The primary objective of the proposed work is to characterize key molecular aspects of sex pheromone-related mating systems in Streptococcus faecalis. Attention will be focussed on both the production of sex pheromones by recipients and the response to such pheromones by certain plasmid-containing donors. Parts of the study will make use of the recently characterized transposons Tn916 and Tn917. More specifically we will: 1) Genetically analyze the pAD1 pheromone response, making use of Tn917 as an insertional mutagen and an E. coli-streptococcus """"""""shuttle vector"""""""" to clone and genetically analyze selected segments of the plasmid; 2) Identify plasmid-encoded (controlled) protein and RNA species that are induced during the cAD1 pheromone response and attempt to relate the proteins to pAD1 determinants revealed from genetic studies; 3) Generate mutants of Streptococcus faecalis that fail to excrete cPD1, examine their ability to excrete other pheromones and to behave as a recipient, and use them to explore the possible existence of pheromone precursor peptides; 4) Clone and sequence segments of DNA believed to contain the structural gene for sex pheromone; 5) Purify and characterize cAD1 and cPD1 and attempt to relate their amino acid sequences to cloned genomic DNA base sequences; 6) Determine the nature by which sex pheromone production is shut off after plasmid acquisition, by analyzing newly discovered plasmid encoded modified forms of cAD1 and cPD1; and 7) Using a panel of S. faecalis """"""""responder"""""""" strains, screen culture filtrates of other bacterial species for pheromone activity.
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