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Program in Molecular Pathogenesis, Skirball Institute, and Departments of Microbiology and Medicine, New York University Medical Center, New York 10016, USA
Correspondence
Richard P. Novick
novick{at}saturn.med.nyu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Subsequent analysis of the svrA mutant, strain P6C63, has revealed an approximately 103-fold attenuation in virulence coupled with a phenotype typical of a strain that does not express agr (Garvis et al., 2002). Such strains fail to produce many of the typical staphylococcal exoproteins, including 
- and 
-toxins, and produce reduced amounts of 
-toxin in comparison with typical agr+ strains. Additionally, they produce sharply increased amounts of protein A (for a review, see Novick, 2003). Consistent with this phenotype, agr transcripts are undetectable in the svrA mutant. These results suggest that svrA is a required upstream activator of agr, and that the lack of agr expression is responsible for the observed attenuation of virulence (Garvis et al., 2002).
SvrA is a large, complex protein predicted to have 12 membrane-spanning domains and is likely localized to the bacterial plasma membrane. In addition to its reported role in virulence, svrA has more recently been determined to be a member of the multi-drug export family and has been alternatively designated mepA (Kaatz et al., 2005; McAleese et al., 2005). Since the svrA phenotype has major implications for the regulation of staphylococcal virulence and does not mirror the properties of any regulatory gene identified to date, we initiated a series of studies in an attempt to define the mechanism by which svrA controls agr function and thereby impacts virulence. In this study, we describe our observations on svrA and report that, unexpectedly, the Tn917 insertion in svrA is not responsible for the agr-defective phenotype of strain P6C63, and that an adventitious secondary mutation in agrC is, in fact, responsible.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. S. aureus strains were grown in CYGP (Novick, 1991), and on GL (Novick, 1991) or commercial (BBL) sheep blood agar (SBA) plates. Escherichia coli strains were grown in Luria–Bertani (LB) broth or on LB agar plates. Antibiotic-resistant S. aureus were selected and maintained on 5 or 10 µg erythromycin ml–1, 5 or 10 µg chloramphenicol ml–1, or 0.1 mM cadmium chloride. Antibiotic-resistant E. coli were selected and maintained on 100 µg ampicillin ml–1.
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). Plasmid DNA was prepared with a kit from Qiagen. PCR was performed with an MJ Research Peltier thermal cycler using Vent polymerase (New England Biolabs).
Transduction.
Staphylococcal phage 80 was used to produce phage lysates for transduction, as described by Novick (1991).
Construction of plasmids.
Plasmid pJC1125 is a derivative of pJC1079 with the temperature-sensitive pT181 repC3 replicon replacing the pT181 repC4.
Plasmid pJC1202 allelic-exchange vector was constructed by a three-way ligation of the ApaI–XhoI fragment (Pbla promoter) of pJC1200 and the XhoI–AvrII (rpsL+) fragment of pJC1201 into the ApaI and AvrII sites of pJC1125. pJC1200 is a pUC18 clone of a PCR product using primers JCO176 (5'-GGGCCCAGCTTACTATGCC-3') and JCO177 (5'-CTCGAGAATAAACCCTCCG-3') at HincII. Plasmid pJC1201 is a pUC18 clone of a PCR product generated with primers JCO178 (5'-CTCGAGCGTCAATGCGACAATAGTAGCATTG-3') and JCO136 (5'-CCTAGGTGCTGTTCCACGTTTACCATCTAAC-3') at HincII.
To construct pJC1206, a PCR product obtained with primers JCO113 (5'-GGTACCCTGCAATTGTCCGACGCG-3') and JCO120 (5'-GATATCCTCGAGCTACCAGTCACACTTACC-3') and chromosomal S. aureus DNA as template was cloned into the HincII site of pUC18 to generate pJC1051. A PCR product obtained with primers JCO118 (5'-TCTAGAGGTCGTCGTGGACCTGCAGG-3') and JCO121 (5'-GGATCCCGTACACTTCTGGCTGAG-3') and the same template was cloned into the HincII site of pUC18 to generate pJC1052. The KpnI–BamHI fragment of pJC1052 was replaced with the KpnI–BamHI fragment of pJC1051 to generate pJC1053. The BamHI–XhoI fragment of pJC1053 was replaced with the BamHI–XhoI fragment (ermC) of pJC1124 to generate pJC1127.
The multi-copy plasmid containing the complete coding sequence of svrA, transcribed by the native svrA promoter, was constructed by replacing the KpnI–SphI fragment of pJC1075 with the KpnI–SphI fragment (svrA) from pJC1050 to generate pJC1059. DNA for a full clone of the svrA gene was amplified in two parts. A PCR product obtained with forward primer JCO113 (5'-GGTACCCTCCAATTGTCCGACGCG-3') and reverse primer JCO115 (5'-GGTGCCCCAATTGCACGTGC-3') and chromosomal S. aureus DNA as template was cloned into the HincII site of pUC18 to generate pJC1041. A PCR product using forward primer JCO114 (5'-GCGATGATGCATTTCTCATTGCC-3') and reverse primer JCO117 (5'-GTCGACCTCAGCCAGAAGTGTACG-3') was cloned into the HincII site of pUC18 to generate pJC1042. A NsiI–KpnI fragment from pJC1042 was replaced with the NsiI–KpnI fragment from pJC1041 to generate pJC1050.
The AvrII–SacII fragment from pCN33 was replaced with the AvrII–SacII fragment (cadCA) from pJC1071 to generate pJC1075. A DNA fragment carrying the cadCA genes was PCR-amplified using primers JCO124 (5'-CCTAGGGTCATACCCTGGTCAAAACCGTTCG-3') and JCO125 (5'-CCGCGGCCGCAGCTGCTGTAAGTATCG-3') and cloned into the HincII site of pUC18 to generate pJC1071.
Construction of svrA deletion mutant.
Allelic exchange was performed in a spontaneously Smr (rpsL) mutant of RN6734, JCSA18. JCSA18 was generated by selection for spontaneous Smr mutants of RN6734 on GL agar with 300 µg streptomycin ml–1.
Strain RN4220 was electroporated with plasmid pJC1206, with selection on GL agar containing 5 µg chloramphenicol ml–1 at 30 °C. Strain JCSA18 was then transformed with RN4220-propagated pJC1206 at 30 °C. Cmr transformants of JCSA18 were restreaked on GL agar containing 10 µg erythromycin ml–1 at 30 °C. Cmr Emr colonies were restreaked on GL agar containing 5 µg erythromycin ml–1 at 42 °C. Emr colonies were streaked on GL agar containing 5 µg erythromycin ml–1+300 µg streptomycin ml–1+0.1 µg cefoxitin ml–1 at 42 °C. Emr Smr colonies were tested for Cms and Emr on GL agar containing 5 µg ml–1 chloramphenicol or erythromycin. Phage 80 was used to produce phage lysates of colonies that were Emr Smr Cms. The phage lysates were then used to transduce RN6390 and RN6734 with selection for Emr to generate JCSA119 and -120, respectively.
PCR analysis.
PCR analysis was used to confirm genotypes. Primers JCO114 and JCO117 anneal to the 5' and 3' ends of svrA, respectively. Primers JCO173 (5'-GACCGGGGACTTATCAGCC-3') and JCO174 (5'-CGATACAAATTCCCACTAAGCGCTC-3') anneal to the terminal inverted repeats of bursa aurealis and Tn917, respectively, facing outward.
DNA sequencing of agrC in P6C63.
The agrC gene of P6C63 was PCR-amplified using primers JCO162 (5'-GAGAGTGTGATAGTAGGTGG-3') and JCO163 (5'-CACATCCTTATGGCTAGTTG-3'), agarose gel-purified and sequenced by the New York University Skirball DNA sequencing core facility using the same primers.
Toxin analysis.
Production of -, - and -toxins was analysed by cross-streaking with RN4220 on SBA (Traber & Novick, 2006).
Exoprotein profiles.
Extracellular protein profiles were determined on 6 h 37 °C culture supernatants by the method of Laemmli (1970). All cultures were grown to equivalent OD600 densities in CY broth. Proteins were precipitated by adjusting filtered supernatants to 10 % TCA. Precipitated proteins were then boiled in 1x Laemmli sample buffer, resolved on 12 % acrylamide SDS-PAGE, and visualized by Coomassie blue staining.
agr-I activation by autoinducing peptide (AIP)-I.
Strain P6C63 was transformed with plasmid pJW7141, which carries AIP-I-responsive luciferase promoter fusion agr-P3-lux. Transformants were grown in CY broth and AIP-I was added to activate the agr-P3 promoter. Luminescence was measured quantitatively using a Molecular Devices LMax II384 microplate luminometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We readily confirmed the agr-expression defect, but because of the well-known genotypic instability of the agr locus (Bjorklind & Arvidson, 1980; Somerville et al., 2002; R. P. Novick and others, unpublished results), we felt it necessary to confirm the reported linkage of the transposon to the agr-defective phenotype. Accordingly, we transduced the Tn917 insertion, using selection for the transposon-carried erythromycin resistance, to several different S. aureus strains with known agr genotypes. For each of these crosses, several independent transductants were screened on SBA and the insertion confirmed by PCR. Surprisingly, in no case did the svrA insertion have any impact on agr expression (Fig. 1), which was inconsistent with the complementation results of Garvis et al. (2002). We recloned svrA and tested the new clone for complementation of the original mutation. In contrast to the results of Garvis et al. (2002), our svrA clone failed to restore agr expression to the original mutant strain, P6C63 (Fig. 2A).
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). The streptomycin-sensitivity selection system takes advantage of the well-known fact that streptomycin-resistance mutations in rpsL, which encodes ribosomal protein S12, are recessive. This provides positive selection against the wild-type (wt) allele when a mutated allele is present, which can readily be adapted to select for elimination of the gene duplications resulting from a Campbell insertion. Following allelic exchange in a streptomycin-resistant derivative of RN6734, the mutation was transduced into wt backgrounds and the genotypes were confirmed by PCR analysis (data not shown). Fig. 1
shows that a complete deletion of the svrA-coding region had no detectable effect on the haemolytic activity of strains RN6390 or RN6734 on SBA, nor did it or other svrA mutations have any effect on the exoprotein profile (data not shown), confirming that svrA does not influence agr regulation.
Given the above results suggesting that svrA does not regulate agr, we attempted to localize the defect in P6C63 within the agr-activation pathway. We introduced into strain P6C63 a plasmid containing a transcriptional fusion of Photorhabdis luciferase to the agr-P3 promoter and measured luciferase activity of the resulting derivative strain. This strain had very low luciferase activity, comparable to that of an agr-null strain derived from the same lineage as that of the svrA mutant (data not shown). This low level of activity is attributable to the basal activity of the P3 promoter and amounts to 1–2 % of the activity seen with the same fusion in an agr wt strain. This result, consistent with the reported absence of the agr transcripts, means that P6C63 cannot activate its own agr locus. We next tested the mutant for its ability to respond to the addition of exogenous AIP, using the same luciferase readout, and found that exogenous AIP-I did not detectably stimulate the P3 promoter (data not shown). Thus, the svrA mutant is defective in its response to exogenous AIP, possibly representing an inability of the peptide to interact productively with its receptor, AgrC. Such phenotypic patterns are characteristic of adventitious mutations in agrA or -C genes (Peng et al., 1988; K. T. Traber & R. P. Novick, unpublished results).
Since spontaneous agr mutations frequently map to agrC, we attempted to complement the P6C63 strain with a multi-copy plasmid encoding agrC. As shown in Fig. 2(A), agrC is sufficient for phenotypic restoration of P6C63 to wt haemolytic activity on SBA. This result strongly suggests that the second-site mutation responsible for the P6C63 phenotype is a spontaneous mutation in agrC. Indeed, sequencing of the agrC gene in P6C63 revealed a single-base deletion of nucleotide A415 of the coding region, resulting in a frame-shift mutation that is predicted to alter the primary sequence beginning at residue 138 of AgrC, and to truncate the peptide at residue 143 (Fig. 2B). This result supports the complementation of strain P6C63 by agrC, confirming the prediction of a secondary agrC mutation and defining it as a frame-shift that results in a truncation of AgrC within the N-terminal sensor domain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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for this strain.
Although the agrC mutation accounts for the attenuation of virulence attributed to the svrA mutation in P6C63, it may not account for the entire effect, since it was reported by Garvis et al. (2002) that P6C63 was slightly more attenuated for virulence than was an agr-null mutant used as a control. Given that strain P6C63 is essentially a double mutant of svrA and agrC, the adventitious agr defect may not account entirely for the attenuation of virulence seen with this strain. To determine whether svrA might have an independent effect on virulence, we tested svrA deletion mutants of RN001 in a murine subcutaneous abscess model (Barg et al., 1992). Despite multiple attempts, we were unable to demonstrate a significant and reproducible defect in the ability to form a skin abscess in hairless SKH-1 mice inoculated with strains deleted for svrA, compared to wt bacteria.
One question of interest is that of when the agrC mutation occurred in the history of P6C63. Although it seems most likely to have occurred early and to be responsible for the identification of P6C63 in the original STM screen (Mei et al., 1997), it is conceivable that loss of a protein with an important role in trans-membrane trafficking could impact on the ability of S. aureus to survive in vivo. It may be that strains lacking SvrA are limited in their nutrient-scavenging ability or are unable to export substances toxic to the bacteria. Such mild non-virulence attenuation may have been sufficient for an in vivo selection, but would likely not display a phenotype in a murine skin abscess model. Therefore, the agrC mutation could have occurred later, during handling of the strain, as has often been the case with the agr locus; this could be resolved only by analysis of the actual organisms recovered from the animal in the screen.
We note that this is not the first reported case of an adventitious agr mutation generating spurious results for a transposon insertion in an unrelated gene: an earlier example has been described by McNamara and Iandolo for their xpr strain (McNamara & Iandolo, 1998).
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ACKNOWLEDGEMENTS |
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Edited by: G. M. Dunny
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Barg, N., Bunce, C., Wheeler, L., Reed, G. & Musser, J. (1992). Murine model of cutaneous infection with Gram-positive cocci. Infect Immun 60, 2636–2640.
Bjorklind, A. & Arvidson, S. (1980). Mutants of Staphylococcus aureus affected in the regulation of exoprotein synthesis. FEMS Microbiol Lett 7, 203–206.
Charpentier, E., Anton, A. I., Barry, P., Alfonso, B., Fang, Y. & Novick, R. P. (2004). Novel cassette-based shuttle vector system for Gram-positive bacteria. Appl Environ Microbiol 70, 6076–6085.
Dean, D. (1981). A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene 15, 99–102.[CrossRef][Medline]
Garvis, S., Mei, J. M., Ruiz-Albert, J. & Holden, D. W. (2002). Staphylococcus aureus svrA: a gene required for virulence and expression of the agr locus. Microbiology 148, 3235–3243.
Ji, G., Beavis, R. & Novick, R. P. (1997). Bacterial interference caused by autoinducing peptide variants. Science 276, 2027–2030.
Kaatz, G. W., McAleese, F. & Seo, S. M. (2005). Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrob Agents Chemother 49, 1857–1864.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
McAleese, F., Petersen, P., Ruzin, A., Dunman, P. M., Murphy, E., Projan, S. J. & Bradford, P. A. (2005). A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline. Antimicrob Agents Chemother 49, 1865–1871.
McNamara, P. J. & Iandolo, J. J. (1998). Genetic instability of the global regulator agr explains the phenotype of the xpr mutation in Staphylococcus aureus KSI9051. J Bacteriol 180, 2609–2615.
Mei, J. M., Nourbakhsh, F., Ford, C. W. & Holden, D. W. (1997). Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26, 399–407.[CrossRef][Medline]
Novick, R. P. (1967). Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33, 155–166.[CrossRef][Medline]
Novick, R. P. (1991). Genetic systems in staphylococci. In Methods in Enzymology (Bacterial Genetics), vol. 204, pp. 587–636. Edited by J. Miller. Orlando, FL: Academic Press.
Novick, R. P. (2003). Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 48, 1429–1449.[CrossRef][Medline]
Novick, R. P., Ross, H. F., Projan, S. J., Kornblum, J., Kreiswirth, B. & Moghazeh, S. (1993). Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12, 3967–3975.[Medline]
Novick, R. P., Projan, S., Kornblum, J., Ross, H., Kreiswirth, B. & Moghazeh, S. (1995). The agr P-2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. Mol Gen Genet 248, 446–458.[CrossRef][Medline]
Peng, H.-L., Novick, R. P., Kreiswirth, B., Kornblum, J. & Schlievert, P. (1988). Cloning, characterization and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J Bacteriol 179, 4365–4372.
Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R., Cole, R. L., Hoff, J. S. & Musser, J. M. (2002). In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence. J Bacteriol 184, 1430–1437.
Traber, K. & Novick, R. P. (2006). C-terminal frameshift in agrA causes delayed activation of RNAIII and is responsible for the agr– phenotype of certain standard laboratory strains and certain clinical isolates. Mol Microbiol (in press).
Wright, J. S., III, Jin, R. & Novick, R. P. (2005). Transient interference with staphylococcal quorum sensing blocks abscess formation. Proc Natl Acad Sci U S A 102, 1691–1696.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Received 19 January 2007;
accepted 28 January 2007.
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