One of the most serious medical problem facing the world is the diminishing efficacy of our armamentarium of antimicrobial agents. One of the ways in which enteric bacteria such as E. coli can become resistant to a broad spectrum of antibiotics is through induction of the multiple antibiotic resistance (mar) locus. We have begun to dissect the regulation of the mar operon by purification of its two principle genetic products, MarR and MarA. MarR is a 16,000 dalton protein that binds tot he promoter region in dimeric form at two sites; Site I lies between the -35 and -10 transcription initiation signals and is sufficient for repression of mar transcription by MarR. The second site, which contains the ribosome binding site and is located between the -10 signal and the first codon of MarR, is not essential for repression of transcription. MarR can bind salicylate but not antibiotics. MarR-promoter DNA complexes are disrupted when MarR binds to salicylate. We have also obtained pure MarA, a protein of 15,500 that binds to, and stimulates the transcription of a dozen promoters dispersed throughout the E. coli chromosome. MarA induces bending of the DNA of these promoters. Some of the promoters require native RNA polymerase subunit, alpha; others can tolerate deletion of the carboxy-terminal domain of this subunit. Remarkably, we find that MarA also stimulates its own promoter. The binding site of MarA has been defined by footprint analysis and deletion mapping. The deletions have been analyzed both in vitro and in vivo for their promoter activity and for their ability to be stimulated by MarA and the MarA-related transactivator, SoxS. Although MarR and MarA bind to different sites, there is competition between their binding to promoter DNA. Goal - To define in molecular terms how the mar operon like other molecular triage systems such as heat shock and s.o.s. repair alert the bacterium tot he appearance of a noxious agent in the environment and stimulate the appropriate response.
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