Enterococcus faecalis is an important opportunistic pathogen that is now the second leading cause of bacteremia and third leading cause of endocarditis in humans. Conjugative plasmids encoding a mating response to peptide sex pheromones are ubiquitous in E. faecalis and probably contribute significantly to the dissemination of antibiotic resistance and cytolysin (e.g. hemolysin/bacteriocin) production in this species. The cytolysin-encoding plasmid pAD1 is an example of such a plasmid and has been a subject of intense scrutiny in the laboratory of the PI for a number of years. Recent identification of key regulatory genes and determination of their nucleotide sequence, along with related physiological studies, has enabled the formulation of a working hypothesis to explain the circuitry which may be utilized during induction of the conjugation response. A key aspect of the model concerns the control of expression of traE1 by a negative regulator encoded by traA that influences transcriptional readthrough of the termination site TTS1/TTS2. The bulk of the proposed study is designed to test this model and further characterize related processes. More specifically the proposed studies will: 1) determine the nature of transcription events that occur between the iad promoter and TTS1 with an emphasis on examining the relationship between transcripts that have been designated m3, m4, and m5; 2) examine the role TraA plays in regulating transcription beyond TTS1/TTS2 and into the traE1 determinant including a determination of whether TraA directly binds to the cAD1 peptide; 3) examine additional factors (i.e. other than TraA) operating at TTS1/TTS2 and affecting transcriptional readthrough; 4) determine if TraE1 plays a role in its own regulation by controlling initiation of the transcript designated m3'; 5) examine the kinetics of shutdown of the pheromone response; 6) characterize the basis of the Dry+/Dryc phase variation mechanism that facilitates a bypass of the physiological response to cAD1; 7) determine if TraA, TraE1, RepA, RepB or other proteins interact with each other; and 8) continue our efforts to clone and characterize cad, the chromosomal determinant for cAD1.
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