MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein
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《抗菌试剂及化学方法》
1.The John D. Dingell Department of Veterans Affairs Medical Center,2.Department of Medicine, Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, Michigan 48201
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.
INTRODUCTION
Antimicrobial agent resistance in bacteria is an increasingly serious threat to modern medical care (16). Such resistance can occur by a number of mechanisms, including modification of the drug to an inactive form, modification of the drug target such that interaction with the drug does not occur, and removal of the drug from its site of action by membrane-based efflux proteins, hereafter referred to as efflux pumps. In recent years, many efflux pumps, from both gram-positive and -negative bacteria, capable of conferring multidrug resistance (MDR) have been described (4). Some of these MDR-conferring pumps have impressive substrate profiles that include mono- and divalent cationic antiseptic and disinfectant compounds, as well as antimicrobial agents.
MDR-conferring efflux pumps belong to one of five families based on structural characteristics and energy requirements. These include the ATP-binding cassette, major facilitator, multidrug and toxin extrusion (MATE), resistance-nodulation-division, and small multidrug resistance (SMR) families (4, 20). The MATE family is the most recently described and the least well characterized. We recently identified and characterized in a preliminary fashion the mepRAB gene cluster in Staphylococcus aureus (GenBank accession number AY661734) (10, 18). These genes encode MepR, which on the basis of homology is a MarR family regulatory protein; MepA, a MATE family multidrug efflux pump; and MepB, a protein the function(s) of which is currently unknown. In wild-type strains, only mepR transcripts are seen, but in mutants in which mepRAB is overexpressed the main transcript produced is mepRAB. However, in these mutants individual mepR, mepAB, and mepB transcripts also are observed. MepR is a repressor of mepA expression, but further details of the regulation of mepRAB expression are not known. Herein we establish that MepR represses mepR and mepA expression by binding upstream of both genes. MepR-mediated repression of mepA, and in a more limited fashion mepR, is abrogated in the presence of MepA substrates, consistent with MepR being a substrate-responsive, and most likely a substrate-binding, regulatory protein.
MATERIALS AND METHODS
Bacterial strains, plasmids, media, and reagents. The strains and plasmids employed in this study are listed in Table 1. Unless otherwise noted, all reagents were the highest grade available and were obtained from Sigma Chemical Co. (St. Louis, Mo.) or, in the case of some antimicrobial agents, their respective manufacturers. Growth media were obtained from BD Biosciences (Sparks, Md.) (brain heart infusion broth [BHIB] and tryptic soy broth) and Invitrogen (Carlsbad, Calif.) (Luria-Bertani broth).
pK434 was constructed as described previously (10). Briefly, the mepR open reading frame, including its ribosome-binding site, was amplified from S. aureus NCTC 8325-4. The PCR product was cloned into pALC2073, placing mepR under the control of the xyl/tetO tetracycline-inducible promoter (3). The plasmid was electroporated into S. aureus RN4220 and then transferred into SA-K2916 (see below) by transduction to reconstitute mepR in trans, producing strain SA-K2916-R (5). SA-K2916-R was grown in the presence of chloramphenicol (10 μg/ml) to ensure plasmid maintenance and, where appropriate, tetracycline to induce plasmid-based mepR expression.
Construction of mepR::lacZ and mepA::lacZ fusions. Internal fragments of the 5' portions of mepR and mepA were amplified by PCR, followed by cloning of each product into pAZ106, a plasmid conferring resistance to ampicillin (Escherichia coli) and erythromycin (S. aureus) and containing a multiple cloning site upstream of a promoterless lacZ gene (13). pAZ106 replicates only in E. coli, and when introduced into S. aureus RN4220 and selected for by erythromycin it will integrate into the chromosome at sites of homology provided by the mepR or mepA fragments. The integration disrupts the native genes and creates transcriptional fusions between those genes and lacZ. DNA sequencing was used to verify fusions in S. aureus RN4220, and phage 85 was used to transduce the fusions into S. aureus SH1000, producing strains SA-K2916 (mepR::lacZ) and SA-K2982 (mepA::lacZ) (5, 21).
Disruption of a chromosomal gene may have a polar effect on a downstream gene(s) that is cotranscribed with it. Analysis of genome data reveals that the open reading frame immediately downstream of mepRAB is oriented in the opposite direction, eliminating the possibility of a polar effect on its transcription. Additionally, we have shown previously that MepB plays no role in MepA-mediated MDR and thus a polar effect of mepR::lacZ or mepA::lacZ fusions on its transcription is unlikely to affect the data presented herein (10).
?-Galactosidase assay. Expression of mepR and mepA in lacZ fusion strains was quantitated by employing a fluorescent ?-galactosidase assay as described previously, with 4-methylumbelliferyl-?-D-galactopyranoside (MUG) as a substrate (11, 12). A range of concentrations of 4-methylumbelliferone were used to prepare a standard curve, and ?-galactosidase activity (expressed in MUG units; 1 U = 1 pmol of MUG cleaved per min per unit of optical density at 600 nm) was determined with a Bio-Tek FLx800 plate reader (Bio-Tek Instruments, Inc., Winooski, Vt.). Gene expression over the course of each experiment (10 h) was quantitated by integrating the areas beneath expression curves with SigmaPlot 9.0 (Systat Software, Inc., Point Richmond, Calif.).
Effect of MepR on mepR expression and of substrate exposure on the expression of mepR and mepA. SA-K2916-R was grown in BHIB containing chloramphenicol (10 μg/ml) without (control) or with a range of concentrations of tetracycline (6.25 to 50 ng/ml) to determine if MepR is autoregulatory. The effect of exposure to MepA substrates on mepR expression was determined by growing SA-K2916-R in BHIB containing chloramphenicol, 10 ng/ml of tetracycline to induce mepR expression from pK434 (where appropriate), plus one-quarter or one-half of the MIC of benzalkonium chloride (BAC), dequalinium, ethidium bromide (EtBr), or pentamidine. In a similar fashion, the effect of these compounds on mepA expression was determined with SA-K2982. ?-Galactosidase activity and cumulative mepR and mepA expression were determined as described previously. Each experiment was repeated a minimum of three times. Results for mepR were expressed as the ratio of expression data in the presence of tetracycline without or with a MepA substrate to that of SA-K2916-R uninduced with tetracycline, where this ratio was 1.0. For mepA, results were expressed as the ratio of drug-exposed to nonexposed SA-K2982. The significance of the reversal of the MepR repressive effect on mepR expression by substrate and the increase in mepA expression in the presence of substrate were analyzed with the Mann-Whitney rank-sum test. A P value of <0.05 was considered significant.
Purification of MepR. MepR was overproduced in E. coli with the Champion pET Directional Expression kit (Invitrogen). This kit includes the pET101/D-TOPO E. coli expression vector, which has a strong T7 isopropyl-?-D-thiogalactopyranoside (IPTG)-inducible promoter that controls the expression of cloned genes (Table 1). The mepR gene was amplified from S. aureus NCTC 8325-4 with primers TOPO-fwd and TOPO-rev (Table 2), producing a 421-bp product consisting of the entire mepR coding region but lacking the native stop codon. To ensure the proper orientation of mepR when cloned into pET101/D-TOPO, TOPO-fwd introduced a CACC sequence immediately 5' to the ATG initiation codon that mates with a GTGG sequence provided by the vector. Cloning into this vector also results in fusion of the mepR coding sequence with that for a linker peptide plus a six-histidine tag at its 3' terminus, followed by a stop codon. The coding region for the linker peptide was removed by PCR-based overlap extension, resulting in MepR with the six-histidine tag attached directly to its C terminus (MepR-H6) (8). Following the appropriate manufacturer's instructions, mepR was overexpressed and then large quantities of purified MepR were obtained by nickel affinity chromatography with the HisTrap kit (Amersham Biosciences AB, Uppsala, Sweden). The homogeneity of the recovered protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15).
Gel MSAs. Target DNAs used in gel mobility shift assays (MSAs) were amplified by PCR with the primer pairs shown in Table 2, one of which was biotinylated by Invitrogen as indicated. Primers mepR fwd and mepR rev produced a 167-bp product consisting of 109 bp of upstream and 58 bp of 5' mepR sequence. Primers mepRA fwd and mepRA rev(A) generated a 244-bp product including 88 bp of 3' mepR sequence, all of the mepRA intergenic region (106 bp), and 50 bp of 5' mepA sequence. Primers mepAB fwd and mepAB rev resulted in a 256-bp product that contained 45 bp of 3' mepA sequence, all of the mepAB intergenic region (103 bp), and 108 bp of 5' mepB sequence. MSAs were performed, and biotin-labeled DNA was detected with the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, Ill.) by following the manufacturer's guidelines as previously described, with modifications (23). Where indicated, DNA targets were incubated with 25 ng of purified MepR-H6 in 20 μl of binding buffer (10 mM Tris-HCl, 50 mM NaCl, 0.5 mM dithiothreitol, pH 7.5) to which glycerol, poly(dI-dC), and bovine serum albumin (final concentrations of 5%, 0.05 μg/ml, and 0.75 μg/ml, respectively) were added. Specificity of the MepR-DNA interaction was established by including a 200-fold molar excess of either nonbiotinylated target DNA (specific competitor) or salmon sperm DNA (nonspecific competitor) in binding reaction mixtures. To assess the impact of substrate on the MepR-DNA interaction, various concentrations of MepA substrates (BAC, cetrimide, chlorhexidine, ciprofloxacin, dequalinium, norfloxacin, pentamidine, and tetraphenylphosphonium bromide [TPP]) were included in the binding reaction mixtures. The effect of salicylate also was assessed.
To estimate the relative affinities of MepR for its identified binding sites (see below), competition MSAs were carried out. These experiments were performed as described above with MepR-H6, the biotin-labeled mepR or mepA upstream sequence to which it specifically bound, and doubling dilutions of the unlabeled alternate MepR-binding site (i.e., excess mepA upstream sequence included in MepR-mepR upstream sequence binding reaction mixtures and vice versa). For these experiments, the mepA upstream sequence used was generated by employing mepRA fwd and mepRA rev(B) (Table 2). The resultant 183-bp product included 88 bp of 3' mepR sequence and 95 bp of the mepRA intergenic region. This fragment contains the entire MepR-binding site identified in this region (see below).
DNase I footprinting. Footprinting was performed by previously described methods, with several modifications (6). Primers were the same as those used for MSAs (Table 2), except that biotinylated forward and reverse primers were employed as necessary to identify sense and antisense MepR footprints. MepR-DNA binding was performed in a 20-μl reaction mixture volume with the same buffer system as described for MSAs, except that 50 ng of MepR-H6 was used and MgCl2 and CaCl2 (final concentrations, 5 and 1 mM, respectively) were included in the buffer. The reaction mixture was incubated for 20 min at room temperature, followed by the addition of DNase I (final concentration, 5 U/ml; Amersham) and an additional 1-min incubation period. The reaction was terminated by adding an equal volume of stop solution (0.6 M sodium acetate, 25 mM EDTA, 40 μg/ml yeast tRNA, pH 5.0), followed by ethanol precipitation. The pellet was washed with ethanol and then resuspended in 5 μl of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol, 0.05% bromphenol blue). Aliquots of DNA were analyzed with a 6% polyacrylamide gel containing 7 M urea. Sequencing ladders were generated with the same primers as were biotinylated for the PCRs. Biotin-labeled DNA was detected with the Chemiluminescent Nucleic Acid Detection Module of the LightShift Chemiluminescent EMSA kit (Pierce).
RESULTS AND DISCUSSION
MepR strongly represses the transcription of its gene. We have shown previously that MepR represses mepA transcription (10). Employing a range of inducing concentrations of tetracycline, ?-galactosidase assays with SA-K2916-R revealed a dose-dependent reduction in chromosomal mepR transcription. Reductions of 12, 39, 57, and 79% were observed in the presence of 6.25, 12.5, 25, and 50 ng/ml of tetracycline, respectively. These data support the conclusion that MepR is autoregulatory. A direct self-repressive effect is most likely and is supported by MSA and footprinting data (see below).
Substrate exposure augments mepR and mepA expression. Growth of SA-K2916-R in the presence of 10 ng/ml of tetracycline repressed chromosomal mepR transcription by approximately 25%. Significant reversal of this repression was not observed with exposure to one-quarter the MIC of any tested MepA substrate, but a trend was evident for pentamidine at this concentration (Table 3). Higher concentrations of BAC and dequalinium (one-half of the respective MICs) did significantly reverse this repression, whereas EtBr did not. Pentamidine at one-half of its MIC could not be evaluated in this in vivo system due to inhibitory effects on test strain growth.
The transcription of mepA in SA-K2982 was low compared to that of mepR in SA-K2916. Under these circumstances, mepR expression was approximately a thousand times that of mepA. These data are consistent with previous Northern blot analyses of S. aureus NCTC 8325-4 revealing the presence of mepR but no mepA or mepB transcripts (10). In contrast to the effect of substrate exposure on mepR expression just presented, exposure of SA-K2982 to one-quarter of the respective MIC of BAC, dequalinium, EtBr, or pentamidine resulted in substantially increased mepA transcription (Table 3). Regulatory proteins for other MDR efflux pumps that have been studied, including BmrR of Bacillus subtilis and QacR of S. aureus, have been shown to have their activity altered by the binding of substrate to them (7, 22, 24). In fact, MarR also is a ligand-binding protein (salicylate), and such binding results in abrogation of its inhibitory effect on marRAB expression (2, 7, 17). Increased mepR and mepA expression in the presence of MepA substrates is consistent with a substrate-MepR interaction and resultant loss of MepR repression. The lesser effect of substrate on increasing mepR versus mepA expression would result in a relative reduction in MepR repression of mepA, a conclusion also supported by gel shift assay data (see below).
MepR binds specifically to the mepR and mepA upstream regions. MSAs revealed that MepR binds upstream of its gene (Fig. 1). This binding is specific, as excess unlabeled target reverses it but excess nonspecific DNA does not. Similarly, MSAs also revealed specific binding of MepR to a 244-bp target that encompasses the entire mepRA intergenic region (described previously). In this case, reversal of the observed shift by excess unlabeled specific DNA revealed the presence of intermediately shifted bands, most plausibly representing the binding of variable numbers of MepR molecules to its cognate DNA target. MarR family proteins typically bind as dimers; MepR may behave in a similar manner but also may bind in other multimeric forms (2, 7, 17).
No interaction between MepR and the mepB upstream region was observed (data not shown). Analysis of the nucleotide sequence upstream of mepB reveals no motifs that may be important for MepR binding (inverted repeats and GTTAG sequences; see below). Thus, the lack of a MepR interaction with this region is not surprising. The reason(s) for the inclusion of mepB in this gene cluster and the function(s) of MepB remain unknown. The structural and functional characterization of the protein may clarify these issues.
Competition MSA experiments revealed that MepR binding to the 167-bp mepR upstream region was reversed significantly by 200-, 100-, and 50-fold molar excesses of the unlabeled 183-bp mepA upstream region, whereas only a 200-fold molar excess of the mepR upstream region minimally competed with the mepA fragment (Fig. 2). These data are consistent with a greater affinity between MepR and the mepA upstream sequence than with the mepR upstream region. These data provide a plausible explanation for the relative "leakiness" of MepR repression of mepR versus its more complete repression of mepA expression, exemplified by earlier work showing mepR but no mepAB transcripts in wild-type strains (10).
Substrate abrogates MepR binding to its operator sites. Inclusion of MepA substrates in MSAs reversed the MepR-target interaction with the mepA upstream region (Fig. 1). Reversal was observed when drug and protein were combined first and then added to target DNA, or when protein and DNA were incubated together, followed by drug addition (data not shown). Various drug concentrations were evaluated to determine the relative potency of each substrate in reversing the MepR interaction with the mepA operator. Under the assay conditions employed, pentamidine and TPP appeared to have the weakest effects whereas chlorhexidine, cetrimide, dequalinium, and BAC had similar, and much greater, effects in that concentrations 6- to 80-fold lower disrupted MepR binding.
No reversal of the MepR interaction with the mepA target fragment occurred with a very high concentration of ciprofloxacin, norfloxacin, or salicylate (4,000 μM), indicating that these compounds have no or a very minimal ability to interact with MepR (data not shown). The absence of a salicylate effect differentiates MepR from its homologue MarR, which does bind this compound (2, 17). The absence of an interaction among ciprofloxacin, norfloxacin, and MepR was surprising given our earlier observation of reproducible two- to fourfold increases in the MICs of these compounds when mepA was overexpressed, establishing that they are MepA substrates (10). The explanation for this apparent paradox simply may be that not all MepA substrates necessarily must be inducers and MepA may be more promiscuous than MepR. A relevant example of such behavior is given by QacA, which effluxes compounds that do not induce its expression or bind to QacR (6). That the order in which MSA components were combined did not alter the observed reversal of the MepR-mepA operator interaction suggests that the presence of substrate can not only interfere with the ability of free MepR to bind to but can also dissociate bound MepR from its cognate DNA.
Similar to the MepR-mepA operator interaction, high concentrations of salicylate, ciprofloxacin, and norfloxacin had no effect on the MepR-mepR autoregulatory operator binding (data not shown). Concentrations of TPP, chlorhexidine, cetrimide, and BAC that were effective at reversing the MepR-mepA operator interaction had a minimal or no effect on MepR binding to the mepR operator. Higher concentrations of each of these compounds (6,000 μM [TPP] and 250 μM [chlorhexidine, cetrimide, BAC]) did result in incomplete reversal (data not shown). Only pentamidine and dequalinium were equipotent in reversing the MepR interaction at both operator sites.
The differential effect of substrate on the MepR interaction with its two operators is intriguing. Our data suggest that substrate and MepR interact, and at the mepA operator the presence of MepA substrates results in dissociation of MepR and relief of mepA repression. This is consistent with our observed effect of substrate increasing mepA expression in SA-K2982 (SH1000 mepA::lacZ; Table 3). The reduced substrate effect on the MepR-mepR operator interaction may allow derepression of mepA with a relative maintenance of mepR repression. In this situation, mepA expression could proceed unimpeded by MepR, when more MepA is needed to detoxify the cell.
Unifying the differential substrate effect and the reduced MepR affinity for the mepR compared to the mepA operator is difficult with the data in hand. It is clear, however, that regulatory control at the mepR operator is complex. A structural analysis of MepR in the presence and absence of drugs and its DNA-binding sites will assist in clarifying these issues.
MepR operator sequences include promoter motifs and inverted repeats. The footprints of MepR in the mepR and mepA upstream regions are shown in Fig. 3 and 4, respectively. The MepR-binding site in the mepA upstream region is rather large (43 [sense strand] and 39 [antisense strand] bp) and includes the –35 and –10 promoter motifs, the mepA transcription start site, and two pairs of inverted repeats. The binding site upstream of mepR is smaller (27 bp on both the sense and antisense strands) and includes a portion of the –10 motif, the mepR transcription start site, and a single inverted repeat. Close examination of the MepR footprints reveals conservation of the sequence GTTAG in inverted repeats found in both (Fig. 3 and 4). This sequence occurs a second time in the mepA upstream footprint just 5' to the –10 promoter motif, suggesting that it may be a signature recognition sequence for MepR.
Competition MSA experiments described previously indicated an apparent greater affinity of MepR for its binding site(s) upstream of mepA compared to that upstream of mepR (Fig. 2). Sequence differences within each operator may provide an explanation for the observed differential MepR affinity and subsequent greater repression of mepA versus mepR transcription in wild-type strains in the absence of substrate (where only mepR transcripts are observed) (10). The large size of the mepA upstream operator and its plurality of inverted repeats and GTTAG sequences compared to the mepR operator, which has only one of each of these sequences, may result in the binding of more MepR molecules and thus more complete repression of the downstream gene than occurs at the mepR operator. Determination of the stoichiometry of binding between MepR and its cognate DNA upstream of mepR and mepA will provide the necessary data to support or refute this hypothesis. The presence of intermediate bands in MSAs for the mepA operator suggests that more than one MepR molecule is capable of binding at this site but does not differentiate between binding of a monomer, dimer, or higher-degree multimer.
Concluding remarks. To the best of our knowledge, the data presented herein are the first to document that an S. aureus regulatory protein can differentially regulate the expression of two genes within the same gene cluster with independent operator sequences upstream of both genes. In addition, the variable effect of substrate on the interaction of MepR with each of its operators has not been described previously for any S. aureus regulatory protein. The apparent augmented affinity of MepR for the mepA operator in the absence of substrate accounts for the presence of mepR but no mepAB transcripts in wild-type strains.
Several regulatory proteins affecting the expression of multidrug efflux pumps have been found to be capable of binding substrates of those pumps. Relevant examples already have been mentioned and include BmrR and QacR. BmrR is an activator of transcription of bmr, which encodes the Bmr MDR efflux pump (1). Substrate binding by BmrR facilitates its binding to its operator site, augmenting bmr transcription (24). QacR is a repressor of transcription of the QacA MDR pump and does so by binding to an operator site upstream of qacA, preventing transcription (6). Drug binding results in a conformational change in QacR, the result of which is dissociation from its operator and relief of qacA repression (22). MepR may behave in a similar fashion, and the determination of its crystal structure in the presence and absence of each operator and with or without drug will reveal its mechanism of multidrug recognition and how drug binding differentially changes the affinity of MepR for its operator sites.
ACKNOWLEDGMENTS
This work was supported by VA Research Funds.
We thank Tim Foster and Ambrose Cheung for supplying pAZ106 and pALC2073, respectively.
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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.
INTRODUCTION
Antimicrobial agent resistance in bacteria is an increasingly serious threat to modern medical care (16). Such resistance can occur by a number of mechanisms, including modification of the drug to an inactive form, modification of the drug target such that interaction with the drug does not occur, and removal of the drug from its site of action by membrane-based efflux proteins, hereafter referred to as efflux pumps. In recent years, many efflux pumps, from both gram-positive and -negative bacteria, capable of conferring multidrug resistance (MDR) have been described (4). Some of these MDR-conferring pumps have impressive substrate profiles that include mono- and divalent cationic antiseptic and disinfectant compounds, as well as antimicrobial agents.
MDR-conferring efflux pumps belong to one of five families based on structural characteristics and energy requirements. These include the ATP-binding cassette, major facilitator, multidrug and toxin extrusion (MATE), resistance-nodulation-division, and small multidrug resistance (SMR) families (4, 20). The MATE family is the most recently described and the least well characterized. We recently identified and characterized in a preliminary fashion the mepRAB gene cluster in Staphylococcus aureus (GenBank accession number AY661734) (10, 18). These genes encode MepR, which on the basis of homology is a MarR family regulatory protein; MepA, a MATE family multidrug efflux pump; and MepB, a protein the function(s) of which is currently unknown. In wild-type strains, only mepR transcripts are seen, but in mutants in which mepRAB is overexpressed the main transcript produced is mepRAB. However, in these mutants individual mepR, mepAB, and mepB transcripts also are observed. MepR is a repressor of mepA expression, but further details of the regulation of mepRAB expression are not known. Herein we establish that MepR represses mepR and mepA expression by binding upstream of both genes. MepR-mediated repression of mepA, and in a more limited fashion mepR, is abrogated in the presence of MepA substrates, consistent with MepR being a substrate-responsive, and most likely a substrate-binding, regulatory protein.
MATERIALS AND METHODS
Bacterial strains, plasmids, media, and reagents. The strains and plasmids employed in this study are listed in Table 1. Unless otherwise noted, all reagents were the highest grade available and were obtained from Sigma Chemical Co. (St. Louis, Mo.) or, in the case of some antimicrobial agents, their respective manufacturers. Growth media were obtained from BD Biosciences (Sparks, Md.) (brain heart infusion broth [BHIB] and tryptic soy broth) and Invitrogen (Carlsbad, Calif.) (Luria-Bertani broth).
pK434 was constructed as described previously (10). Briefly, the mepR open reading frame, including its ribosome-binding site, was amplified from S. aureus NCTC 8325-4. The PCR product was cloned into pALC2073, placing mepR under the control of the xyl/tetO tetracycline-inducible promoter (3). The plasmid was electroporated into S. aureus RN4220 and then transferred into SA-K2916 (see below) by transduction to reconstitute mepR in trans, producing strain SA-K2916-R (5). SA-K2916-R was grown in the presence of chloramphenicol (10 μg/ml) to ensure plasmid maintenance and, where appropriate, tetracycline to induce plasmid-based mepR expression.
Construction of mepR::lacZ and mepA::lacZ fusions. Internal fragments of the 5' portions of mepR and mepA were amplified by PCR, followed by cloning of each product into pAZ106, a plasmid conferring resistance to ampicillin (Escherichia coli) and erythromycin (S. aureus) and containing a multiple cloning site upstream of a promoterless lacZ gene (13). pAZ106 replicates only in E. coli, and when introduced into S. aureus RN4220 and selected for by erythromycin it will integrate into the chromosome at sites of homology provided by the mepR or mepA fragments. The integration disrupts the native genes and creates transcriptional fusions between those genes and lacZ. DNA sequencing was used to verify fusions in S. aureus RN4220, and phage 85 was used to transduce the fusions into S. aureus SH1000, producing strains SA-K2916 (mepR::lacZ) and SA-K2982 (mepA::lacZ) (5, 21).
Disruption of a chromosomal gene may have a polar effect on a downstream gene(s) that is cotranscribed with it. Analysis of genome data reveals that the open reading frame immediately downstream of mepRAB is oriented in the opposite direction, eliminating the possibility of a polar effect on its transcription. Additionally, we have shown previously that MepB plays no role in MepA-mediated MDR and thus a polar effect of mepR::lacZ or mepA::lacZ fusions on its transcription is unlikely to affect the data presented herein (10).
?-Galactosidase assay. Expression of mepR and mepA in lacZ fusion strains was quantitated by employing a fluorescent ?-galactosidase assay as described previously, with 4-methylumbelliferyl-?-D-galactopyranoside (MUG) as a substrate (11, 12). A range of concentrations of 4-methylumbelliferone were used to prepare a standard curve, and ?-galactosidase activity (expressed in MUG units; 1 U = 1 pmol of MUG cleaved per min per unit of optical density at 600 nm) was determined with a Bio-Tek FLx800 plate reader (Bio-Tek Instruments, Inc., Winooski, Vt.). Gene expression over the course of each experiment (10 h) was quantitated by integrating the areas beneath expression curves with SigmaPlot 9.0 (Systat Software, Inc., Point Richmond, Calif.).
Effect of MepR on mepR expression and of substrate exposure on the expression of mepR and mepA. SA-K2916-R was grown in BHIB containing chloramphenicol (10 μg/ml) without (control) or with a range of concentrations of tetracycline (6.25 to 50 ng/ml) to determine if MepR is autoregulatory. The effect of exposure to MepA substrates on mepR expression was determined by growing SA-K2916-R in BHIB containing chloramphenicol, 10 ng/ml of tetracycline to induce mepR expression from pK434 (where appropriate), plus one-quarter or one-half of the MIC of benzalkonium chloride (BAC), dequalinium, ethidium bromide (EtBr), or pentamidine. In a similar fashion, the effect of these compounds on mepA expression was determined with SA-K2982. ?-Galactosidase activity and cumulative mepR and mepA expression were determined as described previously. Each experiment was repeated a minimum of three times. Results for mepR were expressed as the ratio of expression data in the presence of tetracycline without or with a MepA substrate to that of SA-K2916-R uninduced with tetracycline, where this ratio was 1.0. For mepA, results were expressed as the ratio of drug-exposed to nonexposed SA-K2982. The significance of the reversal of the MepR repressive effect on mepR expression by substrate and the increase in mepA expression in the presence of substrate were analyzed with the Mann-Whitney rank-sum test. A P value of <0.05 was considered significant.
Purification of MepR. MepR was overproduced in E. coli with the Champion pET Directional Expression kit (Invitrogen). This kit includes the pET101/D-TOPO E. coli expression vector, which has a strong T7 isopropyl-?-D-thiogalactopyranoside (IPTG)-inducible promoter that controls the expression of cloned genes (Table 1). The mepR gene was amplified from S. aureus NCTC 8325-4 with primers TOPO-fwd and TOPO-rev (Table 2), producing a 421-bp product consisting of the entire mepR coding region but lacking the native stop codon. To ensure the proper orientation of mepR when cloned into pET101/D-TOPO, TOPO-fwd introduced a CACC sequence immediately 5' to the ATG initiation codon that mates with a GTGG sequence provided by the vector. Cloning into this vector also results in fusion of the mepR coding sequence with that for a linker peptide plus a six-histidine tag at its 3' terminus, followed by a stop codon. The coding region for the linker peptide was removed by PCR-based overlap extension, resulting in MepR with the six-histidine tag attached directly to its C terminus (MepR-H6) (8). Following the appropriate manufacturer's instructions, mepR was overexpressed and then large quantities of purified MepR were obtained by nickel affinity chromatography with the HisTrap kit (Amersham Biosciences AB, Uppsala, Sweden). The homogeneity of the recovered protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15).
Gel MSAs. Target DNAs used in gel mobility shift assays (MSAs) were amplified by PCR with the primer pairs shown in Table 2, one of which was biotinylated by Invitrogen as indicated. Primers mepR fwd and mepR rev produced a 167-bp product consisting of 109 bp of upstream and 58 bp of 5' mepR sequence. Primers mepRA fwd and mepRA rev(A) generated a 244-bp product including 88 bp of 3' mepR sequence, all of the mepRA intergenic region (106 bp), and 50 bp of 5' mepA sequence. Primers mepAB fwd and mepAB rev resulted in a 256-bp product that contained 45 bp of 3' mepA sequence, all of the mepAB intergenic region (103 bp), and 108 bp of 5' mepB sequence. MSAs were performed, and biotin-labeled DNA was detected with the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, Ill.) by following the manufacturer's guidelines as previously described, with modifications (23). Where indicated, DNA targets were incubated with 25 ng of purified MepR-H6 in 20 μl of binding buffer (10 mM Tris-HCl, 50 mM NaCl, 0.5 mM dithiothreitol, pH 7.5) to which glycerol, poly(dI-dC), and bovine serum albumin (final concentrations of 5%, 0.05 μg/ml, and 0.75 μg/ml, respectively) were added. Specificity of the MepR-DNA interaction was established by including a 200-fold molar excess of either nonbiotinylated target DNA (specific competitor) or salmon sperm DNA (nonspecific competitor) in binding reaction mixtures. To assess the impact of substrate on the MepR-DNA interaction, various concentrations of MepA substrates (BAC, cetrimide, chlorhexidine, ciprofloxacin, dequalinium, norfloxacin, pentamidine, and tetraphenylphosphonium bromide [TPP]) were included in the binding reaction mixtures. The effect of salicylate also was assessed.
To estimate the relative affinities of MepR for its identified binding sites (see below), competition MSAs were carried out. These experiments were performed as described above with MepR-H6, the biotin-labeled mepR or mepA upstream sequence to which it specifically bound, and doubling dilutions of the unlabeled alternate MepR-binding site (i.e., excess mepA upstream sequence included in MepR-mepR upstream sequence binding reaction mixtures and vice versa). For these experiments, the mepA upstream sequence used was generated by employing mepRA fwd and mepRA rev(B) (Table 2). The resultant 183-bp product included 88 bp of 3' mepR sequence and 95 bp of the mepRA intergenic region. This fragment contains the entire MepR-binding site identified in this region (see below).
DNase I footprinting. Footprinting was performed by previously described methods, with several modifications (6). Primers were the same as those used for MSAs (Table 2), except that biotinylated forward and reverse primers were employed as necessary to identify sense and antisense MepR footprints. MepR-DNA binding was performed in a 20-μl reaction mixture volume with the same buffer system as described for MSAs, except that 50 ng of MepR-H6 was used and MgCl2 and CaCl2 (final concentrations, 5 and 1 mM, respectively) were included in the buffer. The reaction mixture was incubated for 20 min at room temperature, followed by the addition of DNase I (final concentration, 5 U/ml; Amersham) and an additional 1-min incubation period. The reaction was terminated by adding an equal volume of stop solution (0.6 M sodium acetate, 25 mM EDTA, 40 μg/ml yeast tRNA, pH 5.0), followed by ethanol precipitation. The pellet was washed with ethanol and then resuspended in 5 μl of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol, 0.05% bromphenol blue). Aliquots of DNA were analyzed with a 6% polyacrylamide gel containing 7 M urea. Sequencing ladders were generated with the same primers as were biotinylated for the PCRs. Biotin-labeled DNA was detected with the Chemiluminescent Nucleic Acid Detection Module of the LightShift Chemiluminescent EMSA kit (Pierce).
RESULTS AND DISCUSSION
MepR strongly represses the transcription of its gene. We have shown previously that MepR represses mepA transcription (10). Employing a range of inducing concentrations of tetracycline, ?-galactosidase assays with SA-K2916-R revealed a dose-dependent reduction in chromosomal mepR transcription. Reductions of 12, 39, 57, and 79% were observed in the presence of 6.25, 12.5, 25, and 50 ng/ml of tetracycline, respectively. These data support the conclusion that MepR is autoregulatory. A direct self-repressive effect is most likely and is supported by MSA and footprinting data (see below).
Substrate exposure augments mepR and mepA expression. Growth of SA-K2916-R in the presence of 10 ng/ml of tetracycline repressed chromosomal mepR transcription by approximately 25%. Significant reversal of this repression was not observed with exposure to one-quarter the MIC of any tested MepA substrate, but a trend was evident for pentamidine at this concentration (Table 3). Higher concentrations of BAC and dequalinium (one-half of the respective MICs) did significantly reverse this repression, whereas EtBr did not. Pentamidine at one-half of its MIC could not be evaluated in this in vivo system due to inhibitory effects on test strain growth.
The transcription of mepA in SA-K2982 was low compared to that of mepR in SA-K2916. Under these circumstances, mepR expression was approximately a thousand times that of mepA. These data are consistent with previous Northern blot analyses of S. aureus NCTC 8325-4 revealing the presence of mepR but no mepA or mepB transcripts (10). In contrast to the effect of substrate exposure on mepR expression just presented, exposure of SA-K2982 to one-quarter of the respective MIC of BAC, dequalinium, EtBr, or pentamidine resulted in substantially increased mepA transcription (Table 3). Regulatory proteins for other MDR efflux pumps that have been studied, including BmrR of Bacillus subtilis and QacR of S. aureus, have been shown to have their activity altered by the binding of substrate to them (7, 22, 24). In fact, MarR also is a ligand-binding protein (salicylate), and such binding results in abrogation of its inhibitory effect on marRAB expression (2, 7, 17). Increased mepR and mepA expression in the presence of MepA substrates is consistent with a substrate-MepR interaction and resultant loss of MepR repression. The lesser effect of substrate on increasing mepR versus mepA expression would result in a relative reduction in MepR repression of mepA, a conclusion also supported by gel shift assay data (see below).
MepR binds specifically to the mepR and mepA upstream regions. MSAs revealed that MepR binds upstream of its gene (Fig. 1). This binding is specific, as excess unlabeled target reverses it but excess nonspecific DNA does not. Similarly, MSAs also revealed specific binding of MepR to a 244-bp target that encompasses the entire mepRA intergenic region (described previously). In this case, reversal of the observed shift by excess unlabeled specific DNA revealed the presence of intermediately shifted bands, most plausibly representing the binding of variable numbers of MepR molecules to its cognate DNA target. MarR family proteins typically bind as dimers; MepR may behave in a similar manner but also may bind in other multimeric forms (2, 7, 17).
No interaction between MepR and the mepB upstream region was observed (data not shown). Analysis of the nucleotide sequence upstream of mepB reveals no motifs that may be important for MepR binding (inverted repeats and GTTAG sequences; see below). Thus, the lack of a MepR interaction with this region is not surprising. The reason(s) for the inclusion of mepB in this gene cluster and the function(s) of MepB remain unknown. The structural and functional characterization of the protein may clarify these issues.
Competition MSA experiments revealed that MepR binding to the 167-bp mepR upstream region was reversed significantly by 200-, 100-, and 50-fold molar excesses of the unlabeled 183-bp mepA upstream region, whereas only a 200-fold molar excess of the mepR upstream region minimally competed with the mepA fragment (Fig. 2). These data are consistent with a greater affinity between MepR and the mepA upstream sequence than with the mepR upstream region. These data provide a plausible explanation for the relative "leakiness" of MepR repression of mepR versus its more complete repression of mepA expression, exemplified by earlier work showing mepR but no mepAB transcripts in wild-type strains (10).
Substrate abrogates MepR binding to its operator sites. Inclusion of MepA substrates in MSAs reversed the MepR-target interaction with the mepA upstream region (Fig. 1). Reversal was observed when drug and protein were combined first and then added to target DNA, or when protein and DNA were incubated together, followed by drug addition (data not shown). Various drug concentrations were evaluated to determine the relative potency of each substrate in reversing the MepR interaction with the mepA operator. Under the assay conditions employed, pentamidine and TPP appeared to have the weakest effects whereas chlorhexidine, cetrimide, dequalinium, and BAC had similar, and much greater, effects in that concentrations 6- to 80-fold lower disrupted MepR binding.
No reversal of the MepR interaction with the mepA target fragment occurred with a very high concentration of ciprofloxacin, norfloxacin, or salicylate (4,000 μM), indicating that these compounds have no or a very minimal ability to interact with MepR (data not shown). The absence of a salicylate effect differentiates MepR from its homologue MarR, which does bind this compound (2, 17). The absence of an interaction among ciprofloxacin, norfloxacin, and MepR was surprising given our earlier observation of reproducible two- to fourfold increases in the MICs of these compounds when mepA was overexpressed, establishing that they are MepA substrates (10). The explanation for this apparent paradox simply may be that not all MepA substrates necessarily must be inducers and MepA may be more promiscuous than MepR. A relevant example of such behavior is given by QacA, which effluxes compounds that do not induce its expression or bind to QacR (6). That the order in which MSA components were combined did not alter the observed reversal of the MepR-mepA operator interaction suggests that the presence of substrate can not only interfere with the ability of free MepR to bind to but can also dissociate bound MepR from its cognate DNA.
Similar to the MepR-mepA operator interaction, high concentrations of salicylate, ciprofloxacin, and norfloxacin had no effect on the MepR-mepR autoregulatory operator binding (data not shown). Concentrations of TPP, chlorhexidine, cetrimide, and BAC that were effective at reversing the MepR-mepA operator interaction had a minimal or no effect on MepR binding to the mepR operator. Higher concentrations of each of these compounds (6,000 μM [TPP] and 250 μM [chlorhexidine, cetrimide, BAC]) did result in incomplete reversal (data not shown). Only pentamidine and dequalinium were equipotent in reversing the MepR interaction at both operator sites.
The differential effect of substrate on the MepR interaction with its two operators is intriguing. Our data suggest that substrate and MepR interact, and at the mepA operator the presence of MepA substrates results in dissociation of MepR and relief of mepA repression. This is consistent with our observed effect of substrate increasing mepA expression in SA-K2982 (SH1000 mepA::lacZ; Table 3). The reduced substrate effect on the MepR-mepR operator interaction may allow derepression of mepA with a relative maintenance of mepR repression. In this situation, mepA expression could proceed unimpeded by MepR, when more MepA is needed to detoxify the cell.
Unifying the differential substrate effect and the reduced MepR affinity for the mepR compared to the mepA operator is difficult with the data in hand. It is clear, however, that regulatory control at the mepR operator is complex. A structural analysis of MepR in the presence and absence of drugs and its DNA-binding sites will assist in clarifying these issues.
MepR operator sequences include promoter motifs and inverted repeats. The footprints of MepR in the mepR and mepA upstream regions are shown in Fig. 3 and 4, respectively. The MepR-binding site in the mepA upstream region is rather large (43 [sense strand] and 39 [antisense strand] bp) and includes the –35 and –10 promoter motifs, the mepA transcription start site, and two pairs of inverted repeats. The binding site upstream of mepR is smaller (27 bp on both the sense and antisense strands) and includes a portion of the –10 motif, the mepR transcription start site, and a single inverted repeat. Close examination of the MepR footprints reveals conservation of the sequence GTTAG in inverted repeats found in both (Fig. 3 and 4). This sequence occurs a second time in the mepA upstream footprint just 5' to the –10 promoter motif, suggesting that it may be a signature recognition sequence for MepR.
Competition MSA experiments described previously indicated an apparent greater affinity of MepR for its binding site(s) upstream of mepA compared to that upstream of mepR (Fig. 2). Sequence differences within each operator may provide an explanation for the observed differential MepR affinity and subsequent greater repression of mepA versus mepR transcription in wild-type strains in the absence of substrate (where only mepR transcripts are observed) (10). The large size of the mepA upstream operator and its plurality of inverted repeats and GTTAG sequences compared to the mepR operator, which has only one of each of these sequences, may result in the binding of more MepR molecules and thus more complete repression of the downstream gene than occurs at the mepR operator. Determination of the stoichiometry of binding between MepR and its cognate DNA upstream of mepR and mepA will provide the necessary data to support or refute this hypothesis. The presence of intermediate bands in MSAs for the mepA operator suggests that more than one MepR molecule is capable of binding at this site but does not differentiate between binding of a monomer, dimer, or higher-degree multimer.
Concluding remarks. To the best of our knowledge, the data presented herein are the first to document that an S. aureus regulatory protein can differentially regulate the expression of two genes within the same gene cluster with independent operator sequences upstream of both genes. In addition, the variable effect of substrate on the interaction of MepR with each of its operators has not been described previously for any S. aureus regulatory protein. The apparent augmented affinity of MepR for the mepA operator in the absence of substrate accounts for the presence of mepR but no mepAB transcripts in wild-type strains.
Several regulatory proteins affecting the expression of multidrug efflux pumps have been found to be capable of binding substrates of those pumps. Relevant examples already have been mentioned and include BmrR and QacR. BmrR is an activator of transcription of bmr, which encodes the Bmr MDR efflux pump (1). Substrate binding by BmrR facilitates its binding to its operator site, augmenting bmr transcription (24). QacR is a repressor of transcription of the QacA MDR pump and does so by binding to an operator site upstream of qacA, preventing transcription (6). Drug binding results in a conformational change in QacR, the result of which is dissociation from its operator and relief of qacA repression (22). MepR may behave in a similar fashion, and the determination of its crystal structure in the presence and absence of each operator and with or without drug will reveal its mechanism of multidrug recognition and how drug binding differentially changes the affinity of MepR for its operator sites.
ACKNOWLEDGMENTS
This work was supported by VA Research Funds.
We thank Tim Foster and Ambrose Cheung for supplying pAZ106 and pALC2073, respectively.
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