. Antibody is being generated to nodA which has been overexpressed in E. coli, and two synthetic peptides predicted from the DNA sequence of nodC have been constructed and coupled to carrier. We will continue our studies of nod gene expression and nod gene products by constructing further operon and gene fusions between nod genes and indicator genes lacZ, CAT, and lux. In vitro fusions will be based on the complete DNA sequence for these symbiotic genes. Random operon fusions will take advantage of Tn5 derivatives which place lacZ or CAT indicator genes under control of promoters at the insertion site. We will use gene fusions as assays for locating and mutating regulatory nodulation genes. The nod gene proteins will be produced in E. coli and purified for generating antibody, which will be used to confirm the identity of the genes, to follow gene expression in free-living growth and during infection, and to determine the cellular localization of the gene products in vivo. The timing of expression and cellular location of these gene products will provide insight into their possible function during invasion of and nodule induction in their host.
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