9600766 Gray The purpose of this research is to characterize the quorum sensing regulatory system of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum. Quorum sensing is a conserved mechanism of population density-dependent gene activation in Gram-negative bacteria. In quorum sensing, a self-produced extracellular signal molecule called autoinducer interacts with a transcriptional activator protein to activate the expression of specific genes. R. Ieguminosarum produces a unique autoinducer that, together with the transcriptional activator RhiR, activates an operon of rhizosphere-expressed genes (rhiABC) and an unidentified locus that causes an inhibition of cell growth. Both rhiR and rhiABC are encoded by Sym plasmids unique to pea-nodulating biovars of R. Ieguminosarum. The growth-inhibiting function appears to be unique to one specific isolate of the Sym plasmid, pRLlJI, which also encodes a repressor function that blocks normal production of autoinducer. We have recently discovered two additional R. Ieguminosarum autoinducers, the production of which is unaffected by this pRLlJI-encoded repressor. The newly-identified R. Ieguminosarum signals activate rhiABC with RhiR, but do not activate growth inhibition. We have also discovered a unique autoinducer produced by the related species R. meliloti, for which no function is currently known. The specific objectives of this research are: 1) to characterize the molecular structures of the two newly-identified R. Ieguminosarum autoinducers, as well as the autoinducer produced by R. meliloti; 2) to identify the pRLlJI-encoded gene that blocks autoinducer synthesis and elucidate its mechanism of action; and 3) to investigate the role of quorum sensing in the conjugal transfer of pRLlJI. This research addresses fundamental questions of intercellular communication, environmental sensing, and gene regulation in Rhizobium. Due to the extreme conservation of quorum sensing regulators, the results of this research may have direct ap plications to other symbiotic or pathogenic bacterial systems as well. The results of this work will be integrated into an existing course in Microbial Physiology & Genetics, and represent an essential component in the development of a new course on the biology of Bacteria as Multicellular Organisms. As part of a career commitment to innovative instruction, the P.I. is also developing an independent research exercise as part of the laboratory curriculum for Microbial Physiology & Genetics. %%% Bacteria of the genus Rhizobium are of considerable ecological and economic importance as a biological source of fixed nitrogen. These bacteria typically form complex symbiotic associations with the roots of specific host plants such as peas and alfalfa. This research project is an investigation into the molecular aspects of intercellular signaling, cell-cell communication, and gene activation in one species of Rhizobium, R. Ieguminosarum. The molecular structures of specific gene-activating signal molecules produced by the bacteria will be determined, and the genes that regulate production of these different signals will be studied. The possible role of these signal molecules in mediating genetic exchange among populations of bacteria will also be determined. The results of this work should increase our understanding of the regulatory biology of these important bacteria and provide new insights into the effects of extracellular signals on their symbiotic lifestyle. ***
