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.

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

“…MepR must have significant structural plasticity, allowing a shift in conformation in the presence of DNA. The ability of MepR to interact with MepA substrates, resulting in dissociation from its operator sites, is another example of structural plasticity (8). In addition to the predicted deleterious structural effects of the functionally significant substitutions we identified, these residue changes also could have detrimental effects on plasticity.…”

“…Cetrimide is a known MepA substrate and has been shown previously to mediate efficient dissociation of MepR WT from the mepA operator (8). The amount of input mutant MepR in these assays was the smallest amount resulting in a complete shift of 1 fmol of target DNA in the absence of substrate, which was 200 ng for K4375 and 25 ng for K4624.…”

“…Modifications included use of the Probond purification system and the native-condition purification procedure provided by the manufacturer (Invitrogen, Carlsbad, CA). EMSAs employing MepR WT and each derivative and the WT mepR (167 bp) or mepA (244 bp) operator site, established previously by DNase I protection assays, were performed at least twice essentially as described previously, modified by use of a concentration range of MepR derivatives to assess binding affinity and elimination of salmon sperm DNA as a nonspecific competitor (8). Excess (200-fold) unlabeled WT-specific target DNAs were used as specificity controls, as these sequences were WT in all strains possessing either MepR F27L or A103V.…”

“…MepR WT with a six-histidine tag on its carboxy terminus was produced and purified essentially as described previously, as were F27L, P62A, and A103V derivatives (8). Modifications included use of the Probond purification system and the native-condition purification procedure provided by the manufacturer (Invitrogen, Carlsbad, CA).…”

“…Binding affinity is 4-fold greater for the mepA site than that of mepR (K d [dissociation constant] of 6.3 versus 24.3 nM, respectively). Additionally, binding to the mepA site is readily reversed by substrates, compared to a much more attenuated effect at the mepR operator (8). A seven-base-pair conserved "signature" motif (consensus sequence, 5=-GTTAGAT-3=) is found once within the mepR operator and twice within the mepA oper-ator and clearly plays a role in the differing binding affinities of MepR for each site ( Fig.…”