TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant genotype and/or phenotypea | Reference or source |
|---|---|---|
| Strains | ||
| S. aureus | ||
| BB255 | NCTC8325 derivative, rsbU | 3 |
| Newman | Clinical isolate (ATCC 25904), rsbU+ | 22 |
| COL | mec, high-Mcr clinical isolate; Mcr Tcr | 40 |
| RN4220 | NCTC8325-4 r− m+rsbU | 41 |
| GP266 | RN4220 rsbU+sigB1(Am), Tcr | 4 |
| GP267 | RN4220 (rsbU-V-W-sigB)+-tet(L), Tcr | 27 |
| GP268 | BB255 (rsbU-V-W-sigB)+-tet(L), Tcr | 27 |
| MB25 | RN4220 asp23+ (asp23p::luc+)-pEC-ermB, Emr | 27 |
| MB32 | Newman asp23+ (asp23p::luc+)-pEC-ermB Emr | 27 |
| MB55 | RN4220 (rsbU-V-W-sigB-luc+)+-erm(B), Emr | This study |
| MB58 | Newman (rsbU-V-W-sigB-luc+)+-erm(B), Emr | This study |
| MB89 | MB25 (rsbU-V-W-sigB)+-tet(L), Tcr, Emr | This study |
| MS64 | Newman sigB1(Am), Tcr | This study |
| E. coli | ||
| XL1Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15Tn10 (Tcr)] | Stratagene |
| Yeast | ||
| YRG-2 | MATaura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS::UASGAL1-TATAGAL1- HIS3 URA3::UASGAL4 17mers(×3)-TATACYC1lacZ | Stratagene |
| Plasmids | ||
| pAD-GAL4-2.1 | Yeast two-hybrid target plasmid, harboring the GAL4 activation domain, Apr | Stratagene |
| pBD-GAL4 Cam | Yeast two-hybrid bait plasmid, harboring the GAL4 DNA-binding domain, Cmr | Stratagene |
| pADrsbU | pAD-GAL4-2.1 derivative, harboring the rsbU ORF of COL cloned into the MCS in frame with the GAL4 DNA-binding domain, Apr | This study |
| pADrsbV | pAD-GAL4-2.1 derivative, harboring the rsbV ORF of COL cloned into the MCS in frame with the GAL4 DNA-binding domain, Apr | This study |
| pADrsbW | pAD-GAL4-2.1 derivative, harboring the rsbW ORF of COL cloned into the MCS in frame with the GAL4 DNA-binding domain, Apr | This study |
| pADsigB | pAD-GAL4-2.1 derivative, harboring the sigB ORF of COL cloned into the MCS in frame with the GAL4 DNA-binding domain, Apr | This study |
| pAW8 | S. aureus-E. coli shuttle vector harboring tet(L), Tcr | 27 |
| pBDrsbU | pBD-GAL4 Cam derivative, harboring the rsbU ORF of COL cloned into the MCS in frame with the GAL4 activation domain, Cmr | This study |
| pBDrsbV | pBD-GAL4 Cam derivative, harboring the rsbV ORF of COL cloned into the MCS in frame with the GAL4 activation domain, Cmr | This study |
| pBDrsbW | pBD-GAL4 Cam derivative, harboring the rsbW ORF of COL cloned into the MCS in frame with the GAL4 activation domain, Cmr | This study |
| pBDsigB | pBD-GAL4 Cam derivative, harboring the sigB ORF of COL cloned into the MCS in frame with the GAL4 activation domain, Cmr | This study |
| pBus1 | pAW8 with multicloning site from pBluescript II SK (Stratagene) and the rrnT14 terminator sequence from pLL2443, Tcr | 61 |
| pBus1-luc+ | pBus1 with a 1.7-kb Kpnl/EcoRI fragment of pSP-luc+, Tcr | This study |
| pBus1-sigBp2-luc+ | pBus1 with a 363-bp PCR fragment of the upstream region of rsbU fused to the reporter gene luc+, Tcr | This study |
| pCX15 | Xylose-inducible expression vector for staphylococci, Cmr | 71 |
| pEC4 | pBluescript KS+ (Stratagene) containing the 1.45-kb Clal erm(B) fragment of Tn551, Apr Emr | 10 |
| pIK7 | pUC18 with 6.6-kb Pstl/EcoRI S. aureus sigB fragment, Apr | 42 |
| pPG4 | plK7 with 1.45-kb Clal erm(B) fragment of pEC4 into blunted Bsp119I site downstream of sigB; Apr Emr | This study |
| pSP-luc+ | Firefly luciferase cassette vector, Apr | Promega |
| pSPslt | pSP-luc+ with rsbW and sigB upstream of luc+ and the sigB-sa2053 intergenic region containing the sigB terminator and erm(B) downstream of luc+; Apr Emr | This study |
| pXyl | pAW8 with 1.7-kb HindIII-BamHI fragment of pCX15 containing the xylR and the xylA promoter-operator region from S. xylosus, Tcr | This study |
| pXylrsbU | pXyl derivative containing rsbU from COL under the control of the xylose-inducible promoter Pxyl, Tcr | This study |
a
Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Mcr, methicillin resistant; MCS, multiple cloning site; Tcr, tetracycline resistant.