Overexpression of the marA or soxS regulatory gene in clinical topoisomerase mutants of Escherichia coli

Abstract

The contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia coli strains bearing mutations in gyrA and parC and with different levels of fluoroquinolone resistance. Expression of marA and soxS was 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. coli strains used as controls. Comparison of the marOR sequences of clinical Mar mutants with that of E. coli K-12 (GenBank accession no. M96235) revealed point mutations in marR in all mutants which correlated with loss of repressor function as detected in a marO::lacZ transcriptional 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 marOR fragments, identified a variant genotype of marOR. Isolates with the same gyrA and parC mutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marA or soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.

FIG. 1

Northern blot analysis of marRAB mRNAs prepared from clinical E. coli strains incubated without (−) and with (+) 5 mM sodium salicylate for 45 min. RNA samples were transferred to Hybond-N⁺ membranes and probed with radioactively labelled marA. Arrows point to prominent transcripts of ∼1.1 and ∼0.9 kbp.

FIG. 2

Reporter gene assay for MarR function. The host strain ASS121, which lacks the mar and lac loci, carries a chromosomal marO::lacZ transcriptional fusion (40). The effect of the introduction of pSPOK carrying different marR genes on β-galactosidase activity was determined. Cells were grown for 5 h at 30°C in the presence of IPTG, and β-galactosidase activity was measured in triplicate cultures. Results are expressed as percentages of values determined for the control (ASS121 bearing pSPOK without the insert) and are the means and standard deviations of results from three to five consecutive assays. The origins of the cloned marR genes were as follows: bar 1, none; bar 2, AG100 (wild type); bar 3, HO17 (R94→H, G103→S, Y137→H); bar 4, M19 (G31 stop codon); bar 5, NH52 (G103 frameshift); and bar 6, HO4 (G103→S, Y137→H).

FIG. 3

Northern blot analysis of soxS mRNAs prepared from clinical E. coli strains incubated without (−) and with (+) 1.3 mM paraquat for 45 min. RNA samples were transferred to Hybond-N⁺ membranes and probed with radioactively labelled soxS. The arrow designates the ∼400-bp hybridizing band.