The research involves a detailed analysis of the plasmid-mediated response of certain Streptocococus faecalis cells to sex pheromones (termed clumping inducing agents, or CIAs). The proposed studies are based upon ongoing studies using a conjugative, pheromone-responsive tetracycline resistance plasmid called pCF-10 as a model. During the proposed funding period, experiments in three general categories will be done. 1) A collection of mutant derivatives of the plasmid obtained during the current funding period will be subjected to analysis of several phenotypic traits related to drug resistance, pheromone response, and plasmid transfer. Molecular analysis of the DNA and the cell surface antigens determined by these mutants will be carried out also. 2) Recombinant DNA and transposon mutagenesis techniques will be used to determine the minimum amount of DNA necessary for a wild type pheromone response and plasmid transfer phenotype. 3) The ability of cloned fragments of the wild type plasmid to compliment various mutant derivatives will be tested. 4) Regions of the plasmid that contain important regulatory and structural genes will be sequenced and the transcription of these regions in the presence and absence of pheromone stimulation will be analyzed: 5) Biochemical, immunological, and functional analyses of pheromone-induced antigens will be continued. This combined approach is designed to elucidate the genetic control mechanisms and the biochemical nature of the pheromone response process. The results should improve our understanding of the continuing spread of antibiotic resistance in streptococci. This system is also a useful model for studying intercellular chemical communication and the bacterial cell surface.
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