The purpose of this study is to investigate and characterize the molecular basis of the sex-pheromone-induced mating response in Enterococcus (formerly Streptococcus) faecalis, with special emphasis on the conjugative plasmid pAD1. The pAD1 plasmid encodes a hemolysin (which also has bacteriocin activity), as well as resistance to ultraviolet light; and studies in mice have shown that the plasmid contributes to virulence. A significant percentage of human parenteral enterococcal infections involve hemolytic strains of E. faecalis bearing similar or closely related plasmids. In recent years we have learned a great deal about the pAD1 pheromone response; the proposed genetic studies constitute a continuation of our efforts and should provide further insight into mechanisms controlling the intercellular transfer of this widely disseminated plasmid. Specifically we will: 1) Perform a transcriptional analysis of the entire pAD1 Tra region, utilizing the transponson Tn917lac to generate gene fusions and analyzing the production of RNA during the pheromone response; 2) Characterize (sequence) the determinant on pAD1 for """"""""aggregation substance""""""""; 3) Determine the sequence of the chromosome-borne determinant for the sex pheromone cAD1 and the plasmid-borne determinant for the pheromone inhibitor iAD1; 4) Examine genetic aspects of pheromone production by generating and analyzing mutants altered in cAD1 production; 5) Conduct a detailed analysis of the 7 kb region on an effort to completely sequence this region; 6) Analyze a newly discovered """"""""switching phenomenon"""""""" that is independent of the pheromone response and involves spontaneously activating or deactivating transfer functions; 7) Locate the transfer origin (oriT) on pAD1; and 8) Determine the structure of cAM373 from Staphylococcus aureus.
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