A. Multiply antibiotic resistant bacteria are an increasingly important problem in the treatment of infectious disease. In Escherichia coli and other enteric bacteria, expression of the marRAB operon confers resistance to a variety of antibiotics and superoxide- generating agents. We previously purified MarR, the repressor of the operon, described its binding to the mar promoter and the reduction of this DNA binding when MarR is bound by salicylate, an inducer of the operon. We have also purified the MarA product and have shown that it is a transcriptional activator for a number of genes of the mar regulon in vitro. We have now begun a systematic analysis of the mar promoter by synthesizing (24) 5 base-pair (bp) transversion mutations that span 120 bp of the promoter. This has allowed us to identify: the putative -35 and -10 RNA polymerase sigma 70 recognition hexamers; the site of MarA binding; the requirement for proper phasing between the MarA site and the -35 hexamer; and a new site which binds Fis (a small DNA-binding and -bending protein). Furthermore, we have demonstrated roles for Rob and SoxS, two MarA-like transcriptional activators, and for Fis in activating the mar operon in vivo. The ability to bind these activators presumably attunes the operon to diverse internal and external stimuli. B. We previously demonstrated that isoniazid (INH), a clinically important antituberculosis drug, induced an SOS-like response and lethality in peroxidase mutants of E.coli and Salmonella typhimurium. We are now isolating and sequencing INH-resistant mutants of these strains to help elucidate this mechanism of INH action.
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