Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences.
Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics. Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics. The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO. MarR formed 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 within marO from digestion by DNase I. Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of the -10 signal. Site II begins 13 bp downstream of site I, ending immediately before the first base pair of marR. Site II, approximately 80% homologous to site I, is not required for repression since a site II-deleted mutant (marO133) was repressed in trans by wild-type MarR. The absence of site II did not prevent MarR from complexing with the site I of marO133. Salicylate bound to MarR (Kd approximately 0.5 mM) and weakened the interaction of MarR with sites I and II. Thus, repression of the mar operon, which curbs the antibiotic resistance of E. coli, correlates with the formation of MarR-site I complexes. Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism.
Martin
R G
RG
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
Rosner
J L
JL
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C080004:MarR protein, E coli; D012097:Repressor Proteins