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Journal of Bacteriology, April 2007, p. 2996-3005, Vol. 189, No. 8
0021-9193/07/$08.00+0     doi:10.1128/JB.01819-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and ß-Lactams in Staphylococcus aureus{triangledown}

Que Chi Truong-Bolduc and David C. Hooper*

Division of Infectious Diseases and Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114-2696

Received 4 December 2006/ Accepted 25 January 2007


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four ß-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MgrA, a multifunctional MarR-like regulator, was first identified by its ability to bind directly to the norA promoter, leading to altered expression of norA, which encodes the NorA efflux pump in Staphylococcus aureus, an important pathogen responsible for infections in hospital and community settings (28). Infections caused by S. aureus can be difficult to treat because of resistance to multiple antibiotics and multiple virulence factors (2). One of the most interesting mechanisms of resistance in S. aureus is its ability to express several multidrug resistance efflux pumps, which constitute a major defense against diverse families of toxins and antimicrobial agents (17, 18).

The complete genome of S. aureus N315 is 2.81 Mb in length, containing genes predicted to encode 210 transporters. Sixty-seven of these transporters are predicted to be ATP dependent, representing 31% of the total. The majority of the ATP-dependent transporters belong to the ABC family (63 ABC transporters, or 94% of the total of 67). Among the 114 (54.3%) secondary transporters energized by ion gradients across the membrane, there are 28 efflux pumps (24.6%) belonging to the major facilitator superfamily (MFS) and fewer that are members of other families. A similar distribution is found in the genomes of S. aureus strains COL, Mu50, and NCTC 8325 (11, 19). NorB, NorC, and Tet38 are three new additions to the MFS of transporters in S. aureus, and recently MepA, a multidrug resistance pump belonging to the multidrug and toxin extrusion family, was identified (9, 26, 27). Efflux pumps can extrude a specific class of antibiotics such as tetracyclines (TetK, TetL, and Tet38) or macrolides (MsrA) or can extrude diverse unrelated compounds, such as quinolones, ethidium bromide, and cetrimide (NorA, NorB, and MepA) (1, 9, 22, 26).

AbcA is an ATP-dependent transporter of the ABC family, members of which use the energy liberated by ATP hydrolysis rather than the energy generated by transmembrane ion gradients, which is used by the members of the MFS to extrude their substrates (3). AbcA was shown to participate in cell wall autolysis, but no relation was established between its overexpression and resistance to ß-lactam antibiotics (4, 6, 24). This transporter shares an overlapping promoter region with the structural gene (pbpD) encoding the PBP4 protein, a transpeptidase/carboxypeptidase, which is involved in cell wall synthesis and confers a decrease in sensitivity to ß-lactam drugs (4). The expression of abcA and that of pbpD, however, appear to be independent of each other and to require different regulatory factors. The transcription of abcA depends on the agr regulatory system (24).

MgrA affects resistance to antibiotics by controlling the expression of at least four efflux pumps, NorA, NorB, NorC, and Tet38, which are responsible for decreases in susceptibility to hydrophilic (norfloxacin and ciprofloxacin) and hydrophobic (moxifloxacin and sparfloxacin) quinolones, tetracycline, and chemical compounds (ethidium bromide, cetrimide, and tetraphenylphosphonium [TPP]) (26-28). In addition to modulating the expression of efflux transporters, MgrA also regulates autolytic activity and the expression of several virulence factors, including alpha-toxin, nuclease, protein A, and capsular polysaccharides (7, 8, 14).

In this report, we have identified and characterized an additional regulatory factor, NorG, a new member of the GntR (gluconate regulatory protein) family that regulates expression of the NorB and AbcA efflux pumps and affects resistance to both quinolones and ß-lactam antimicrobial agents. We have further identified AbcA as a transporter that can confer resistance to ß-lactams.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, plasmids, growth media, and other materials. Bacterial strains and plasmids used in this study are listed in Table 1. S. aureus strains were cultivated in brain heart infusion (BHI) broth (Difco, Sparks, MD) at 37°C unless otherwise stated. Escherichia coli strains were grown in Luria-Bertani (LB) medium. Lysostaphin was obtained from AMBI Products Inc., New York, NY; ciprofloxacin and moxifloxacin from Bayer Corp., Westhaven, CT; sparfloxacin from Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI; and 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole (Hoechst 33342), norfloxacin, ethidium bromide, cetrimide, tetracycline, TPP, rhodamine, nafcillin, methicillin, penicillin G, cefotaxime, and chloramphenicol from Sigma Chemical Co., St. Louis, MO. All primers used in this study were synthesized at the Tufts University Core Facility, Boston, MA.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
MIC determinations. MICs of quinolones, ß-lactams, TPP, rhodamine, ethidium bromide, cetrimide, and Hoechst 33342 were carried out by serial agar dilution on BHI agar. All plates were incubated at 30°C or 37°C for 24 h before reading. Determinations of MICs of quinolones, ß-lactams, and other chemical compounds for transformants containing pQT13 or pQT14 were done on BHI agar containing 5 µg tetracycline per ml to ensure maintenance of the plasmid, with incubation at 30°C.

RNA analysis. Total S. aureus RNA was prepared by extraction from lysostaphin-treated cells grown to exponential phase at 37°C or 30°C, using the RNeasy minikit (QIAGEN, Valencia, CA). The concentration of RNA was determined spectrophotometrically as the absorbance at 260 nm. For Northern blot analysis, 10 µg of total RNA was electrophoresed through a 0.9% agarose-0.66 M formaldehyde gel in morpholinepropanesulfonic acid (MOPS) and blotted onto Hybond-N+ membranes as previously described (26, 28). DNA probes were amplified from the ISP794 chromosome and labeled with psoralen for the detection of specific transcripts, using the Northern maxi kit (Ambion, Inc., Austin, TX) as recommended by the manufacturer. Blots were hybridized with probes overnight at 42°C, washed, and autoradiographed with Kodak X-Omat film. The reverse transcription-PCR (RT-PCR) analyses were performed using the SuperScript one-step RT-PCR kit (Invitrogen Inc.) with 10 picograms of total RNA as the template. Primers for norB (5'-GAAGATAGTTTCAATACAGA-3' and 5'-ATTATAAATGATAGGATGAA-3') generated a 370-bp amplicon. The running conditions were 1 cycle for 30 min at 45°C; 1 cycle for 2 min at 94°C; 30 cycles for 45 s at 94°C, 45 s at 48°C, and 30 s at 72°C; and 1 cycle for 10 min at 72°C. The 16S rRNA was used as an internal control to normalize the RT-PCR data as described previously (5, 27).

Cloning and overexpression of norG. To clone the norG gene, primers based on flanking sequences (NCTC8325, Oklahoma University) were synthesized by the Tufts University Core Facility (Boston, MA). A 1,321-bp fragment was amplified by PCR from S. aureus ISP794 chromosomal DNA with sense primer 5'-ATGGACAGCTGATGAAGATA-3') (the PstI site is underlined) and antisense primer 5'-CGAATTAGAATTCTTGTTTTAA-3' (the EcoRI site is underlined), which generated flanking PstI and EcoRI sites, respectively. The amplified norG gene was digested with PstI and EcoRI, ligated into the PstI and EcoRI sites of the plasmid pGEM3-zf(+) to yield pGEM3-zf(+)-norG, and introduced into E. coli DH5{alpha}. Plasmids extracted from ampicillin-resistant colonies were screened for the norG fragment insertion by restriction endonuclease digest patterns and confirmed by DNA sequencing.

To generate a plasmid for overexpression of norG in S. aureus, the norG gene was amplified by PCR from S. aureus ISP794 chromosomal DNA with sense primer 5'-ATGGAGGATCCATGAAGATA-3' (the BamHI site is underlined) and antisense primer 5'-CGAATTAGAATTCTTGTTTTAA-3' (the EcoRI site is underlined), which generated flanking BamHI and EcoRI sites, respectively. The amplified norG gene was digested with BamHI and EcoRI and ligated into the BamHI and EcoRI sites of the temperature-sensitive shuttle plasmid pSK950 to yield pQT13. This plasmid was then electroporated into S. aureus RN4220 (8325 r–) to generate transformants, and the structure of pQT13 in S. aureus was confirmed by restriction mapping. Electrocompetent ISP794 was then transformed with this plasmid isolated from RN4220. Tetracycline-resistant colonies isolated at 30°C were confirmed to have intact pQT13 by restriction mapping.

Construction of an abcA overexpressor. The abcA gene was amplified by PCR from S. aureus ISP794 chromosomal DNA with sense primer 5'-GGATCCTTAATCTGTTAATTTTTGA-3' (the BamHI site is underlined) and antisense primer 5'-GAATTCATGAAACGAGAAAATCCAT-3' (the EcoRI site is underlined). The amplified abcA gene was digested with BamHI and EcoRI and ligated into the BamHI and EcoRI sites of the temperature-sensitive shuttle plasmid pSK950 to yield pQT14. This plasmid was electroporated into S. aureus RN4220 (8325 r–), reextracted, and then introduced into ISP794 by electroporation. Tetracycline-resistant colonies isolated at 30°C were confirmed to have intact pQT14 by restriction mapping.

Construction of a norG mutant by allelic exchange. The 800-bp DNA fragment containing the cat gene was amplified from plasmid pLI50 using primers catpvu1 and catpvu2 (23, 28). The PCR product was digested with PvuII and then ligated into an EcoRV site within the putative norG coding region of plasmid pGEM-3zf(+)-norG. The resultant plasmid containing the 2.1-kb norG::cat was subcloned into the temperature-sensitive shuttle plasmid pCL52.2 to yield pCL52.2-(norG::cat). The allelic exchange procedure was then carried out as described previously(28). pCL52.2-(norG::cat) was first introduced into RN4220 by electroporation, and chloramphenicol-resistant (5 µg/ml) colonies of RN4220 were grown at 30°C in the presence of 5 µg/ml tetracycline and used for reisolation of pCL52.2-(norG::cat), which was then electroporated into ISP794. ISP794 harboring pCL52.2-(norG::cat) was grown in BHI broth with tetracycline (3 µg/ml) at 30°C, diluted 1:1,000 in fresh medium, and propagated at 42°C for 24 h. The culture was diluted and grown again at 30°C without selection for 48 h. Chloramphenicol-resistant, tetracycline-sensitive colonies, representing possible double-crossover events, were tested for cat insertion into norG by PCR and sequencing. To construct the mgrA::cat norG::cat double mutant, we carried out a second allelic exchange using the same plasmid construct pCL52.2-(norG::cat) and QT1 (mgrA::cat) as the recipient. Since QT1 already had one chromosomal copy of the cat gene, we increased the chloramphenicol concentration to 10 µg/ml for the selection of the double mutant. DNA sequencing was performed to confirm the presence of the chromosomal insertion of the norG:: cat and mgrA::cat genes.

DNA mobility shift analysis. Primers designed to amplify the putative promoter regions of norA, norB, norC, norG, abcA, pbpD, tet38, and mgrA are listed in Table 2. One of the primers was biotinylated at the Tufts University Core Facility (Boston, MA). The gel mobility shift assay was carried out using the LightShift chemiluminescent EMSA kit (Pierce, Rockford, IL), as recommended by the manufacturer. The biotin-labeled DNA was incubated with the indicated amount of cell extract or purified proteins from S. aureus in 20 µl of binding buffer (10 mM HEPES [pH 8], 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 0.1 mg/ml of bovine serum albumin, 0.25 mM dithiothreitol) containing 1 µg of poly(dI-dC), 200 ng of sheared herring sperm DNA, and 10% glycerol. The reaction mixture was incubated for 20 min at room temperature and analyzed by 5% nondenaturing polyacrylamide gel electrophoresis (PAGE). For the competition assays, a 100-fold excess of specific or nonspecific unlabeled DNA was added to the reaction mixture prior to the incubation.


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  		TABLE 2. Primers used to amplify putative promoter regions

 
Identification of the NorG protein from cell extracts. Cell extracts collected from 1 liter of overnight culture of S. aureus QT1 were used to purify the NorG protein, as previously described for purification of MgrA protein (28). The 150-bp biotinylated norA DNA fragment was immobilized on magnetic beads with covalently coupled streptavidin (Dynabeads M-280; Dynal) according to the manufacturer's protocol. DNA bound to beads was incubated with protein extract in binding buffer containing herring sperm DNA (200 ng) for 20 min at room temperature. Beads were washed twice with binding buffer containing herring DNA and twice with binding buffer without DNA. Proteins were then eluted in binding buffer containing 0.5 M NaCl. Eluted proteins were dialyzed against water, concentrated, and separated by sodium dodecyl sulfate (SDS)-PAGE. The 47-kDa protein was blotted onto a polyvinylidene difluoride membrane for N-terminal amino acid sequencing by the Edman degradation method (Tufts Core Facility, Boston, MA).

Purification of NorG protein. The norG gene was subcloned into the plasmid pTrcHisA (Invitrogen, Carlsbad, CA) to yield pTrcHisA-norG and then introduced into E. coli BL21. The purification of histidine-tagged NorG was carried out as recommended by the manufacturer. E. coli BL21 harboring pTrcHisA-norG was grown to mid-log phase in LB medium, at which time IPTG (isopropyl-ß-D-thiogalactopyranoside) (1 mM) was added to the culture. After 3 h, the cells were harvested by centrifugation and then resuspended in 20 mM sodium phosphate buffer, pH 7.4. The cells were lysed with lysozyme (0.02%) and then centrifuged (100,000 x g) for 90 min. The supernatant was applied to a nickel affinity column (iminodiacetic acid-Sepharose-Ni) (Amersham Pharmacia Biotech, Uppsala, Sweden) and then washed with buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5% glycerol) supplemented with concentrations of imidazole increasing from 10 to 60 mM. NorG protein was eluted with 100 mM imidazole. The homogeneity of the eluted protein was verified by SDS-PAGE.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of norG. We determined the presence of a protein in addition to MgrA that binds to the norA gene promoter, by the pattern of gel shift of a 150-bp DNA fragment containing the norA promoter DNA, with and without incubation with cell extracts of strain QT1 (mgrA), which lacks MgrA. Using magnetic beads coupled to the 150-bp DNA fragment containing the norA promoter as an affinity agent, we incubated cell extracts of QT1 and eluted a single protein that was bound, in a manner similar to that that led to the isolation of MgrA from wild-type cell extracts (28). After separation by SDS-PAGE, the protein band was transferred to a polyvinylidene difluoride membrane, and N-terminal amino acid sequencing identified the sequence KIPPQRQLATQY, which matched with that encoded by a 1,321-bp open reading frame (ORF) designated SA0104 in the genome of S. aureus N315. This ORF is predicted to encode a protein of the FadR subfamily of the GntR-like family of regulators. Based on its role in regulation of resistance to quinolones and ß-lactams as outlined below, we named this ORF norG (Fig. 1).


Figure 1
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  		FIG. 1. Nucleotide and amino acid sequences of the 1,321 bp of S. aureus DNA containing the norG gene from ISP794 (complete sequence shown). The –35 and –10 sequences of the putative promoter region and the putative helix-turn-helix (H-T-H) region are shown. The coding region of norG, preceded by a putative ribosome-binding site (RBS), is demarcated by the ATG start codon and the TAA stop codon.

 
NorG binds to efflux pump promoters. We cloned the norG gene into plasmid pTrcHisA, a His tag expression vector. After induction with IPTG (1 mM) and purification using nickel affinity chromatography, we isolated a protein of ~50 kDa (47 kDa predicted for native NorG plus 3 kDa for the His tag region) to ~95% apparent homogeneity by SDS-PAGE (data not shown). The purified protein was then used to perform gel shift assays of DNA fragments containing the putative promoters of several genes known to encode efflux pumps.

After incubation of NorG with the 150-bp DNA fragment containing the norA promoter, a clear shift was shown in the DNA mobility pattern on agarose gels, a shift that was abolished in the presence of a 100-fold excess of unlabeled norA promoter DNA but not with a 100-fold excess of herring DNA. These data indicated that NorG bound specifically to the norA promoter fragment, as expected based on the affinity purification procedure used for its identification (Fig. 2A).


Figure 2
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  		FIG. 2. (A) Gel mobility shift analyses of the interactions of the crude cell extracts (CE) from ISP794 and QT1 and the purified NorG protein with the biotinylated 150-bp norA promoter fragment. Competing unlabeled herring sperm DNA (nonspecific) and norA promoter DNA (specific) were used to determine the specificity of promoter binding. The amount of labeled DNA used was 2 ng per reaction. The amount of protein used was 50 ng per reaction for the NorG protein and 100 ng per reaction for the crude cell extracts. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and the purified NorG protein with the biotinylated 150-bp norB P1 and P2 promoter fragments. (C) Schematic representation of the positions of norB and the three adjacent ORFs on the S. aureus N315 published genome (11). The two putative promoters and the putative rho-dependent terminator are indicated.

 
Similar experiments were carried out using DNA fragments containing the putative promoters of norB, norC, and tet38, which encode efflux pumps, as well as the putative promoter of norG itself. In addition, the two identified promoters of mgrA were amplified together on a 200-bp fragment (8). As previously reported, norB may have two promoters, P1 and P2, with P2 located upstream of the ORF SA1272, encoding a putative alanine dehydrogenase, and P1 directly upstream of norB itself (26) (Fig. 2C).

Purified NorG-mediated DNA fragment shifts were found with both the P1 and P2 putative promoters of norB, shown as separate DNA fragments in gel shift assays (Table 2 and Fig. 2B and C), since P1 and P2 were separated by three ORFs totaling approximately 5 kb. Similar promoter DNA band shift patterns were found associated with the promoters of norC and norG, but no change in DNA fragment mobility was detected with the promoters of tet38 and mgrA. Interestingly, purified MgrA protein also caused a shift in mobility of the putative norG promoter fragment, suggesting that MgrA could affect expression of norG (Fig. 3A). The DNA fragment shifts were each shown to be specific by competition experiments using 100-fold excesses of specific and nonspecific unlabeled DNAs (data not shown), as was done to establish the specificity of the interaction of NorG with the norA promoter.


Figure 3
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  		FIG. 3. (A) Gel mobility shift analyses of the interactions of the purified NorG and MgrA proteins with the biotinylated promoters of mgrA, norG, and norC. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and QT1 and the purified NorG and MgrA proteins with the biotinylated 150-bp abcA promoter. (C) Schematic representation of the overlapping promoter region abcA-pbpD as published by Domanski et al. (4). Primers designed to amplify abcA DNA generated a fragment that included the inverted repeat located 8 bp downstream from the abcA transcriptional start (+1). This region was shown to be essential for the expression of both genes. The –35 and –10 consensus sequences of the abcA and pbpD promoters are underlined and/or in bold. The inverted repeat region is underlined and in bold. The asterisk indicates the transcription start site for abcA. The boxed DNA region indicates the abcA promoter that was used in the gel mobility shift binding assay. The pbpD promoter region used for the gel mobility shift assay contains the inverted repeat as well as the –10 and –35 regions (underlined). The inverted repeat is located at a distance of 46 bp from the transcription start site of the pbpD gene (T).

 
The abcA gene, which encodes a putative ABC transporter, is transcribed divergently from pbpD, the structural gene encoding the penicillin-binding protein PBP4. The published overlapping promoter region abcA-pbpD as well as the specific regions harboring either abcA or pbpD promoters were amplified and labeled with biotin and used in the gel mobility shift assays. The inverted repeat that was shown previously to affect the expression of abcA and pbpD was present in both DNA fragments (4) (Table 2 and Fig. 3C). NorG bound to the abcA promoter and the overlapping region, but no binding to the putative pbpD promoter was detected. MgrA also bound to the abcA promoter and the overlapping region but not to the pbpD promoter (Fig. 3B). We performed the competition experiments using 100-fold excesses of specific and nonspecific unlabeled DNAs, which demonstrated the specificity of the interaction of NorG and MgrA with the abcA promoter (data not shown).

Phenotype of norG mutants. We constructed a norG knockout mutant, QT11, by allelic exchange of a disrupted copy of norG::cat for the wild-type copy of norG on the chromosome of strain ISP794 (28). We also constructed a norG mgrA double mutant, QT12, using the same allelic exchange procedure with strain QT1 (mgrA::cat). QT12 carried two copies of the cat gene on its chromosome and was able to be constructed by its ability to grow in the presence of 10 µg chloramphenicol per ml, a concentration at which neither QT1 nor QT11 grew. The growth curves of the two mutants QT11 and QT12 are similar to that of the wild-type ISP794 (data not shown). We determined the MICs of quinolones, ß-lactams, and dyes for ISP794, QT1, QT11, and QT12. The MICs of the quinolones (norfloxacin, ciprofloxacin, moxifloxacin, and sparfloxacin) showed no change for mutant QT11 (norG::cat) relative to its parent strain. Mutant QT12 (norG::cat mgrA::cat), however, showed an increase of fourfold for the four quinolones tested compared to those of the wild-type ISP794 but twofold less than those of the single mutant QT1 (mgrA). Thus, intact norG is needed for the full effect of mutation in mgrA on resistance to quinolones.

We also determined the MICs of four ß-lactams (nafcillin, penicillin G, methicillin, and cefotaxime) and five dyes (cetrimide, TPP, rhodamine, ethidium bromide, and Hoechst 33342) for all strains. Interestingly, QT11 (norG) and QT12 (norG mgrA), respectively, showed fourfold and twofold increases in the MICs of the four ß-lactams, while QT1 (mgrA) was twofold more sensitive to these ß-lactams than the wild-type strain ISP794. Both QT11 and QT12 showed increases in MICs of dyes, but QT12 was twofold more resistant than QT11. The MIC data are summarized in Table 3.


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  		TABLE 3. Susceptibilities of strains to quinolones and other agents

 
Alterations in gene expression in norG mutants. We carried out Northern blotting using RNAs extracted from ISP794, QT1, QT11, and QT12, which were hybridized with biotin-labeled probes generated from 200-bp internal regions of either abcA or pbpD (encoding PBP4). The level of abcA transcripts from QT11 was at least threefold greater than that from QT12 and was almost undetectable for ISP794 and QT1 (Fig. 4A). In contrast, no increases in the level of transcription of pbpD in QT11 and QT12 were detected (data not shown). In order to confirm these findings, RT-PCRs were carried out using the same primers designed for amplification of the two probe DNAs used for Northern hybridizations. We found a similar increase (threefold) in the intensity of the abcA amplicon for QT11 compared to that of QT12, confirming the results of the Northern blot assays (data not shown).


Figure 4
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  		FIG. 4. (A) Northern blot analyses of RNAs isolated from the specified S. aureus strains. The same amount of RNA (10 µg) was loaded in each lane, and loading was verified by ethidium bromide staining of 16S rRNA before RNAs were transferred onto a nylon membrane and hybridized with specific probes (abcA or norG). (B) RT-PCR using RNA extracted at exponential phase. Each reaction used 10 picograms of total RNA as the template and primers specific to an internal region of norB. We used 16S rRNA as an internal control for the RT-PCR assays as described previously (27). Photographs of ethidium bromide-stained gels were scanned and analyzed using the NIH Scion Image program as described previously (5).

 
Because the low copy number of the norA, norB, and norC transcripts in the wild-type strain ISP794 made it difficult to perform Northern blotting, we performed RT-PCR assays to detect whether any differences occurred in transcript levels between strains. The 16S rRNA was used as an internal control as described previously (5, 27). RT-PCRs were carried out for norA, norB, and norC in ISP794 (parent strain), QT11, and QT12. An increase of twofold was found associated with norB and norC amplicons in QT12 (norG mgrA), an increase that was similar to that with the mgrA mutation alone (26, 27). In contrast, there was no change in the levels of these transcripts for QT11 (norG) relative to those for the wild-type strain. No change in levels of norA mRNA was detected in either mutant (data not shown).

Alterations in gene expression and phenotype by overexpression of norG. We cloned the 1,321-bp norG gene into plasmid pSK950 to generate pQT13. This plasmid was introduced first into RN4220 and then into ISP794 to study the effects of overproduction of norG. As expected, in Northern blots there was an increase (threefold) in the level of norG transcripts in ISP794(pQT13) compared to ISP794 (Fig. 4A). In complementation experiments pQT13 was introduced into mutant QT11 (norG::cat), resulting in an increase of norG mRNA and a decrease in abcA mRNA. RT-PCR assays using specific primers of an internal region of 200 bp of the norA, norB, and norC genes showed a 2.5-fold increase in norB transcript levels in ISP794(pQT13) compared to ISP794 (Fig. 4B). In contrast, no differences in the levels of norA and norC transcripts were detected between the two strains.

We then determined the MICs of quinolones, ß-lactams, and dyes for ISP794 and ISP794(pQT13). The MICs of norfloxacin, ciprofloxacin, moxifloxacin, and sparfloxacin showed an increase of fourfold for ISP794(pQT13), while no change occurred for the other drugs tested (Table 3). The transformant QT11(pQT13) showed a MIC profile for quinolones, ß-lactams, and dyes that was identical to that of ISP794(pQT13) and increased for quinolones relative to ISP794. The presence of norG overexpression from the plasmid also did not affect the growth rate (data not shown).

Overexpression of abcA causes increased resistance to ß-lactams. Susceptibility to ß-lactams was not affected by expression of the genes encoding NorA, NorB, NorC, and Tet38. To assess whether the overexpression of abcA seen in QT11 (norG) contributed to the ß-lactam resistance phenotype of this strain, we cloned the 1,728-bp abcA gene into the plasmid pSK950 to generate plasmid pQT14. This plasmid was introduced into RN4220 and then into the parental strain ISP794. We assessed the overexpression of abcA from the plasmid construct pQT14 by Northern blotting. There was an increase of fourfold for abcA mRNA of ISP794(pQT14) compared to that of ISP794 or QT11, with or without pQT13 (Fig. 4A). We then determined the MICs of quinolones, ß-lactams, and dyes for ISP794 and ISP794(pQT14). ISP794(pQT14), relative to plasmid-free ISP794, showed increases in MICs of 128-fold for nafcillin, 64-fold for penicillin, and 8-fold for methicillin and cefotaxime, with the magnitude of the increase correlating with the hydrophobicity of the ß-lactam side chains (Table 3). ISP794(pQT14) also showed increases in MICs of eightfold for TPP, fourfold for rhodamine, and twofold for ethidium bromide and the Hoechst 33342 dye. No change in the MICs of cetrimide or quinolones was detected. The presence of abcA overexpression from the plasmid also did not affect the bacterial growth rate (data not shown). Thus, in the norG mutant, ß-lactam resistance is attributable at least in part to overexpression of abcA.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NorG directly activates the expression of norB, encoding the NorB efflux pump. NorG belongs to the GntR family of regulators. GntR is itself a repressor of the gluconate operon in Bacillus subtilis. GntR-like regulators possess a helix-turn-helix region, an N-terminal DNA-binding domain, and a highly variable C-terminal domain, which contains the effector-binding sites and an oligomerization region. Based on the heterogeneity of the C-terminal regions of this family, NorG was classified in the FadR subfamily, which consists of proteins with an all-helical C-terminal domain, often involved in the regulation of various metabolic pathways or oxidized substrates in amino acid metabolism (21). NorG protein bound specifically to norA, norB, and norC promoters, but the transcription level of only norB was increased when norG was overexpressed. These data correlated with a fourfold increase in MICs of moxifloxacin and sparfloxacin for the strain overexpressing norG from plasmid pQT13 and are consistent with the previously reported substrate profile of the NorB efflux pump (26).

As reported in our previous study, upstream of norB are three ORFs, encoding a putative amino acid permease (N315-SA1270), a putative threonine deaminase (N315-SA1271), and a putative alanine dehydrogenase (N315-SA1272), all of which showed at least a threefold increase in their transcript levels in microarrays of QT1 (mgrA) compared to those of parental ISP794 (26). The intergenic region between SA1272 and SA1273 contained a putative rho-dependent terminator and a putative promoter, P2, in addition to the putative promoter P1 directly upstream of the norB gene. NorG bound specifically to both putative norB promoters.

The pattern of DNA gel band shifts showed that MgrA bound specifically to the norG promoter and bound less strongly to the norB promoter (26). In contrast, NorG bound specifically to the norB promoter but not to the mgrA promoter. These data taken together with our earlier microarray data showing a threefold increase in the transcripts of norG and norB in QT1 (mgrA) suggested that NorG regulates amino acid metabolism via expression of ORFs SA1270 to SA1272 coordinately with the expression of the NorB transporter, which transports small hydrophobic molecules, such as moxifloxacin and sparfloxacin, across the cytoplasmic membrane. The exact role of NorB, if any, in amino acid metabolism or transport is not known, but its coordinated expression with that of components of certain metabolic pathways may reflect a response to environmental conditions in which removal of toxins by efflux and changes in amino acid metabolism are both advantageous to cell survival.

We postulate that MgrA acts as an indirect repressor of norB via repression of the expression of NorG, which acts as a direct activator of transcription of norB. A similar regulatory system has been demonstrated for the farAB-encoded efflux pump in Neisseria gonorrhoeae, which confers resistance to antibacterial fatty acids. The farAB operon was indirectly activated by the MtrR regulator, which is a direct repressor of FarR, a repressor of the farAB system (12). The role of NorG in regulation of expression of norA- and norC-encoded efflux pumps remains to be determined. The ability of the protein to bind to both promoters without an apparent effect on gene transcript levels in vivo suggests participation of other proteins or other pathways in this regulation. This hypothesis is supported by our finding that disruption of the norG gene did not lead to any detectable effects on the transcription of norA or norC in mutant QT11 (norG).

NorG represses the expression of genes involved in ß-lactam susceptibility and resistance. The norG::cat mutant had no change in quinolone resistance phenotype, but this mutant showed a fourfold increase in the MICs of ß-lactam drugs, including nafcillin, penicillin G, methicillin, and cefotaxime. By Northern blotting and RT-PCR assays, we found that the transcription level of abcA increased fourfold in this mutant. AbcA is an ATP-dependent transporter, involved in cell autolysis, and its transcription is dependent on the agr regulatory system (3, 24). pbpD is the structural gene encoding the transpeptidase PBP4 of S. aureus, a native low-molecular-weight penicillin-binding protein that participates in the synthesis of highly cross-linked muropeptide components of the cell wall (13). Although abcA and pbpD are divergently transcribed, they share an intergenic region with overlapping promoters. An inverted repeat region of 26 bp that plays an important role in the expression of these two genes was found 8 bp from the transcription initiation point (+1) of abcA, while it was at a distance of 46 bp from the transcription initiation point of pbpD (4). Schrader-Fischer and Berger-Bachi (24) found no connection between resistance to methicillin and AbcA expression in their studies, but the cloned abcA structural gene in those experiments lacked approximately 4% of its full sequence. Exposure of cells to methicillin, however, led to an increase in expression of abcA transcripts (24). We found an increase in abcA transcripts without a change in pbpD transcripts in the norG mutant, strengthening the earlier suggestion that abcA and pbpD are regulated differently (6). Consistent with this notion, NorG bound to the putative abcA promoter, including the inverted repeat region, in a specific manner, but no binding occurred in the DNA region bearing the putative pbpD promoter (data not shown).

AbcA and resistance to ß-lactams. Cloning and overexpression of the complete abcA gene on plasmid pQT14 resulted in increased ß-lactam resistance. Thus, it appears that ß-lactams may be substrates of AbcA and that the ß-lactam resistance phenotype of a norG mutant can be attributed at least in part to overexpression of abcA.

AbcA is classified in the group A family of ATP-dependent transporters, whose members have two membrane-spanning domains and an ATP-binding domain in a single polypeptide. Exporting substrates is the principal function of this family (24). AbcA shows similarity with efflux transporters LmrA of Lactococcus lactis (65% amino acid similarity) and MsbA of Escherichia coli (57% amino acid similarity). The substrate profiles of these two transporters both include the dyes ethidium bromide and Hoechst 33342 (20). Overexpression of abcA from pQT14 also produced increases in the MICs of the dyes, TPP, rhodamine, ethidium bromide, and Hoechst 33342. In E. coli, MsbA is an essential transporter involved in the trafficking of lipids, including lipid A. Since the targets of ß-lactams are extracellular transpeptidases, a role of multidrug transporters located in the cytoplasmic membrane in ß-lactam resistance might be unexpected. LmrA in L. lactis, however, has been previously shown to confer resistance to lipophilic but not hydrophilic ß-lactam antibiotics, suggesting a link between the ability of the antibiotics to partition into the cytoplasmic membrane and LmrA-mediated drug resistance (29). Similarly, the multidrug efflux pump AcrAB of Salmonella enterica serovar Typhimurium confers resistance to ß-lactams with more lipophilic side chains, possibly due to side chain partitioning in the membrane (16). The observation that AbcA confers resistance to ß-lactam drugs with a preference toward the more lipophilic ones such as nafcillin or penicillin G supports this hypothesis. Further study of the transport properties of AbcA is ongoing.

AbcA was previously shown to be involved in cell autolysis and is under the control of the agr regulatory system (24). In our early microarray experiments leading to the identification of the NorB, NorC, and Tet38 efflux pumps (26, 27), we also observed a fivefold increase in abcA mRNA in a strain overexpressing mgrA compared to that of the wild-type ISP794 and a specific binding of MgrA to the overlapping promoter abcA-pbpD (data not shown), suggesting that MgrA is a direct activator for the expression of abcA. In this study, we demonstrated that abcA was also under the control of NorG and also affects resistance to ß-lactams. Taken together, our data suggest that AbcA is oppositely regulated by MgrA and NorG.

Conclusions. Multiple regulators affect the expression of a variety of efflux pumps that alter antimicrobial susceptibility in S. aureus. Thus far two regulators, NorG and MgrA, have been shown to bind the norA promoter, a property that led to their identification (28). In contrast to MgrA, NorG binds more strongly to the putative norB promoters P1 and P2, as well as to the norC putative promoter. NorG also binds specifically to its own putative promoter and the promoter of abcA. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA behaves as an indirect repressor for norB and a direct activator for abcA, whereas NorG behaves as a direct activator for norB and a direct repressor for abcA. The multiplicity of staphylococcal efflux pumps and the complexity of their regulation imply that such pumps are highly important to the physiology of S. aureus and likely contribute to its ability to survive in diverse environments, an ability that underlies its facility in colonizing, persisting in, and causing disease in mammalian hosts.


   ACKNOWLEDGMENTS
 
This work was supported in part by grant R01-AI23988 from the U.S. Public Health Service, National Institutes of Health (to D.C.H.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, Massachusetts General Hospital, 55 Fruit Street, Boston MA 02114-2696. Phone: (617) 726-3812. Fax: (617) 726-7416. E-mail: dhooper{at}partners.org Back

{triangledown} Published ahead of print on 2 February 2007. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, April 2007, p. 2996-3005, Vol. 189, No. 8
0021-9193/07/$08.00+0     doi:10.1128/JB.01819-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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