Article Figures & Data
Figures
- FIG. 1.
Northern blotting analysis of vraS transcripts in wild-type strain RN4220 and the vraS mutant strain (M1) treated with VAN or OXA. (A) Map of the four ORFs contained in the vraSR operon illustrating the probe (line beneath map) used in Northern blotting analysis and the binding sites for primers (short arrows) (PE1 to PE4) used in the primer extension assay. The vraSR probe was PCR amplified using the primers vraSRprobeF and vraSRprobeR. The vraSR mutant (M1) contains an ermC cassette inserted into the EcoRI site of the chromosomal vraS gene, as depicted above the map. (B) Northern blot of RNA isolated from uninduced cultures (U) or cultures induced by VAN (2 μg/ml) or OXA (1 μg/ml). Membranes were hybridized with a vraSR-specific probe (shown beneath the map in panel A). Overnight cultures of S. aureus strains were diluted 1:100 in tryptic soy broth (10 ml in a 50-ml conical Falcon tube), shaken at 250 rpm, and grown at 37°C. After a 1-h incubation, the cultures were treated and incubated for another hour. The vraSR probe was PCR labeled with [α-32P]dATP (Amersham) using the Prime-a-Gene labeling system (Promega). The hybridized blots were washed with 0.1× SSC buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1% sodium dodecyl sulfate four times at 70°C, each time for 30 min. (C) Results from primer extension (PE) analysis of MRSA strain IL-A with the use of primers PE1, PE2, and PE3. The asterisk indicates the major tsp. The reaction product was mixed with half the volume of a sequencing stop solution, denatured, and applied to a 5% sequencing gel. The sequencing ladder (lanes labeled G, A, T, C) was prepared by using an fmol DNA cycle sequencing system (Promega) with the same primer used in the primer extension reaction.
- FIG. 2.
Analysis of vraSR-lacZ fusions by β-galactosidase assay in wild-type strain RN4220 and the vraSR mutant (M1). (A) Map of the DNA fragments (horizontal black bars beneath the map) PCR amplified from the vraSR operon from MRSA strain IL-A, which was used to produce the vraSR-lacZ fusions. PCR primers were designed with KpnI (forward primers) and BamHI (reverse primers) restriction enzyme recognition sequences to facilitate cloning of the fragments. Fragment coordinates are relative to the vraSR tsp determined as shown in Fig. 1C. Each square filled with diagonal lines at the end of each fragment represents the amino terminus of the corresponding ORF to which lacZ was joined. VP-462 and VP-262 terminate with the sixth codon of orf1, VP1 and VP1′ terminate with the sixth codon of vraS, and VP2′ and VP2 terminate with the sixth codon of vraR. To optimize translation of lacZ in S. aureus, each fragment was fused upstream of, and in frame with, the 33rd codon of a 3,051-bp lacZ fragment isolated by PCR from pCMVβ (primer pairs in Table 2). Each PCR fragment was digested with KpnI and BamHI and ligated upstream of the lacZ fragment (Table 2) in pAW8 (18). (B) β-Galactosidase assays of VAN-specific induction of vraSR-lacZ fusions in wild-type strain RN4220 and vraSR mutant strain M1. The data are the means of at least three experiments. Error bars represent the standard deviations of the means. (C) β-Galactosidase assays of OXA (Ox)-specific induction of vraSR-lacZ constructs harbored in RN4220 or strain M1. β-Galactosidase specific activity was expressed as nanomoles of product formed per minute per mg of protein at 37°C.
- FIG. 3.
Analysis of pbp2-lacZ fusions by β-galactosidase assay in wild-type strain RN4220 and the vraSR mutant (M1). (A) Map of the pbp2 operon and PCR fragments (horizontal bars beneath map) encompassing its various regions that were fused upstream of and in frame with lacZ. The three tsp’s (P1, P1′, and P2) and various primers used in this study (half arrowheads) are shown relative to the prf and pbp2 ORFs. Coordinates are relative to the P1 tsp, with accession number AB035448 used as a reference sequence. The operon fragments in pPBP2-b, pPBP2-c, and pPBP2-d each terminate after the eighth codon of pbp2 (square filled with diagonal lines). The fragment in pPBP2-a terminates after the sixth codon of prfA (square filled with horizontal lines). (B) VAN-specific induction pbp2-lacZ fusions pPBP2-a (a), pPBP2-b (b), pPBP2-c (c), and pPBP2-d (d) in wild-type strain RN4220 and in M1. Con, RN4220 harboring vector containing only the lacZ insert. The data represent the means of at least three experiments. Error bars represent the standard deviations of the means. (C) OXA (Ox)-dependent induction of pbp2-lacZ fusions pPBP2-b (b) and pPBP2-c (c) in strains RN4220 and M1. β-Galactosidase specific activity was expressed as nanomoles of product formed per minute per mg of protein at 37°C.
- FIG. 4.
Disk diffusion assay for induction of pbp2- and vraSR-lacZ fusions. Strain RN4220 (Wt) and the vraSR mutant strain (M1) carrying pbp2-lacZ fusions (pPBP2-b, pPBP2-c) or a vraSR-lacZ fusion (pVP-462) were diluted to the optical density of a 0.5 McFarland turbidity standard (BBL), swabbed onto tryptic soy agar containing TET (10 μg/ml) and X-Gal, overlaid with antibiotic-containing disks (Sensi-Disc; BBL), and incubated at 37°C overnight. Subsequent incubation at 4°C for 2 days intensified the blue color. The concentrations of the antibiotics were as follows: VAN, 30 μg; CTX, 30 μg; CEC, 30 μg; OXA (OX), 1 μg; FOX, 30 μg; BAC, 10 μg; ERY, 2 μg.
Tables
- TABLE 1.
Strains and plasmids
Strain or plasmid Relevant featuresa Reference or source S. aureus strains RN4220 S. aureus intermediate host strain (restriction negative) for cloning (derived from strain 8325-4) 6 M1 vraSR mutant of RN4220 with ermC insertion in the EcoRI site of vraS, erythromycin resistant This study IL-A MRSA clinical strain 2 Plasmids pGEM-T Escherichia coli vector, ampicillin resistance (blaZ) Promega pAW8 S. aureus-E. coli shuttle vector, Tetr, intergenic region from pUC118, pAMα1 gram-positive ori and ColE1 ori from E. coli Akihito Wada pCMVβ Vector used as a source for lacZ gene Clontech pALC80 Used as a source for ermC gene; pSPT181 containing a 2.4-kb sar fragment carrying ermC gene, stable at 32°C Ambrose Cheung pCL52.1 E. coli-S. aureus shuttle plasmid; gram-positive Ts ori from pE194; Spxr; Tetr gene from pT181 Chia Lee (10) pVRASR-1 pGEM-T containing cloned vraSR PCR fragment This study pVRSASR-erm pVRASR-1 containing ermC in the EcoRI site of vraS This study pVRASR-erm-pCL52.1 PstI-digested pVRASR-erm ligated with PstI-digested pCL52.1 This study pVP1 pAW8 containing a translational fusion between the vraSR operon fragment, VP1, from strain IL-A and lacZ from pCMVβ This study pVP1′ pAW8 containing a translational fusion between the vraSR operon fragment, VP1′, from strain IL-A and lacZ from pCMVβ This study pVP2 pAW8 containing a lacZ translational fusion between the vraSR operon fragment, VP2, from MRSA strain IL-A and lacZ from pCMVβ This study pVP2′ pAW8 containing translational fusion between vraSR operon fragment, VP2′, from MRSA strain IL-A and lacZ from pCMVβ This study pVP-262 pAW8 containing a translational fusion between the VP-262 fragment from the vraSR operon from MRSA strain IL-A and lacZ from pCMVβ This study pVP-462 pAW8 containing a translational fusion between the VP-462 fragment from the vraSR operon from MRSA strain IL-A and lacZ from pCMVβ This study pPBP2-b pAW8 containing a translational fusion between the pbp2 operon fragment, PBP2-b, from MRSA strain IL-A and lacZ from pCMVβ This study pPBP2-c pAW8 containing a translational fusion between the pbp2 operon fragment, PBP2-c, from MRSA strain IL-A and lacZ from pCMVβ This study ↵a The lacZ fragment in all lacZ fusions begins at the 33rd codon; ori, origin of replication; Ts, temperature sensitive.
- TABLE 2.
Primer pairs used for producing PCR fragments
Fragment Primer Size (bp) Resulting plasmid Forward Reverse lacZ fragment for creating fusions lacF2-BamHI lacR1-PstI pVRASRerm-pCL52.1 vraSR operon fragments vraSR-F vraSR-R 1,700 pVRASRerm vraSR probe vraSRprobeF vraSRprobeR 2,022 None (not cloned) VP1 VraF1-KpnI VraR1-BamHI 1,183 pVP1 VP1′ VraF2-KpnI VraR1-BamHI 1,513 pVP1′ VP2 VraF3-KpnI VraR2-BamHI 1,076 pVP2 VP2′ VraF4-KpnI VraR2-BamHI 2,218 pVP2′ VP-262 VraF5-KpnI VraR3-BamHI 412 pVP-262 VP-462 VraF6-KpnI VraR3-BamHI 612 pVP-462 pbp2 operon fragments PBP2-a PBP2F1-KpnI PBP2R1-PstI 326 pPBP2-a PBP2-b PBP2F1-KpnI PBP2R2-PstI 955 pPBP2-b PBP2-c PBP2F2-KpnI PBP2R2-PstI 647 pPBP2-c PBP2-d PBP2F3-KpnI PBP2R2-PstI 431 pPBP2-d
HTML Page - index.htslp
Files in this Data Supplement:
- Supplemental file 1
-
Table of primer sequences
MS Word document, 44.5K
- Supplemental file 1
-
Table of primer sequences
