Overexpression of the marAor soxSRegulatory Gene in Clinical Topoisomerase Mutants ofEscherichia coli

ABSTRACTThe contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia colistrains bearing mutations in gyrAand parCand with different levels of fluoroquinolone resistance. Expression of marAand soxSwas evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA,soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. colistrains used as controls. Comparison of the marORsequences of clinical Mar mutants with that of E. coliK-12 (GenBank accession no. M96235) revealed point mutations in marRin all mutants which correlated with loss of repressor function as detected in a marO::lacZtranscriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marORfragments, identified a variant genotype of marOR. Isolates with the samegyrAand parCmutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marAor soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.