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.

The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of Staphylococcus aureus

“…Interestingly, Q18P resulted in the most dramatic overexpression of mepA , 198-fold, which is even greater than that observed for mepR truncations and consistent with the complete loss of the repressor function of MepR. Although the initial functional consequences of these MepR mutations have been assessed previously using electrophoretic mobility shift assay (EMSA) and in vitro transcription analysis (18, 19), the mechanisms underlying their loss of function have been elusive in great part due to the lack of germane structural data.…”

“…This proline residue is conserved in MepR (P62), and the identical TTA box is present in the GTTAG signature sequence of the mepR operator binding site. Even though the MepR P62A mutant was found to bind mepR and mepA operator DNA with wild-type-like affinity (19), the rigid prolyl side chain of P62 may still be involved in DNA specificity by its selection against particular DNA sequences, rather than in favor of one. Residue R87 is conserved among MarR family members (see Fig.…”

“…Although this mutation is relatively conservative, it results in at least a 10-fold-weaker repressor activity (19). Residue 103 is located in a region of MepR that connects its DNA binding and dimerization domains and is likely to play an important role in the conformational flexibility of the wHTH motif (Fig.…”

“…Moreover, the equivalent positions in other proteins are occupied with large charged residues. A recent study by Schindler et al (19) using an electrophoretic mobility shift assay (EMSA) demonstrated that the impaired repressor function of the A103V mutant is due to diminished affinity for cognate DNA. To characterize quantitatively how the A103V change affected DNA binding activity of MepR, we used isothermal titration calorimetry (ITC).…”

“…Multidrug-resistant strains of S. aureus overexpressing mepA have been discovered in clinical isolates and can be selected in the laboratory (19). Sequencing data demonstrated that mepA overexpression is due frequently to the defective repressor function of MepR that results from either truncation or substitution mutations.…”