Transcriptional and translational regulation of the marRAB multiple antibiotic resistance operon in Escherichia coli

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Abstract
Summary: The marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the −35 and −10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild‐type MarR. When both sites were wild type, transcription in the derepressed marR‐deleted strain was 19‐fold that of the wild‐type strain; when only site I or site II was wild type, this ratio was reduced to 4.3‐ and 2.6‐fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild‐type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine–Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem–loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild‐type strain. However, a single basepair mutation that strengthens the stem–loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5 bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50–75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.