This proposal involves a molecular and genetic analysis of the regulatory mechanisms involved in controlling expression of conjugative transfer of the tetracycline-resistance plasmid, pCF10 in Enterococcus faecalis. Analysis of this system has revealed that plasmid-encoded transfer functions in donor cells are induced by very small amounts of a peptide pheromone produced by recipient cells. Currently available data has led to the formulation of two models for important stages of regulation in the system. A model describing a compartmentalization of pheromone internalization machinery accounts for the ability of a donor cell to mount mating response to pheromone produced by recipients, and avoid self-induction by endogenous pheromone. A model for the involvement of pheromone at an intracellular stage, which involves positive control of translation is also presented. The experiments proposed are designed to test important predictions of the models, and include the following aims: i) to identify the putative propheromone molecule that generates endogenous activity; ii) use a combination of genetics, immunoelectron microscopy, and biochemistry to study the cellular location of various components of the internalization machine and their interactions; iii) use a combined genetic and biochemical approach to analyze the interactions of a plasmid-encoded regulatory RNA molecule with its predicted ribosomal target, and other components of the system and iv) use biochemical techniques to elucidate the mechanisms of action of a key negative regulatory protein, PrgX, in the system.
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