Role of the multidrug resistance regulator MarA in global regulation of the hdeAB acid resistance operon in Escherichia coli

Abstract

MarA, a transcriptional regulator in Escherichia coli, affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of hdeAB and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of hdeAB by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of hdeAB. Moreover, repression of hdeAB by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of hdeAB by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on hdeAB under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of hdeAB expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of hdeAB by MarA depends on pH, growth phase, and other regulators of hdeAB and is associated with reduced resistance to acid conditions.

FIG. 1.

Regulation of the hdeAB operon. The figure has been produced using data from the literature (8, 13, 16, 20, 22, 24, 26, 27, 30, 33, 37, 39, 42, 43, 44, 46) as well as the results obtained in this study. Thick arrows represent genes. Direct regulation is shown as a continuous thin arrow. Indirect regulation, or regulation in which a direct effect has not been confirmed, is shown as a dotted thin arrow. See the supplemental material for a more detailed explanation of this figure. cAMP, cyclic AMP; CRP, cAMP receptor protein.

FIG. 2.

Binding sites in the hdeAB promoter for the selected regulators. The hdeAB promoter was analyzed from positions −168 to +54 as described in Materials and Methods. The binding sites of the selected transcriptional regulators are shown below the corresponding sequence as continuous arrows (known) or dotted arrows (putative). In parentheses are the number of nucleotides that match the consensus sequence/number of nucleotides of the consensus sequence. Even though hdeAB is transcribed from RNA polymerase containing σ⁷⁰ or σ³⁸ (39), its promoter does not have the −10 sequence (CTATACT) found in many promoters regulated by σ³⁸ that replaces the −10 sequence (XTATAAT) found in σ⁷⁰ promoters (12). TSS, transcription start site.

FIG. 3.

Effect of MarA on the expression of hdeAB. The β-galactosidase activity in Miller units of the hdeABp-lacZ chromosomal reporter was determined using HdeA100 cells grown in LB at pH 7.0 (left) or in LB at pH 5.5 (right) to exponential or stationary phase. The assays were performed in the absence of MarA (without IPTG) or in the presence of MarA (with IPTG). The results are presented as the averages ± the standard errors of the means (n ≥ 3). Statistically significant differences (P < 0.01) between related conditions (exponential versus stationary phase at the same pH; pH 7.0 versus pH 5.5 in the same growth phase; and presence versus absence of MarA under the same pH and growth phase) are indicated by asterisks.

FIG. 4.

Effect of MarA on the acid resistance of E. coli. The acid challenge at pH 2.5 was performed as described in Materials and Methods, using early-stationary-phase cells grown in LB broth at pH 7.0 without (gray) or with (black) 5 mM sodium salicylate to induce marRAB. Two different strains were compared: the ΔmarRAB strain JHC1096 (ΔmarRAB) and the wild-type strain GC4468 (WT). The percent survival at pH 2.5 after incubation for 2 h was determined in comparison with untreated samples. The results are presented as the averages ± the standard errors of the means (n = 3). The n-fold decrease in survival produced by sodium salicylate in each strain (ratio between gray and black bars) is indicated above the bars.