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Antimicrobial Agents and Chemotherapy, May 2009, p. 1832-1839, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01255-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the ^B-Dependent yabJ-spoVG Operon in Staphylococcus aureus: Role in Methicillin and Glycopeptide Resistance

Bettina Schulthess,¹ Stefan Meier,¹ Dagmar Homerova,² Christiane Goerke,³ Christiane Wolz,³ Jan Kormanec,² Brigitte Berger-Bächi,^1* and Markus Bischoff⁴

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland,¹ Institute of Molecular Biology, Centre of Excellence for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republic,² Institute of Medical Microbiology and Hygiene, University of Tübingen, Tübingen, Germany,³ Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Homburg/Saar, Germany⁴

Received 19 September 2008/ Returned for modification 7 December 2008/ Accepted 31 January 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The alternative sigma factor ^B of Staphylococcus aureus controls the expression of multiple genes, including virulence determinants and global regulators; promotes capsule production; and increases the resistance levels of methicillin-resistant S. aureus (MRSA) and glycopeptide-intermediate-resistant S. aureus (GISA) strains. We show here that deletion of the ^B-controlled yabJ-spoVG operon, which codes for potential downstream regulators of ^B, abolished capsule synthesis and reduced resistance in MRSA and GISA to the same extent that ^B inactivation did. Introduction of the yabJ-spoVG operon in trans restored the original phenotype. By genetic manipulations, we show that SpoVG but not YabJ is required for complementation. We therefore postulate that SpoVG is the major factor of the yabJ-spoVG operon required in S. aureus for capsule formation and antibiotic resistance.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A large set of virulence factors allows Staphylococcus aureus to cause a wide spectrum of diseases, which range from superficial to life-threatening infections (26). The production of the virulence determinants is tightly controlled by a network of global regulators, including the alternative transcription factor ^B (for reviews, see references 8 and 30). Besides its role in the general stress response (7, 12, 13, 19, 24, 25), ^B affects methicillin and glycopeptide resistance (2, 28, 34, 39, 40), pigment production (13, 21, 25, 28, 32), internalization into endothelial cells (29), S. aureus-induced apoptosis (16), and capsule formation (27) and modulates the expression of a variety of virulence factors and regulatory elements (for a review, see reference 3). Recent microarray analyses showed that ^B affects the expression of a large number of genes/operons (3, 33). However, a surprisingly large fraction of these genes/operons are not preceded by an apparent ^B promoter (18), including e.g., the cap operon, which codes for capsular polysaccharide production (for a review, see reference 31), and fnbA, which codes for fibronectin binding protein A. This indicates that downstream-acting regulators mediating the effect of the alternative transcription factor ^B should exist. The way ^B exerts its impact on the resistance to cell wall antibiotics and capsule formation as well as the postulated ^B effectors remain unknown.

We recently showed that inactivation of the staphylococcal ^B-controlled yabJ-spoVG operon, which codes for Bacillus subtilis YabJ and SpoVG sequence homologs, significantly reduces the level of transcription of the cap operon and impedes capsule formation in capsular polysaccharide-producing strain Newman (27), suggesting that products of the yabJ-spoVG locus might serve as ^B effectors that modulate ^B-dependent genes lacking an apparent ^B promoter.

In this study, we deleted the yabJ-spoVG operon and analyzed its effect on the resistance levels of methicillin-resistant S. aureus (MRSA) and glycopeptide-intermediate-resistant S. aureus (GISA) strains. We present here data showing that the deletion of the yabJ-spoVG operon decreases resistance in MRSA and GISA and that SpoVG is the major effector molecule of the yabJ-spoVG locus with respect to resistance and capsule formation.

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Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. The bacteria were grown on sheep blood agar or in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI) with shaking (180 rpm) at 37°C. When required, the media were supplemented with either 100 µg ampicillin, 10 µg erythromycin, 50 µg kanamycin, or 10 µg tetracycline per ml.

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TABLE 1. Strains and plasmids used in this study

Molecular biological methods. General molecular biology techniques were performed according to the standard protocols described by Sambrook et al. (37) and Ausubel et al. (1).

Strain construction. To construct S. aureus yabJ-spoVG mutants, fragments covering 0.8 kb of the yabJ upstream region (up-fragment) and 1.0 kb of the spoVG downstream region (down-fragment) were amplified by PCR with primer pairs oSTM49-oSTM50 and oSTM03-oSTM04 and S. aureus COL DNA as the template (Tables 1 and 2). The products were digested with KpnI-BamHI and PstI-HindIII, respectively, and cloned into plasmid pEC1 (6), with the up-fragment preceding the erm(B) cassette and the down-fragment following the resistance marker. The resulting plasmid, pSTM14, was digested with KpnI and HindIII, and the insert was cloned into the suicide vector pBT (13), yielding plasmid pSTM15, which was electroporated into RN4220 (23) (Fig. 1). Allelic replacement mutants were selected with erythromycin and screened for the loss of tetracycline resistance, resulting in strain SM133(RN4220 yabJ-spoVG::erm(B)), which served as a donor for the transduction of the yabJ-spoVG deletion into S. aureus strains of different genetic backgrounds (Table 1). S. aureus rsbUVW-sigB mutants BS126 and BS128 were constructed by transducing the rsbUVW-sigB::erm(B) mutation of strain IK184 (25) into strains NM143 (38) and COLn (20), respectively. The deletion of the yabJ-spoVG and the sigB operons was confirmed by PCR and Southern analyses, and the absence of major rearrangements after genetic manipulation of the strains was confirmed by whole-genome SmaI pulsed-field gel electrophoresis.

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TABLE 2. Primers used in this study

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FIG. 1. Genetic organization of the S. aureus yabJ-spoVG locus. Schematic representations of the yabJ-spoVG region of S. aureus COL and its yabJ-spoVG mutant, strain SM154, are shown. ORFs, promoters, and terminators are indicated. ORF notations and nucleotide (nt) numbers correspond to those of the respective genomic regions of strain COL (RefSeq accession no. NC_002951).

Construction of reporter gene plasmids pSTM19, pSTM20, and pSTM21. DNA fragments covering different parts of the yabJ-spoVG region were amplified by PCR with primer pairs oSTM66-oSTM69 (for pSTM16), oSTM66-oSTM67 (for pSTM17), and oSTM68-oSTM69 (for pSTM18) and with S. aureus COL DNA as the template (Tables 1 and 2). The resulting PCR products were digested with KpnI-NcoI and cloned into pSP-luc⁺ (Promega) upstream of the luciferase reporter gene luc⁺. The yabJ-spoVG regions fused to luc⁺ in the resulting plasmids were excised with KpnI and XbaI and cloned into the Escherichia coli-S. aureus shuttle plasmid pBus1 (36) to obtain plasmids pSTM19, pSTM20, and pSTM21, respectively. The identities of the inserts were confirmed by sequencing.

Construction of complementation plasmids pSTM08, pSTM09, and pSTM10. A DNA fragment covering yabJ-spoVG and the preceding ^B-dependent promoter, P_yabJ, was amplified by PCR with primer pair oSTM07-oSTM08 and S. aureus COL DNA as the template (Tables 1 and 2). The resulting product was digested with BamHI and KpnI and cloned into shuttle plasmid pBus1 (36), yielding plasmid pSTM08. To mutagenize either yabJ or spoVG in pSTM08, the following strategies were applied. For pSTM09 [P_yabJ-yabJ-spoVG1(Am)], a mutation was introduced into spoVG with primer pairs oSTM07-oSTM42 and oSTM43-oSTM08 on pSTM08 to amplify P_yabJ-yabJ, including the 5' part of spoVG and the 3' part of spoVG, respectively. The resulting PCR products were digested with BamHI-SacI and SacI-KpnI and cloned into pBus1, thereby introducing a frame shift into spoVG, resulting in an amber mutation at amino acid 26. For pSTM10 [P_yabJ-yabJ1(Am)-spoVG], which harbors a point mutation in yabJ, primer pairs oSTM07-oSTM40 and oSTM41-oSTM08 were used together with pSTM08 to amplify DNA fragments covering P_yabJ, including the 5' part of yabJ and the 3' part of yabJ followed by spoVG, respectively. The resulting products were digested with BamHI-SacI and SacI-KpnI and cloned into pBus1, thereby introducing a frame shift into yabJ, yielding in an amber mutation at amino acid 22. The identities of the inserts were confirmed by sequencing.

Luciferase assay. Luciferase activity was measured as described earlier (4) by using the luciferase assay substrate and a Turner Designs TD-20/20 luminometer (Promega).

Primer extension experiments. The transcriptional start points were mapped by primer extension analysis, as described by Harraghy et al. (15). DNA fragments were amplified by PCR with S. aureus COL DNA as the template. The probes yabJ-pe and spoVG-pe were prepared by using the forward primers yabJpe-F and spoVGpe-F, respectively, together with the 5'-end-labeled reverse primers yabJpe-R and spoVGpe-R, respectively. The oligonucleotides were 5' end labeled with [-³²P]ATP (4,500 Ci mmol^–1; ICN) and T4 polynucleotide kinase (Biolabs).

Capsule determination. Capsular polysaccharide type 5 (CP-5) production was determined by indirect immunofluorescence from cultures grown for 8 h in LB broth, as described earlier (38), using mouse immunoglobulin M monoclonal antibodies to CP-5 (17).

Susceptibility testing. Plates containing an antibiotic gradient were prepared as described before (38). Cells of the strains to be tested were resuspended in physiological NaCl solution to a density of a 0.5 McFarland standard and swabbed onto the plate along the gradient. Growth was read after 24 h and 48 h of incubation at 35°C. Teicoplanin and vancomycin MICs were determined by using Etests, according to the manufacturer's instructions (AB Biodisk, Solna, Sweden). Oxacillin MICs were determined by broth microdilution, as recommended by the Clinical and Laboratory Standards Institute (9), except that LB broth instead of cation-adjusted Mueller-Hinton broth with 2% NaCl was used. Population analysis profiles were established by plating appropriate dilutions of an overnight culture on LB agar plates containing increasing concentrations of oxacillin or teicoplanin. The numbers of CFU were determined after 48 h of incubation at 35°C.

SpoVG-His₆ expression and antibody preparation. For the construction of SpoVG-His₆-overexpressing plasmid pSTM33, a 0.33-kb DNA fragment covering the open reading frame (ORF) of spoVG was amplified by PCR with primer pair oSTM05-oSTM06 and S. aureus Newman DNA as the template (Tables 1 and 2). The fragment was cloned into the expression vector pET28a(+) (Novagen) by using the NdeI and XhoI restriction sites, such that the hexahistidine tag was fused in frame to the C terminus of SpoVG. pSTM33 was electroporated into E. coli DH5, and the insert was verified by sequencing and was finally introduced into strain BL21(DE3) (Novagen) for overexpression. Cells were grown in LB broth to an optical density at 600 nm of 0.5 and induced with 0.3 mM isopropyl-β-D-thiogalactopyranoside. After 3 h, cells were collected by centrifugation and resuspended in 30 ml phosphate-buffered saline (pH 7.4) supplemented with a complete EDTA-free protease inhibitor cocktail tablet (Roche) and 0.1 mg ml^–1 DNase. The cells were disrupted (Cell Cracker; EMBL, Heidelberg, Germany), and the debris was separated by centrifugation at 13,000 x g for 10 min. Purification of the His-tagged protein was performed on nickel-nitriloacetic acid columns, according to the recommendations of the manufacturer (Qiagen, Basel, Switzerland). The protein sample was further purified by analytical gel filtration on a Superdex 75 HR column on a Pharmacia Biotech fast-performance liquid chromatography system. The correct molecular weight of the purified protein was confirmed by nano-electrospray ionization mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One milligram of the purified SpoVG-His₆ was used to raise polyclonal chicken antibodies (Davids Biotechnologie, Regensburg, Germany). The resulting crude antiserum was purified against the immobilized SpoVG antigen.

Western blot analysis. Cytoplasmic protein extracts were obtained by lysing S. aureus, grown in LB broth at 37°C for 5 h, in phosphate-buffered saline (pH 7.4) containing 2 mM phenylmethylsulfonyl fluoride and 40 µg each of lysostaphin, lysozyme, DNase, and RNase per ml. Protein fractions (50 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and subjected to Western blot analysis with anti-SpoVG antibodies.

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Transcriptional control of the yabJ-spoVG locus. The ^B-dependent yabJ-spoVG operon is transcribed as a bicistronic mRNA, with smaller yabJ- and spoVG-specific bands appearing later in growth and peaking toward early stationary phase (27). To search for a potential transcriptional start point (TSP) for the smaller spoVG transcript, we performed a primer extension analysis and included the recently characterized yabJ promoter (P_yabJ) as a positive control (3, 18). This analysis confirmed the TSP of the bicistronic yabJ-spoVG transcript previously identified by S1 nuclease mapping at position 548659 of the S. aureus COL genome (GenBank accession no. NC_002951), 144 bp upstream of the yabJ ORF. The TSP is preceded by a perfectly conserved ^B consensus promoter sequence (GTTTAA-₁₄-GGGTAT) (Fig. 2). A second potential TSP candidate, at position 549247 of the S. aureus COL genome, that mapped only 8 bp upstream of the spoVG ORF and immediately downstream of the proposed ribosomal binding site (Fig. 2C) was identified. However, the sequence preceding the spoVG TSP candidate shared no homology with either a ^A consensus promoter (TTGACA-_16/18-TATAAT) (10, 35) or a ^B consensus promoter (GTTTAA-_12/15-GGGTAT) (18), suggesting that the small spoVG transcript may possibly be a result of processing and not a product of a specific promoter.

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FIG. 2. Determination of TSP candidates of the yabJ-spoVG locus of S. aureus by primer extension analysis. (A) Map of the yabJ-spoVG region showing the positions of the two putative TSPs obtained with primers yabJ-pe (diamond) and spoVG-pe (circle). Proposed ORFs, promoters, and terminators are indicated. (B) Primer extension products (PEP) were analyzed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders. Horizontal arrows indicate the positions of the primer extension products. (C) Nucleotide sequences of the putative S. aureus yabJ-spoVG operon promoters. Vertical arrows indicate the nucleotides corresponding to the putative TSPs. The predicted –35 and –10 promoter boxes are shown, and the ribosomal binding site is underlined.

To verify a putative promoter function suggested by the signal preceding the spoVG ORF and to measure the activities of the yabJ promoter and the candidate spoVG promoter, we constructed a series of promoter-luc⁺ reporter gene plasmids covering different parts of the yabJ-spoVG locus (Fig. 3A) and introduced them into strain Newman and its rsbUVW-sigB derivative, strain IK184. Plasmid pSTM19, whose sequence covers the region preceding the spoVG ORF up to the yabJ promoter region, as well as plasmid pSTM20, whose sequence covers the yabJ upstream region, including P_yabJ, produced, as expected, comparable and high levels of luciferase activity in strain Newman after 8 h of growth (Fig. 3B). However, plasmid pSTM21, which harbors the 3' part of yabJ, including the putative spoVG TSP and the spoVG ATG but which lacks P_yabJ, yielded no luciferase activity, signaling that this region indeed does not likely harbor a promoter. The two smaller bands appearing in later growth stages are therefore degradation products of the bicistronic yabJ-spoVG transcript. No luciferase activities were found in IK184 transformed with either of the plasmids, confirming the ^B dependence of the yabJ-spoVG transcripts.

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FIG. 3. yabJ-spoVG promoter activity. (A) Schematic representation of the yabJ-spoVG locus of S. aureus and of the reporter gene plasmids pSTM19, pSTM20, and pSTM21 showing the portion of the yabJ-spoVG region fused to the luciferase gene (luc+). ORFs, promoters, and terminators are indicated. (B) Luciferase activities of plasmids pSTM19, pSTM20, and pSTM21 in strains Newman and IK184 (Newman rsbUVW-sigB). The strains were grown in LB broth supplemented with 10 µg ml–1 tetracycline at 37°C and 180 rpm and were sampled after 5 h of growth. The values represent the means ± standard deviations from three independent assays.

Influence of yabJ-spoVG on capsule formation in strain Newman. Deletion of spoVG abolishes yabJ-spoVG transcription and CP-5 production in strain Newman, as shown earlier (27). In order to determine whether yabJ or spoVG was responsible for capsule production, we substituted the complete operon with an erm(B) cassette to be able to complement the resulting yabJ-spoVG mutant, strain SM148, with a series of recombinant plasmids harboring different parts of the yabJ-spoVG operon (Fig. 4A). Confirming our earlier findings, the SM148 mutant produced no capsule after 8 h of growth (Fig. 4B). The capsule could be restored by trans complementation with plasmid pSTM08 harboring yabJ-spoVG but not with plasmids carrying either yabJ (pSTM11) or spoVG (pSTM13) alone. From these findings, we hypothesized that the yabJ-spoVG mRNA or both products of the operon may be required for CP-5 formation. To test this hypothesis, we introduced a frameshift mutation in either yabJ [yabJ1(Am)-spoVG] or spoVG [yabJ-spoVG1(Am)], thereby maintaining the bicistronic transcript but truncating and inactivating either YabJ or SpoVG. Interestingly, the trans complementation of SM148 restored the capsule only with the yabJ1(Am)-spoVG construct, pSTM10, but not with the yabJ-spoVG1(Am) construct, pSTM09 (Fig. 4B). Western blots showed that only the complementing plasmids pSTM08 (yabJ-spoVG) and pSTM10 [yabJ1(Am)-spoVG] produced SpoVG; but pSTM09 [yabJ-spoVG1(Am)], pSTM11 (yabJ), and pSTM13 (spoVG) did not (Fig. 4C), even though they produced abundant amounts of mRNA (data not shown). The apparent inability of the P_yabJ-spoVG fusion (pSTM13) to produce the SpoVG protein, despite the presence of spoVG transcripts, may have been caused by a 2-nucleotide exchange between the ribosomal binding site and the spoVG start codon due to the cloning strategy. However, a P_yabJ-spoVG fusion construct with a restored wild-type sequence, as well as fusions of spoVG, including its own ribosomal binding site to P_yabJ, failed to produce SpoVG (B. Schulthess, unpublished results), suggesting a complex regulation of SpoVG translation that requires further investigations. Selective translation of the coding mRNA may have various causes, as reviewed by Gualerzi et al. (14), for the translational control of cold shock genes, which may also apply for the yabJ-spoVG operon. This suggests that SpoVG is required for CP-5 production in strain Newman and that the yabJ-spoVG mRNA but not YabJ is necessary for SpoVG production and activity.

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FIG. 4. trans complementation of capsule production of SM148 (Newman yabJ-spoVG). (A) Schematic representation of the complementation plasmids. ORFs, promoters, and terminators are indicated. (B) CP-5 expression determined by indirect immunofluorescence of strain SM148 transformed with either pSTM08, pSTM09, pSTM10, pSTM11, pSTM13, or empty control plasmid pBus1 grown in LB broth for 8 h at 37°C. The CP-5 expression of strain Newman containing pBus1 served as a control for wild-type CP-5 production. Bacteria were stained with 4',6-diamidino-2-phenylindole (DAPI), marked with CP-5-specific monoclonal antibodies, and stained with Cy-3-conjugated antimouse antibodies (CY-3). (C) Western blot analysis of SpoVG in cytoplasmic protein fractions of strain Newman and strain SM148 complemented with either pBus1, pSTM08, pSTM09, pSTM10, pSTM11, or pSTM13 after 5 h of growth in LB broth.

Influence of yabJ-spoVG on levels of antibiotic resistance in MRSA and GISA. The activity of the alternative ^B factor positively influences the levels of oxacillin and glycopeptide resistance in MRSA and GISA strains (2, 28, 34, 39, 40). However, the genes by which ^B affects the resistance levels are not yet known. To see if the effect of ^B on resistance was mediated by the yabJ-spoVG locus, we inactivated yabJ-spoVG in MRSA strain COL and its plasmid-cured derivative, strain COLn, and in GISA strains BB938 and NM143 by transduction of yabJ-spoVG::ermB, yielding strains SM154, SM165, SM159, and SM233. The levels of resistance of these four transductants to oxacillin and teicoplanin, respectively were significantly reduced in a characteristic, strain-specific fashion, as shown by population analysis profiles (Fig. 5), although the difference in absolute MICs may be of debatable clinical relevance (Table 3). The MICs of the yabJ-spoVG mutant were comparable to those of the corresponding rsbUVW-sigB mutants. Interestingly, the reduction in the level of resistance to teicoplanin was more pronounced than that to vancomycin. This may point to an altered interaction of the lipophilic teicoplanin anchor with the membrane, while vancomycin, whose activity is enhanced by dimerization, was apparently less affected.

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FIG. 5. Antibiotic susceptibilities of MRSA strains COL (A) and COLn (B) and GISA strains BB938 (C) and NM143 (D) and their isogenic yabJ-spoVG mutants. Population analysis profiles for oxacillin and teicoplanin, respectively are shown for each wild type (squares) and the corresponding yabJ-spoVG mutant (triangles).

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TABLE 3. MICs

Since strain COL harbors the tetracycline resistance plasmid pT181, which interferes with our complementing plasmids, the cured plasmid-less strain COLn was used for the trans complementation assays. Surprisingly, COLn had a higher level of oxacillin resistance than COL, and the COLn yabJ-spoVG mutant (strain SM165) had a smaller effect on oxacillin resistance (Fig. 5A and B) that was, however, sufficient for visualization on gradient plates and performance of trans complementation assays (Fig. 6). Similar to what was observed for CP-5 formation, the trans complementation of oxacillin resistance in SM165 and of teicoplanin resistance in SM159 was successful only with plasmids pSTM08 and pSTM10, which contained yabJ-spoVG and yabJ1(Am)-spoVG, respectively. Plasmids pSTM11, pSTM13, and pSTM09 as well as the empty plasmid, pBus1, had no effect, demonstrating that neither yabJ, spoVG, nor yabJ-spoVG1(Am) was able to complement the resistance.

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FIG. 6. trans complementation of oxacillin and teicoplanin resistance. The yabJ-spoVG mutants SM165 and SM159 were complemented with either pBus1 (empty plasmid), pSTM08 (yabJ-spoVG), pSTM09 [yabJ-spoVG1(Am)], pSTM10 [yabJ1(Am)-spoVG], pSTM11 (yabJ), or pSTM13 (spoVG); and their levels of resistance were compared with those of their corresponding wild-type strains, strains COLn and BB938, respectively, on gradient plates containing oxacillin (A) or teicoplanin (B).

This study showed that the deletion of the ^B-dependent yabJ-spoVG operon in MRSA and GISA strains decreased the resistance levels in a way similar to that found for the deletion of ^B, suggesting that ^B exerts its effect on methicillin and glycopeptide resistance via the gene products of the yabJ-spoVG locus, in particular, via SpoVG. Which of the multiple chromosomal genes affecting either methicillin or glycopeptide intermediate resistance levels are controlled by the ^B-SpoVG cascade remains to be identified.

 
This study was supported by the Bonizzi-Theler Foundation, the Roche Research Foundation, Swiss National Science Foundation grants 3100A0-10024 and 31-117707, and European Community grant EU-LSH-CT2003-50335 (BBW 03.0098). J.K. is supported by grants 2/6010/26 and 2/0104/09 from the Slovak Academy of Sciences.

 
* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Zurich, Gloriastr. 32, 8006 Zurich, Switzerland. Phone: 41 44 634 26 50. Fax: 41 44 634 49 06. E-mail: bberger{at}imm.uzh.ch

Published ahead of print on 17 February 2009.

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Antimicrobial Agents and Chemotherapy, May 2009, p. 1832-1839, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01255-08
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