MepR, a repressor of the Staphylococcus aureus MATE family multidrug efflux pump MepA, is a substrate-responsive regulatory protein

Abstract

The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

FIG. 1.

Gel mobility shift analysis of the effect of MepA substrates on the binding of MepR to operator sites in the mepR and mepA upstream regions. Competing DNA consisted of a 200-fold molar excess of DNA identical to the target fragment (superscript a, specific competitor) or salmon sperm DNA (superscript b, nonspecific competitor). The specificity of the MepR operator interaction is demonstrated in the first four lanes of each gel, in which reversal of the MepR-mediated shift in the presence of excess specific competitor DNA and the lack of such reversal in the presence of nonspecific competitor DNA are shown. P, pentamidine; T, TPP; CH, chlorhexidine; C, cetrimide; D, dequalinium; BC, BAC; +, present; −, absent.

FIG. 2.

Competition gel MSA. Labeled mepR and mepA upstream target DNA sequences were combined with doubling dilutions of unlabeled alternate target (competing) DNA. Values indicate the molar excesses of competing DNA used. Superscript a, excess unlabeled DNA identical to the indicated target (specificity control). +, present; −, absent.

FIG. 3.

MepR footprint, mepR upstream region. (A) The biotinylated mepR upstream fragment (167 bp) was incubated without (−) and with (+) purified MepR before being subjected to DNase I digestion and subsequent electrophoresis. The MepR-protected regions on the sense and antisense strands are boxed. (B) Nucleotide sequence of the MepR operator site upstream of mepR. An inverted repeat is indicated by bold arrows; the partial −10 promoter motif and the mepR transcription start site (TSS, determined previously) are shown in bold; the conserved GTTAG sequence is underlined.

FIG. 4.

MepR footprint, mepA upstream region. See the Fig. 3 legend for details. The DNA target used was 244 bp (see text). (A) Sense and antisense strand footprints. (B) Nucleotide sequence of the MepR operator sequence upstream of mepA. Note the presence of two inverted repeats and GTTAG sequences.