BINDING OF PURIFIED MULTIPLE ANTIBIOTIC-RESISTANCE REPRESSOR PROTEIN (MARR) TO MAR OPERATOR SEQUENCES

Elevated expression of the marORAB multiple antibiotic-resistance oper on enhances the resistance of Escherichia coil to various medically si gnificant antibiotics, Transcription of the operon is repressed in viv o by the marR-encoded protein. MarR, and derepressed by salicylate and certain antibiotics, The possibility that repression results from Mar R interacting with the marO operator-promoter region was studied in vi tro using purified MarR and a DNA fragment containing marO. MarR forme d at least two complexes with marO DNA bound >30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites wi thin marO from digestion by DNase I. Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of th e -10 signal, Site II begins 13 bp downstream of site I, ending immedi ately before the first base pair of marR. Site Il, approximate to 80% homologous to site I, is not required for repression since a site II-d eleted mutant (mar0133) was repressed in trans by wild-type MarR, The absence of site II did not prevent MarR from complexing with the site I of mar0133. Salicylate bound to MarR (K-d approximate to 0.5 mM) and weakened the interaction of MarR with sites I and II, Thus, repressio n of the mar operon, which curbs the antibiotic resistance of E. coil, correlates with the formation of MarR-site I complexes. Salicylate ap pears to induce the mar operon by binding to MarR and inhibiting compl ex formation, whereas tetracycline and chloramphenicol which neither b ind MarR nor inhibit complex formation, must induce by an indirect mec hanism.