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Antimicrobial Agents and Chemotherapy, March 2006, p. 1104-1107, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.1104-1107.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

NorC, a New Efflux Pump Regulated by MgrA of Staphylococcus aureus

Que Chi Truong-Bolduc, Jacob Strahilevitz, and David C. Hooper*

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

Received 24 October 2005/ Returned for modification 28 November 2005/ Accepted 4 January 2006


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ABSTRACT
 
NorC, a new efflux pump, like NorB, contributes to quinolone resistance that includes resistance to moxifloxacin and sparfloxacin in Staphylococcus aureus. norC expression, like that of norB and tet38, is negatively regulated by MgrA, and overexpression of both norC and norB contributes to the quinolone resistance phenotype of an mgrA mutant.


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TEXT
 
NorA, NorB, and Tet38 are previously described efflux transporters of the major facilitator superfamily in Staphylococcus aureus that are under the control of MgrA, a global regulator that also affects diverse virulence factors (5, 7, 11, 12). MgrA acts as a negative regulator of NorB, and NorB overexpression in an mgrA mutant contributes to low-level quinolone resistance but does not fully account for the quinolone resistance phenotype of the mgrA mutant. We report here the identification of an additional chromosomally encoded multiple-drug resistance efflux pump, termed NorC, which is also negatively regulated by mgrA and which in addition to NorB contributes to quinolone resistance in an mgrA mutant.

S. aureus cells (Table 1) were grown in brain heart infusion (BHI) broth, and Escherichia coli cells were grown in Luria-Bertani (LB) broth. norC and cat genes were amplified by PCR, using primers containing BamHI and EcoRI for norC and PvuII for cat. The conditions were as follows: 1 cycle for 3 min at 94°C; 30 cycles for 45 s at 94°C, 1 min at 48°C, and 1 min at 72°C; and 1 cycle for 10 min at 72°C. norC was cloned into pGEM3-zf(+) and then subcloned into pSK950 to generate pQT11. pGEM3-zf(+)-norC was digested with MscI and ligated to the PvuII-digested cat gene, generating construct pGEM3-zf(+)-norC::cat. norC::cat was subcloned into pCL52.2, generating pQT12. Allelic exchange was carried out as previously described to generate mutant QT9 (norC::cat) (12). To create a norC mgrA double mutant, we transferred norC::cat from QT9 into QT1 using bacteriophage {varphi}85 as described previously (11).


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  		TABLE 1. Bacterial strains and plasmids used in this study

MICs were determined on BHI agar supplemented with serial twofold drug dilutions. Transformants containing pSK950, pQT8, and pQT11 were plated on BHI with 5 µg/ml tetracycline and incubated at 30°C.

Primers amplifying a 400-bp amplicon of SA0098 (5'-GTAGAAACGAATGTCGGACCAC-3' and 5'-AATGGCATC ATTGGCCATA-3') and a 200-bp amplicon of norC (5'-AAA TGGTTCTAAGCGACCAA-3' and 5'-ATAAATACCTGA AGCAACGCCAAC-3') were synthesized. Reverse transcriptase PCR (RT-PCR) used SA0098 as standard RNA and norC as target RNA (4). SA0098 was cloned into pSK950 and then introduced into mutant QT9 (norC::cat). RNA from a transformant (0.01 to 0.20 µg) was added to the RT-PCR mix. ISP794 and QT1 RNA amounts were 0.15 µg for each reaction. Conditions were as follows: 1 cycle for 30 min at 45°C; 1 cycle for 2 min at 94°C; 28 cycles for 45 s at 94°C, 45 s at 48°C, and 30 s at 72°C; and 1 cycle at 10 min for 72°C. Photographs of ethidium bromide-stained gels were scanned and analyzed using the NIH Scion Image program (version 6.1), as described previously (2).

Primers for the norC promoter (5'-GCAGCTGTGGTACCAGATGGTGA-3' and 5'-ACTGCAGTTTCATTCATGTTAGTT A-3') containing KpnI and PstI were synthesized (restriction sites underlined). Conditions were as follows: 1 cycle for 3 min at 94°C; 30 cycles for 45 s at 94°C, 45 s at 48°C, and 20 s at 72°C; and 1 cycle for 10 min at 72°C. The product was digested with KpnI and PstI and cloned into pWN2018, generating pWN2018-PnorC. Cells containing pWN2018-PnorC were grown in Trypticase soy broth at 37°C to an optical density at 600 nm of 0.9. The assays used nitrocefin as the substrate as described previously (1, 2). The activities were expressed in micromoles of nitrocefin hydrolyzed per hour per gram of cell protein.

Because overexpression of norB in mgrA mutant QT1 only partially explained the quinolone resistance phenotype of QT1 (11), we analyzed further microarray data comparing QT1 and its parent strain ISP794 and identified another putative transporter gene, annotated as open reading frame (ORF) SA0099 in the S. aureus N315 genome. This ORF showed a 2.9-fold increase in mRNA in QT1. ORF SA0099 is predicted to encode a protein identical to S. aureus SbtA, which is listed in GenBank (S. Sinjee and L. J. V. Piddock). The predicted protein, which we have named NorC, had 61% amino acid identity with NorB. To confirm the increased expression of norC, we performed noncompetitive RT-PCR under conditions for which no competition occurred between the target and the standard, and the output signals of the amplification products were plotted versus the amount of RNA. norC RNA levels in QT1 were increased 2.75-fold relative to those in ISP794 (Fig. 1). Northern hybridization also showed a similar increase (data not shown).


Figure 1
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  		FIG. 1. (A) Noncompetitive quantitative RT-PCR using ORF SA0098 RNA as standard RNA and norC mRNA as target RNA. The output signals of the two amplification products were plotted versus the amount of standard RNA. (B) Graphic representation of the quantitative RT-PCR. The target signal (norC amplicon) remains constant, and the standard curve (SA0098 amplicon) is linear. The intersection point between the two lines is the equivalence point and indicates the arbitrary value for the norC amplicons.

Upstream of norC is ORF SA0098 (1,179 bp), which is predicted to encode a putative aminoacylase/carboxypeptidase. Using the Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html), we found a putative norC promoter, PnorC, which overlapped the end of SA0098. To assess the role of this promoter in the expression of norC, we constructed pWN2018-PnorC (norC promoter-blaZ fusion) and introduced it into ISP794 and QT1 mgrA. We then compared the ß-lactamase expression from this construct with that from pBF8-30, a norA promoter-blaZ fusion (1), measuring the initial linear rate of nitrocefin color development from 0 to 5 min. In the parent strain ISP794, there was little difference in ß-lactamase expression levels for norC and norA promoter fusions. In QT1, in contrast, a 3.7-fold increase in expression was observed for the norC promoter, with little change in the expression level for the norA promoter relative to expression in ISP794. Thus, PnorC has promoter activity that increases in an mgrA mutant background. Although incubation of a 200-bp DNA fragment upstream of norC containing PnorC with crude cell extracts of ISP794 and QT1 exhibited differing patterns of DNA mobility shift, no mobility shift was seen with 2 µg purified histidine-tagged MgrA protein, which causes a mobility shift of norA promoter DNA (data not shown) (3, 11, 12). Thus, heterologously expressed MgrA appears not to interact directly with PnorC, suggesting that other cellular factors or modified MgrA is the direct regulator of norC expression.

To assess the effect of norC overexpression on resistance to quinolones, plasmid pQT11 with cloned norC and control plasmid pSK950 were introduced separately into ISP794. Relative to ISP794(pSK950), ISP794(pQT11) showed fourfold increases in MICs of norfloxacin, garenoxacin, and moxifloxacin; twofold increases in MICs of ciprofloxacin, sparfloxacin, and premafloxacin; and no change in the MIC of gemifloxacin. Increases in MICs were inhibited by reserpine (Table 2).


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  		TABLE 2. Activity of quinolones against norC and mgrA strains and other mutant strains

In norC knockout mutant QT9 derived from ISP794, there was no change in the MICs of most quinolones, except for a twofold decrease in MICs of sparfloxacin and moxifloxacin relative to ISP794. In contrast, the norC mgrA double mutant QT10 exhibited increased susceptibility to all quinolones tested relative to the mgrA mutant and to within twofold of that of ISP794. Transformants QT9(pQT11) and QT10(pQT11) showed increases in quinolone resistance to levels the same as those for ISP794(pQT11) and QT1, respectively (Table 2). Thus, norC contributes to the resistance phenotype of an mgrA mutant.

NorC represents a third multiple-drug resistance efflux pump, in addition to NorA and NorB, that can cause low-level quinolone resistance when overexpressed. Expression of norC, also like that of norA and norB, is regulated by mgrA. MgrA appears to function as a negative regulator of norC, as it does for norB and tet38, which encodes tetracycline resistance (11). Overexpression of mgrA from a plasmid, in contrast, acts positively on expression of norA in the ISP794 genetic background (12). Thus, MgrA plays a central role in modulating expression of at least four genes encoding efflux pumps and in modulating resistance to quinolones and tetracycline (11, 12).

The phenotypes of NorC-overexpressing strains and mutant QT9 (norC::cat) establish a role for NorC in low-level reduced susceptibility to sparfloxacin and moxifloxacin in S. aureus. NorB overexpression also causes low-level resistance to sparfloxacin and moxifloxacin, but NorB is apparently not expressed in the wild-type strain to a level sufficient to affect susceptibility to these agents, since the susceptibility of mutant QT5 (norB::cat) did not differ from that of its wild-type parent (11).

The resistance profiles of the two double mutants, QT6 (mgrA norB) and QT10 (mgrA norC), further suggest that NorB and NorC efflux pumps act in concert to generate the quinolone resistance phenotype when MgrA is inactivated. Thus, MgrA acts to coordinately regulate the expression of at least four efflux pumps in S. aureus. Although it appears to act directly on the norA promoter (12), the effects of MgrA on other promoters, including PnorC, appear to be indirect (11), indicating that regulatory elements in addition to MgrA are important for controlling expression of several efflux pumps in S. aureus.


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


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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


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Antimicrobial Agents and Chemotherapy, March 2006, p. 1104-1107, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.1104-1107.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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