TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype or relevant characteristicsa | Source or reference(s) |
|---|---|---|
| Strains | ||
| E. coli XL-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqlacZΔM15 Tn10(Tcr)] | Laboratory stock |
| S. aureus | ||
| Oxford (NCTC6571) | Wild-type laboratory strain | Laboratory stock |
| RN4220 | Restriction mutant of 8325-4 (UV-cured of prophages) used as primary recipient for plasmids propagated in E. coli; spontaneous mutant with 11-bp deletion in rsbU gene | 16, 28 |
| SH1000 | Derivative of 8325-4 carrying the intact rsbU gene | Gift of S. Foster |
| MMA6 | RN4220 nrdD::(pMM-1) Emr | This study |
| MM1B | RN4220 nrdD::(pMM-3) Emr Kmr | This study |
| MM1C | RN4220 nrdDG::Ωkm-2 Kmr | This study |
| MM1C+ | MM1C (pMM-5) Emr Kmr | This study |
| MSA6 | φ11/MMA6 × SH1000b Emr | This study |
| MS1C | φ11/MM1C × SH1000, Kmr | This study |
| Plasmids | ||
| pUC18 | Cloning vector; lacZ Apr | MBI Fermentas |
| PMUTIN-4 | pUC18-based suicidal vector for gram-positive bacteria; Pspac−lacZ PpenP-lacI Apr Emr | 51 |
| pAUL-A | Temperature-sensitive shuttle vector; lacZ Emr | 43 |
| pBR322::Ωkm-2 | pBR322 carrying the Ωkm-2 cassette; Apr Kmr | 38 |
| pMM-1 | PacI-NarI-digested pMUTIN-4 containing ∼900-bp PacI-NarI PCR-amplified nrdD fragment; Apr Emr | This study |
| pMM-2 | Product of the triple ligation of the PacI-NarI-digested pMUTIN-4, ∼1-kb PacI-EcoRI PCR-amplified 5′ end of nrdD, and ∼1-kb EcoRI-NarI PCR-amplified 3′ end of nrdG fragments; Apr Emr | This study |
| pMM-3 | EcoRI-digested pMM-2 containing the Ωkm-2 cassette; Apr Emr Kmr | This study |
| pMM-4 | PstI-XbaI-digested pUC18 containing ∼2.8-kb PCR-amplified complete nrdDG operon of RN4220; Apr | This study |
| pMM-5 | HindIII-BamHI-digested pAUL-A containing the HindIII-BamHI fragment of pMM-4; Emr | This study |
a
Abbreviations: Apr, Emr, Kmr, and Tcr, resistance to ampicillin, erythromycin/lincomycin, kanamycin, and tetracycline, respectively.
b
Transduction with phage φ11 from donor × recipient.