From the School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia
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ABSTRACT |
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The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of the qacA and divergently transcribed qacR mRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of the qacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump.
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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.
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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.
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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.
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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 (PqacA; 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 (PqacR; 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.
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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
PqacA 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 PqacA (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.
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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.
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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).
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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.
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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 PqacA-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.
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FOOTNOTES |
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* This work was supported in part by a Project Grant from the National Health and Medical Research Council (Australia).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of an Australian Postgraduate Award.
§ Recipient of the Ernest Fields Scholarship, Faculty of Medicine, Monash University, Australia.
¶ To whom correspondence should be addressed: School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, New South Wales 2006, Australia. Tel.: 61-2-9351-2376; Fax: 61-2-9351-4771; E-mail: skurray{at}bio.usyd.edu.au.
1
The abbreviations used are: CAT, chloramphenicol
acetyltransferase; bp, base pair(s); DTT, dithiothreitol; Eb, ethidium
bromide; IPTG, isopropyl-1-thio--D-galactopyranoside;
IR, inverted repeat; MIC, minimum inhibitory concentration; NTA,
nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR,
polymerase chain reaction; QAC, quaternary ammonium compound; tsp,
transcription start point.
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REFERENCES |
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