Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene

Abstract

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.

Fig 1

mepA operator site. 1 and 2, sites of mutations observed in the mepA-overexpressing strains SA-K4375 (G→A at −1 and −2) and SA-K4624 (A→G at −14 and G→A at −20), respectively. Inverted repeats are identified by arrows, −35 and −10 promoter motifs and the transcription start site (TSS) are indicated in bold, and the consensus GTTAGAT sequences are highlighted in gray.

Fig 2

Repression of chromosomal mepR (black bars) or mepA (gray bars) expression by MepR derivatives. Values are means ± standard deviations. All data were normalized to the activity of wild-type (WT) MepR. Germany, MepR mutant with 10 substitutions; a, significantly reduced repressor activity versus WT MepR at the mepR or mepA operator; b and c, significantly increased repressor activity versus T94P or A103V MepR, respectively, at the mepR and mepA operators (P < 0.05). The expression of all derivatives except those with changes at position 103 was induced with 0.05 μg/ml tetracycline (see the text for details).

Fig 3

Tetraphenylphosphonium bromide (TPP) MIC ratios (gray bars) and ethidium bromide (EB) efflux activity (black circles) of selected MepR derivatives. MIC ratios of less than 1 indicate preserved MepR repressor activity, with 0.5, 0.25, and 0.125 equal to 2-, 4-, and 8-fold MIC reductions, respectively. Numbers within bars represent percent MepR repressor activity (compared to WT) at the mepA operator as determined in β-galactosidase assays. a, efflux activity equivalent to that of the WT; b, efflux activity equivalent to that of empty pALC2073; c, reduced efflux activity compared to that of T94P or A103V, indicative of greater MepR repression of mepA transcription in the presence of T132M (P < 0.01).

Fig 4

Electrophoretic mobility shift assays. Binding of MepR wild type (WT) versus that of MepR A103V or MepR F27L mutant protein to the WT mepR and mepA operators is shown. The amount of input protein used and percent shift of target are indicated. Binding specificity of each protein was established by including a 200-fold excess of unlabeled specific DNA (indicated by + in the final two lanes of each image).

Fig 5

Electrophoretic mobility shift assays. Binding of MepR wild type (WT) versus that of MepR K4375 (T94P+T132M) or MepR K4624 (Germany; 10 substitutions) to WT and cognate mepA DNA targets. The amount of input protein used and percent shift of target are indicated.

Fig 6

Substrate responsiveness of MepR derivatives. Cetrimide (50 μM) was included in the mixtures for all binding reactions shown here. Experiments with K4375 MepR employed 200 ng of input protein, whereas 25 ng was used for K4624.

Fig 7

MepR substitution mutations. Blue and magenta, monomers A and B of the MepR functional dimer, respectively. Helix numbers are indicated for monomer A (α1, α2, etc.), and positions of studied mutations are depicted as spheres (wild-type side chains are shown). Green, no detrimental effect at either the mepR or mepA operator; yellow, function mildly to moderately impaired; red, function markedly impaired or inactivating. (A and B) Apo-MepR face-on and rotated 180^o about its central axis, respectively. The dimerization interface is indicated, as is the DNA binding domain of one monomer.

Fig 8

(A) Structural comparison of reduced or DNA binding-compatible Xanthomonas campestris OhrR (blue) and MepR (red). (B) Conformational differences in helix 5 between reduced (OhrR[r]; blue) and oxidized (OhrR[ox]; green) OhrR, resulting in disruption of the helix into two distinct helices (5A and 5B) and loss of DNA binding affinity. Position A103 in MepR (red) is represented by a yellow sphere. (C) Introduction of potential steric clash when proline is substituted for threonine at MepR position 94. Distances are in angstrom units. The closest approaches for both residues are illustrated; all conformers/ring puckers of the proline side chain approach the peptide backbone at isoleucine 77 more closely than any side chain rotamer of threonine.