INTRODUCTION

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Closely following the discovery of mammalian P-glycoprotein (1, 2), the phenomenon of multidrug resistance was also described for a bacterial system, the Staphylococcus aureus QacA pump (3), and has since been found to be widespread among both Gram-negative and Gram-positive bacteria (4-6). Resistance involves the active transport of a structurally diverse range of toxic compounds, typically hydrophobic cations, from the cell by a single efflux system. In the case of P-glycoprotein, the ability to export many anticancer agents (7) has prompted investigations into its mode of action. Biochemical studies and the generation of mutants with altered drug binding capabilities have suggested P-glycoprotein interacts directly with various substrates (7, 8). Additionally, recent progress has been made toward determining the structure of P-glycoprotein (9). Despite these advances, the basis of multidrug recognition by P-glycoprotein is still not understood. The functional similarities of bacterial multidrug efflux systems with P-glycoprotein, together with their presence in significant human pathogens, such as S. aureus (3), Pseudomonas aeruginosa (10), Neisseria gonorrhoeae (11), and Mycobacterium tuberculosis (12), makes elucidation of their molecular mechanisms an important research goal. Some progress has been made toward delineating the significance that various motifs and individual amino acids hold for determining the specificity of transport and overall mechanism of action for the QacA (13) and Bacillus subtilis Bmr (14) multidrug transporter proteins (reviewed in Ref. 6), but the strong association of efflux pumps with the membrane makes isolation and in depth analysis of these proteins difficult.

Considerable effort has also been directed toward identifying the factors involved in regulation of multidrug transporter gene expression. For mammalian P-glycoprotein (15, 16), as well as the Bmr (17), and Escherichia coli EmrB (18) bacterial multidrug efflux systems, increased gene expression followed the addition of some of the structurally diverse compounds exported by these pumps. Indeed, a certain degree of regulatory control over the genes for membrane transport proteins is to be expected, given their toxic nature toward the cell if overexpressed, as has been observed for the Gram-negative bacterial tetracycline resistance gene, tetA (19). Control of tetA expression by the specific repressor protein, TetR, is the best understood example of the regulation of a gene encoding a drug transporter (19). Induction of tetA expression occurs when TetR binds a tetracycline/divalent metal cation complex, inducing a conformational change in the protein such that TetR no longer binds the tetA operator, thereby liberating the promoter site (20). A similar style of regulation may also be responsible for the induction of expression observed for some bacterial multidrug efflux genes. However, for this to be effective, both the transporter and the regulatory protein would need to recognize the structurally diverse compounds that these systems efflux from the cell. Despite this, regulation of some bacterial multidrug efflux genes involves specific trans-acting regulatory proteins, such as the B. subtilis BmrR (17), E. coli EmrR (18), and N. gonorrhoeae MtrR (21) proteins. Importantly, the B. subtilis BmrR transcriptional activator protein binds directly to at least some of the compounds that both induce bmr expression and are also substrates for the Bmr transporter (17, 22, 23).

Analysis of the region immediately upstream from the S. aureus qacA gene revealed a putative regulatory element, qacR, previously called orf188 (24). Based on homology comparisons, QacR, together with TetR, belong to a family of regulatory proteins which all share common features associated with a multi-helical DNA-binding domain at their N-terminal ends and have highly divergent C termini postulated to be involved in the binding of inducing compounds (24, 25). In this paper, we make the first steps toward understanding how QacR regulates the expression of qacA, by demonstrating it is a negative regulator that both binds to an operator site adjacent to the qacA promoter and also appears to interact directly with a diverse range of inducing compounds.

	EXPERIMENTAL PROCEDURES

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Bacterial Strains and Plasmids-- The bacterial strains, plasmids, and primers used in this study are described in Table I. For all experiments performed in S. aureus, strain SK982 was the host; E. coli strain DH5 was used for CAT¹ assays, QacR overexpression, and in all cloning manipulations. All strains were cultured at 37 °C in LB media containing, where appropriate, 100 µg of ampicillin, 50 µg of kanamycin, 30 µg of chloramphenicol, or 20 µg of gentamicin per ml. The plasmid pSK616, containing qacA-qacR DNA originally derived from pSK1 (24), was the starting material for all manipulations, unless otherwise specified. The vector pSK5201, used to generate promoter fusions to the chloramphenicol acetyltransferase cat reporter gene, was constructed by replacing the 1.4-kilobase pair PvuI-NdeI fragment in pKK232-8 (26) with the 1.9-kilobase pair PvuI-NdeI M13 origin-containing fragment from pMAL-p2 (New England Biolabs), to allow rescue of single-stranded DNA from subsequent constructs.

RNA and DNA Isolation and Manipulations-- Total cellular RNA was purified from S. aureus strains SK982 and SK982 containing the qacA⁺, qacR⁺ plasmid pSK1 by the hot phenol method as described by Miller (29), modified for use with S. aureus by an initial incubation on ice in protoplast lysis buffer (15 mM Tris-HCl, 6 mM EDTA, 450 mM sucrose, pH 8.0) containing 0.25 mg/ml lysostaphin (Sigma) for 45 min to generate protoplasts. Double- and single-stranded plasmid DNA preparations, DNA manipulations, transformations, and site-directed mutagenesis were by standard procedures (30). For dideoxy sequencing of double-stranded DNA. the Sequitherm kit (Epicentre Technologies) was utilized. PCR was performed using Pfu enzyme (Stratagene), according to the manufacturer's instructions.

Identification of Promoters: Primer Extension-- Primer extension analysis was performed essentially as described by Ausubel et al. (31), utilizing the primers 899EcoRI for qacA and 557 and 672BamHI for qacR (Fig. 1). Primers were end-labeled with [-³²P]ATP (Bresatec), mixed with 50 µg of total RNA, denatured by heating at 80 °C for 3 min, and then hybridized at 42 °C for 90 min before being extended by the addition of dNTPs and Moloney murine leukemia virus reverse transcriptase (Promega). The primer extension products were loaded on a 6% polyacrylamide gel and electrophoresed alongside dideoxy sequencing ladders generated with the same primers.

Determination of Promoter Activity: Chloramphenicol Acetyltransferase Assay-- Overnight cultures of E. coli DH5 cells harboring a promoter fusion construct alone, or together with pSK4238 providing qacR in trans, were diluted 1:40 in 200 ml of LB media and grown overnight with the appropriate antibiotics and in some cases the addition of a sub-MIC of a potential qacA inducing compound. Cells were collected by centrifugation, resuspended in 10 ml of 1 M Tris-HCl (pH 8.0) and lysed by sonicating (Branson Sonifier B-12) twice for 30 s at 75 watts. The lysate was cleared by centrifugation before the level of CAT activity was determined using acetyl-CoA (Sigma) according to the method of Shaw (32). Triplicate experiments were performed on 3 separate days.

Expression and Purification of QacR from E. coli-- An overnight culture of E. coli DH5 cells freshly transformed with pSK5210 was diluted 1:50 in 1 liter of prewarmed LB media and grown to an OD₆₀₀ of 0.5, at which stage overexpression of QacR was induced by the addition of 0.5 mM IPTG. After another 2-2.5 h, the cells were harvested by centrifugation and resuspended in 20 ml of cold sonication buffer (50 mM NaH₂PO₄, 300 mM NaCl, 5 mM 2-mercaptoethanol, 20% v/v glycerol, pH 7.8). The cells were frozen overnight at 70 °C and then thawed on ice before addition of lysozyme (Sigma) to 1 mg/ml and incubated on ice for 30 min. Cells were lysed by sonication (75 watts, 30 s, 1 min of cooling, repeated eight times), followed by the addition of RNase A to 10 µg/ml and DNase I to 5 µg/ml and incubated on ice for 30 min. The lysate was cleared by centrifugation at 15,000 rpm for 20 min before being mixed with 10 ml of 50% Ni²⁺-NTA metal chelate affinity resin (Qiagen) for 1.5 h at 4 °C. The resin was then packed into a column at 4 °C and washed at a flow rate of 15 ml/h with 40 ml of sonication buffer and then overnight with 200 ml of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 5 mM 2-mercaptoethanol, 20% v/v glycerol, 50 mM imidazole, pH 7.3). Purified QacR was eluted in wash buffer containing 300 mM imidazole (pH 8.0). The fractions were analyzed by SDS-PAGE (12.5% polyacrylamide) using the buffer system of Laemmli (33) and proteins visualized by staining with Coomassie Brilliant Blue R. Molecular size standards were purchased from Bio-Rad, and protein concentration was estimated by the method of Bradford (34), using bovine serum albumin (New England Biolabs) as a standard. Gel-filtration chromatography was performed on a FPLC Superose 12 HR 10/30 column (Amersham Pharmacia Biotech) with 100 mM KCl, 20 mM Tris-HCl, 20 mM 2-mercaptoethanol, pH 7.5, as the buffer. Gel-filtration molecular weight markers MW-GF-200 were purchased from Sigma. Low pH native PAGE was performed as described in Ref. 35. Western analysis using a 6×His monoclonal antibody to detect hexahistidine-tagged proteins was performed according to the manufacturer's (CLONTECH) instructions.

Treatment of QacR with Divalent Metal Cations-- Purified QacR (6 µg) in a final volume of 40 µl was incubated in 20 mM Tris-HCl (pH 7.5) containing the indicated amounts of divalent metal cations for 3 h at 22 °C. The reaction was stopped by the addition of 20 µl of non-reducing SDS-PAGE sample buffer containing 30 mM EDTA. Samples were heated at 100 °C for 4 min without reducing agent unless otherwise stated, cooled on ice for 2 min, and 5-µl aliquots analyzed by SDS-PAGE followed by silver staining to visualize the proteins.

Gel-mobility Shift Analysis-- Labeled probes obtained by PCR amplification using primers end-labeled with [-³²P]ATP were purified with the Wizard DNA purification kit (Promega). Initial experiments utilized a 212-bp PCR product corresponding to the whole qacR-qacA intergenic region (bp 672-867; Fig. 1) generated with the primers 672BamHI and 867HindIII. Subsequent experiments in which substrates of QacA were added to some of the binding mixtures used a smaller 137-bp PCR product (bp 738-867; Fig. 1), which included the qacA promoter and IR1 portions of the intergenic region, produced with the primers 738 and 867HindIII. A 186-bp PCR fragment from the 3' end of the qacR gene that acted as a specificity control was generated using the primers 108HindIIIH6 and 262. The QacR fractions obtained from the affinity column were immediately exchanged into GMS buffer (15 mM Tris-HCl, 1 mM EDTA, 100 mM KCl, 7.5% v/v glycerol, pH 7.5) containing 10 mM 2-mercaptoethanol by passage through a Sephadex G-50 column previously equilibrated with this buffer and stored in single-use aliquots at 70 °C until required. The ³²P-labeled DNA fragments, approximately 2000 cpm/reaction, were incubated with the indicated amounts of QacR in GMS buffer containing 0.3 mM DTT, 75 µg of bovine serum albumin per ml, and 75 µg of poly(dI-dC) (Sigma) per ml in a total volume of 20 µl. Some reactions also had substrates of QacA or divalent metal cations added at the indicated concentrations. After 15 min of incubation at 22 °C, the reaction mixtures were analyzed by high ionic strength PAGE and autoradiographed as described by Ausubel et al. (31).

DNase I Footprinting-- Footprinting of the qacA coding strand utilized the 137-bp DNA fragment used in gel-mobility shift experiments, generated using primers 738 (end-labeled with [-³²P]ATP) and 867HindIII. For footprinting of the qacA non-coding strand, a 154-bp fragment (bp 738-899; Fig. 1) was prepared using the primers 899EcoRI (end-labeled with [-³²P]ATP) and 738. Approximately 40,000 cpm/reaction of labeled DNA was incubated in GMS buffer containing 0.3 mM DTT, 75 µg/ml bovine serum albumin, and 60 µg/ml poly(dI-dC) for 15 min at 22 °C in a total volume of 20 µl. Selected reactions also contained 600 ng of purified QacR. One unit of DNase I (Promega) in 20 µl of DNase I buffer (10 mM Tris-HCl, 10 mM MgCl₂, 2 mM CaCl₂, 1 mM DTT, 15 mM NaCl, pH 7.5) was then added, mixed, and the digestion allowed to proceed for exactly 1 min at 22 °C before being terminated with the addition of 40 µl of stop solution (0.6 M sodium acetate, 25 mM EDTA, pH 4.8). The reactions were extracted once with phenol-chloroform (3:1 v/v) and the DNA ethanol precipitated overnight at 20 °C. The resulting pellet was washed twice with 70% ethanol and resuspended in 6.5 µl of loading dye (49% formamide, 0.0125% xylene cyanol, 0.0125% bromphenol blue). After heating at 95 °C for 3 min, 2-µl aliquots of the DNA were analyzed on a 6% polyacrylamide gel. Sequencing ladders were generated in each case with the same primer as was end-labeled for the PCR reaction.

	RESULTS

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Localization of the Promoter Elements and Transcription Start Points for qacA and qacR-- Previous inspection of the sequence upstream of qacA had revealed a divergently transcribed open reading frame, qacR, postulated to regulate qacA transcription by binding to a region of dyad symmetry (IR1; Fig. 1) that partially overlapped the most likely sequence for the qacA promoter (P_qacA; Fig. 1) (24). Additionally, a potential promoter for qacR (bp 794-827 in Fig. 1) was identified, which would overlap IR1 and possibly subject qacR expression to auto-regulation (24). However, on further analysis of the qacR-qacA intergenic sequence, we identified another potential promoter-like element for qacR (P_qacR; bp 702-729 in Fig. 1) that lies closer to the start of the gene and has its own distinct region of dyad symmetry (IR2; Fig. 1), suggesting that expression of qacR could be initiated from multiple promoters.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Regulatory region of the qacA and qacR genes. The respective promoters, PqacA and PqacR, their putative "10" and "35" regions, and the tsp for each gene are shown. Bold type delineates the most likely ribosome binding sites. Inverted repeats, IR1 and IR2, that partially overlap each promoter are indicated by bold arrows. Thin arrowed lines denote selected oligonucleotide primers (Table I) used in this work. The positions of only relevant restriction sites are shown. The deduced amino acid sequences for the amino termini of the respective gene products, QacR and QacA, are given adjacent to their coding strands. Boxed amino acids indicate the location of the putative helix-turn-helix motif in QacR. The numbering is according to the previously published qacR-qacA sequence from the S. aureus multiresistance plasmid pSK1 (24).

View larger version (89K): [in this window] [in a new window]   Fig. 2.   Primer extension analysis of the qacA and qacR transcription start points in S. aureus. Primer extension and sequencing reactions utilizing primer 899EcoRI for qacA and primer 557 for qacR were performed as detailed under "Experimental Procedures." The corresponding DNA sequences, with the 10 region boxed and the transcription initiation nucleotide circled, are shown to the left for each gene. Nucleotides are numbered as described in Fig. 1. Primer extension analysis of qacA; S. aureus strain SK982 without (lane 1) and with (lane 2) the qacA+, qacR+ plasmid pSK1. Primer extension analysis of qacR; SK982 pSK1 uninduced (lane 3), Eb (50 µg/ml for 15 min) induced (lane 4), and SK982 alone (lane 5).

View this table: [in this window] [in a new window]   Table II Effect of qacR, promoter mutations, and deletion of one half of IR1 on the CAT activity of various PqacA-cat and PqacR-cat promoter fusions The level of CAT activity was determined as described under "Experimental Procedures" after overnight growth of E. coli DH5 cells harboring the indicated plasmid(s).

The qacR Gene Encodes a Repressor of qacA Expression That Acts via IR1-- To analyze the potential regulation of qacA by the qacR gene product, CAT assays were carried out on E. coli DH5 cells harboring pSK5203 or pSK5212. It was clearly demonstrated that in the case of pSK5212, the presence of the qacR gene in cis to qacA resulted in a significant reduction in transcription levels from P_qacA when compared with pSK5203 (6.41 versus 1.37 units; Table II). When pSK5203 and the compatible plasmid pSK4238, which provided qacR in trans, were present in the same cell, QacR completely repressed expression from the qacA promoter, resulting in no detectable cat activity (Table II). As expected, the presence of the pSK4238 vector, pK184, lacking qacR, had no effect on transcription from P_qacA (data not shown). Taken together, these data indicate that the observed high level of repression afforded by pSK4238 was a result of qacR being placed under the control of the strong lac promoter, leading to abnormally high levels of QacR. The presence of pSK4238 was found to have no significant effect on the level of transcription from the qacR promoter in pSK5202 (Table II), indicating that the expression of qacR is not autoregulatory.

Purification of QacR-- To obtain large quantities of QacR for in vitro studies, the qacR gene was first cloned downstream from a strong ribosome binding site and the IPTG-inducible tac promoter in the E. coli vector pTTQ18, resulting in inducible overexpression of QacR (Fig. 3A, lane 2). Utilization of a His tag incorporated at the C terminus of QacR allowed purification close to homogeneity by Ni²⁺-NTA chromatography (Fig. 3A, lanes 5 and 6). The purified QacR protein migrated in reducing SDS-PAGE gels with an observed molecular mass of approximately 23 kDa, the size predicted for a QacR monomer (24) with a hexahistidine tag. N-terminal amino acid sequencing of the first 12 residues of the purified protein confirmed its identity. The molecular mass of freshly purified QacR was determined by gel-filtration chromatography to be 22.5 kDa, in close agreement with the predicted molecular mass for a QacR-His tag monomer.

View larger version (74K): [in this window] [in a new window]   Fig. 3.   QacR overexpression, purification and divalent metal cation induced formation of disulfide-bonded oligomers. A, Coomassie Brilliant Blue R-stained SDS-PAGE gel of QacR with a C-terminal His tag purified from IPTG-induced E. coli DH5 pSK5210 cells using Ni2+-NTA affinity resin as described under "Experimental Procedures." DH5 pSK5210 cells uninduced (lane 1), and induced with 0.5 mM IPTG for 2.5 h (lane 2), cleared cell lysate before mixing with resin (lane 3), flow-through during column washing (lane 4), purified QacR (lane 5), overloaded QacR (lane 6), and oligomer formation after 3 months of storage at 4 °C in the elution buffer (lane 7). The positions of migration and molecular masses (in kDa) of the fully reduced QacR monomer (QacRI), the QacR dimer observed after long term storage (QacRII), and protein size standards are indicated. B, silver-stained SDS-PAGE gel of divalent metal cation-catalyzed formation of disulfide bonds. Purified QacR was incubated with the indicated concentrations of either CuCl2 or NiCl2, as described under "Experimental Procedures." The reactions were stopped by the addition of half a volume of SDS-PAGE loading buffer containing 30 mM EDTA without () or with (+) 100 mM DTT followed by heating to 100 °C for 4 min and analysis by SDS-PAGE.

Involvement of Metal Cations in the Formation of Disulfide-bonded Oligomers-- Addition of EDTA to a final concentration of 5-10 mM immediately after elution of QacR from the Ni²⁺-NTA column was able to partially suppress the formation of oligomers during storage (data not shown), supporting the involvement of divalent metal cations in catalyzing disulfide-bond formation between purified QacR molecules. To test the role of divalent metal cations, QacR monomer was incubated with various concentrations of NiCl₂ and CuCl₂, which resulted in an increased rate of oligomer formation, reaching a maximum at 10 mM for CuCl₂ and at 1 mM for NiCl₂ (Fig. 3B). In particular, treatment with low concentrations of Ni²⁺ resulted in the appearance of a significant dimer band (QacRII; Fig. 3B), corresponding to the predominant oligomeric species observed after long term storage, which suggested that leaching of Ni²⁺ ions from the affinity column during the purification process could be contributing to the oligomer formation. To reverse the formation of the oligomers, heating in the presence of 100 mM DTT was required, confirming their disulfide-bonded nature. A faster migrating species, presumably representing QacR containing an intramolecular disulfide bond, was also reduced to the non disulfide-bonded monomer (Fig. 3B). Interestingly, addition of CuCl₂ to a concentration of 1 mM or more largely abolished the intramolecular disulfide-bonded species, in favor of oligomeric forms (Fig. 3B). Two further metals tested, ZnSO₄ and MgSO₄, had no effect on the formation of disulfide bonds (data not shown).

QacR Binds to a DNA Fragment Containing IR1 and the qacA Promoter-- Gel-mobility shift assays were used to show QacR bound specifically to a DNA fragment corresponding to the qacA-qacR intergenic region (bp 672-867; Fig. 1), and not to a control fragment from the 3' end of the qacR gene (Fig. 4). Only excess, unlabeled, intergenic competitor DNA, and not excess control DNA, was able to partially titrate out QacR from gel-mobility shift binding reactions (Fig. 4), confirming the specificity of QacR for the intergenic DNA fragment. Additionally, cleavage of the intergenic fragment at the DraI site (bp 751; Fig. 1), to yield two singly end-labeled fragments, resulted in only the qacA promoter-containing fragment being retarded (Fig. 4). This demonstrated that QacR is specific for the qacA promoter and did not bind to a DNA fragment containing the qacR promoter and IR2, lending further support to the postulate that expression of qacR is not autoregulated. In addition, only the crude protein extract from IPTG-induced E. coli DH5 cells harboring the QacR overexpressing plasmid pSK5210, but not the control plasmid pTTQ18, was able to shift labeled intergenic DNA (data not shown), confirming that QacR alone is responsible for the observed retardation of intergenic DNA. The addition of CuCl₂ or NiCl₂ had no observable effect on gel-mobility shift binding reactions (data not shown), indicating that at concentrations which increased the rate of oligomer formation (Fig. 3B) these divalent metal cations do not appear to have any direct effect on the binding of QacR to IR1.

View larger version (96K): [in this window] [in a new window]   Fig. 4.   Gel-mobility shift assays demonstrating specificity of QacR binding to a qacA promoter-containing DNA fragment. Autoradiograph of purified QacR added in the indicated amounts to; the 212-bp labeled DNA fragment corresponding to the entire qacR-qacA intergenic region (a), the 186-bp labeled specificity control DNA fragment from the 3' end of the qacR gene (b), or the labeled 212-bp intergenic fragment cleaved with DraI (Fig. 1) to yield a 121-bp PqacA-containing fragment (c) and a 91-bp PqacR-containing fragment (d). A 50-fold excess of cold, unlabeled, 212-bp intergenic (+I) or 186-bp control (+C) competitor DNA was also added to some assays, prior to incubation for 15 min and non-denaturing PAGE as described under "Experimental Procedures." The position of protein-DNA complexes e and f, that resulted from QacR binding to fragments a and c, respectively, are also indicated.

View larger version (84K): [in this window] [in a new window]   Fig. 5.   Identification of the QacR binding site by DNase I footprinting. A, end-labeled qacA promoter/operator DNA fragments were incubated without () and with (+) 600 ng of purified QacR before being subjected to DNase I digestion and electrophoresis as described under "Experimental Procedures." Footprinting of the qacA sense strand used a PCR fragment generated with labeled primer 738 and unlabeled primer 867HindIII. Footprinting of the qacA antisense strand utilized a PCR fragment generated with labeled primer 899EcoRI and unlabeled primer 738. The regions that QacR protects from DNase I digestion are bracketed in each case. Sequencing reactions were performed with the same primer as labeled for the relevant PCR reaction. B, sequence of the qacA 10 region and the downstream inverted repeat, IR1, with the region QacR protects from DNase I digestion highlighted (white), the qacA tsp circled, the qacA 10 region boxed, and the location of IR1 shown as bold arrows. Nucleotides are numbered as described in Fig. 1.

Substrates of QacA Act as Inducers of qacA Expression-- The QacA protein confers resistance to a wide range of structurally dissimilar compounds from four distinct classes of chemicals; the dyes, biguanidines, diamidines, and QACs. To investigate if these substrates potentiate the expression of qacA, CAT assays after overnight incubation in sub-MICs of potential inducing compounds were carried out with E. coli DH5 cells harboring pSK4150, which contained the qacR gene in cis to the qacA promoter. A significant increase in CAT activity was observed for many of the QacA substrates (Table III). Conversely, except for Eb, which showed a small degree of induction independent of QacR, no increase in CAT activity was observed when the qacA promoter alone was tested against a range of inducing compounds (data not shown). Also tested for their ability to increase qacA gene expression were another diamidine, propamidine isethionate, and another QAC, cetyltrimethylammonium bromide, both of which showed no induction (data not shown). None of the compounds tested were able to overcome the strong repression of the qacA promoter in pSK5203 afforded by qacR being overexpressed in trans from the plasmid pSK4238 (data not shown).

View this table: [in this window] [in a new window]   Table III Induction of expression from a PqacA-cat transcriptional fusion with qacR in cis upon the addition of various inducing compounds The level of CAT activity was determined for E. coli DH5 cells harboring pSK4150, which contained the qacA promoter fused to cat with qacR present in cis. After overnight growth in the indicated sub-MICs of potential inducing compounds, CAT assays were performed as described under "Experimental Procedures."

Structurally Diverse Substrates of QacA Inhibit Binding of QacR to a qacA Operator-containing DNA Fragment-- As for the in vivo induction of expression, many QacA substrates were also able to show an in vitro effect on binding of QacR to the qacA operator site. Fig. 6 demonstrates a strong dissociation of QacR from the operator-containing DNA fragment when any of the dyes Eb, rhodamine 6G, proflavin, or crystal violet were included in gel-mobility shift experiments. To dissociate QacR from the operator containing DNA, a concentration 50 times the MIC for S. aureus of the biguanidine compound chlorhexidine digluconate was required, whereas none of the diamidines, pentamidine isethionate (Fig. 6), propamidine isethionate, or diamidinodiphenylamine-hydrochloric acid, as well as a QAC, cetyltrimethylammonium bromide (data not shown) had any effect. The QACs dequalinium chloride and cetylpyridinium chloride both strongly inhibited binding of QacR to the operator, at a sub-MIC, and 10-fold greater than the MIC for S. aureus, respectively (Fig. 6). Benzalkonium chloride was shown to partially inhibit binding of QacR to operator DNA at a concentration that was 4 times the MIC of this compound for S. aureus (Fig. 6). The addition of QacA substrates at the concentrations used in the above experiments to DNA alone did not affect its mobility.

View larger version (89K): [in this window] [in a new window]   Fig. 6.   Gel-mobility shift analysis of the effect of potential inducing compounds on the binding of QacR to the qacA promoter-containing DNA fragment. Gel-mobility shift analysis was carried out with the 32P-labeled 137-bp (bp 738-867; Fig. 1) qacA promoter/operator-containing DNA fragment (a), incubated in the absence () or presence (+) of 50 ng of purified QacR with the addition of potential inducing compounds (µg/ml) at the indicated concentrations. The position of the protein-DNA complex (b) formed by QacR binding to the qacA promoter/operator DNA is shown.

	DISCUSSION

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This paper provides the first experimental evidence to demonstrate that the expression of the S. aureus multidrug efflux gene qacA is regulated by a repressor protein, QacR, the product of a divergently transcribed open reading frame. By both in vitro gel-mobility shift (Fig. 4) and DNase I protection experiments (Fig. 5), and in vivo analysis by deletion of one half of the dyad symmetry which resulted in constitutive expression of P_qacA-cat fusions (Table II), the operator site for QacR binding was shown to have been correctly identified; located immediately downstream from the qacA promoter (IR1; Fig. 1). By analogy with studies on promoter/operator systems such as that of the E. coli lac operon (39, 40), the binding of QacR to its operator may not inhibit the binding of RNA polymerase, but would prevent the transition of the RNA polymerase-promoter complex into a productively transcribing state, insuring adequate repression of qacA transcription.

The inability of QacR to autoregulate expression of its own gene was demonstrated by qacR overexpressed in trans being able to completely abolish transcription from the qacA promoter, yet having no effect on its own promoter fused to a reporter gene (Table II). This finding was confirmed by showing that QacR did not bind to a DNA fragment containing the qacR promoter (Fig. 4). Most other members of the family of regulatory proteins sharing homology with QacR that are divergently transcribed appear to regulate the expression of their own genes, such as actII-ORF1 from a Streptomyces antibiotic exporting complex (41), CamR, a repressor of D-camphor degradation in Pseudomonas putida (42), TcmR, the repressor of the Streptomyces glaucescens tetracenomycin C resistance gene tcmA (43), and TetR (19). QacR appears to be unusual for this family of proteins in that expression of its own gene was not subject to autoregulation. Although we demonstrated IR2 was not bound by QacR, an alternative role for this potential operator sequence in the control of qacR, and hence qacA gene expression, awaits further detailed investigation.

The repression of qacA transcription by QacR was able to be overcome by the addition of a range of structurally dissimilar QacA substrates, resulting in induction of qacA expression (Table III). Gel-mobility shift assays (Fig. 6) suggested that for many QacA substrates, induction of qacA expression involved QacR interacting directly with the substrates. Direct recognition of structurally dissimilar compounds, rather than the involvement of a secondary messenger signaling cellular damage, has also been shown for BmrR (17, 22, 23). Some in vivo inducers of qacA expression inhibited in vitro binding of QacR to qacA operator DNA only at concentrations greatly exceeding their MIC for S. aureus. This may reflect the fact that all QacA substrates appear to be hydrophobic cations; such hydrophobicity may result in these compounds having significantly elevated intracellular levels compared with the concentration at which they were added to the surrounding medium (17). Unexpectedly, two QacA substrates which showed no in vivo induction of qacA gene expression, chlorhexidine digluconate and cetylpyridinium chloride, were able to inhibit binding of QacR to operator DNA, but only at concentrations vastly in excess of their MIC for S. aureus (Fig. 6). For diamidinodiphenylamine-hydrochloric acid, pentamidine isethionate, and propamidine isethionate (all diamidines), and cetyltrimethylammonium bromide (a QAC), no notable effects on the expression of qacA, or the binding of QacR to operator DNA were observed. These results suggest that the level of transcription occurring from the qacA promoter when qacR is present in cis (Table II) enables QacA-mediated efflux of substrates such as the diamidines, compounds not recognized by the repressor protein QacR.

On the basis of the gel-filtration and native PAGE results, it would appear that QacR is purified as a monomer. The failure to detect any disulfide-bonded oligomers in Western blots of crude cell lysates is a strong indication that the multimers which form during long term storage of purified QacR do not occur naturally in the cell and are therefore unlikely to have any physiological significance. The ability of Cu²⁺ and Ni²⁺ to induce oligomerization (Fig. 3B), together with the requirement for a reducing agent to reverse the process, supports the involvement of divalent metal cations in the formation of intra- and intermolecular disulfide bonds subsequent to the isolation of QacR. Formation of disulfide-bonded oligomers during the storage of purified proteins has been shown to be responsible for the inactivation of human fibroblast growth factor-1 (44), T4 lysozyme (45), and Bacillus -amylases (46). The observed heterogeneity in the banding pattern of both QacR monomers and oligomers is most likely the result of different combinations of intermolecular and intramolecular disulfide bonds, which also occurs for human fibroblast growth factor (44). Furthermore, rapid formation of disulfide-bonded dimers following the purification of the bacterial mercury resistance regulator, MerR, has also been a problem (47). Interestingly, for MerR (48) and also ArsR, the repressor of the E. coli arsenical and antimonial resistance ars operon (49), cysteine residues are required for the binding of the metal cations that act as inducers of these systems, resulting in conformational changes in the respective regulatory proteins, thereby inducing expression. The cysteine residues in QacR could likewise play a role in the apparent ability of this protein to bind the divalent and monovalent cationic compounds that act as inducers of qacA gene expression.

The ability of QacR to interact with multiple and chemically diverse compounds makes it an attractive candidate for future studies. Because of their location in the soluble cytoplasmic fraction of the cell, multidrug efflux regulatory proteins are much easier targets than the corresponding membrane bound transporter for initial studies directed at understanding how structurally diverse compounds are recognized by a single protein. Further work involving the demonstration of a direct interaction between QacR and inducing compounds, mutational analysis of individual residues, and refinement of the purification process toward x-ray crystallography studies on the structure of QacR bound to its operator DNA or inducing compounds is in progress.