TABLE 1.
Bacteria, phage, and plasmids used in this study
| Strain, phage, or plasmid | Relevant characteristic(s)a | Source and/or reference(s) |
|---|---|---|
| Strains | ||
| E. coli | ||
| XL1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqlacZΔM15 Tn10(Tcr)] | Laboratory stock |
| HB101 | supE44 hsdS20(rB− mB−) recA ara-14 proA2 lacY1 galK2 rspL20 xyl-5 mtl-1 | Laboratory stock |
| BL21(DF3) | Laboratory stock | |
| S. aureus | ||
| 8325-4 | Wild-type strain UV cured of prophages; spontaneous mutant with an 11-bp deletion in the rsbU gene (σB negative) | Laboratory stock; 10, 19 |
| SH1000 | Derivative of 8325-4 carrying the intact rsbU gene (σB positive); rsbU+ | Gift of S. Foster; 15 |
| RN4220 | Restriction mutant of 8325-4 used as a primary recipient for plasmids propagated in E. coli | Laboratory stock |
| RN4220 ΔnrdH | RN4220 in which the arcR gene was deleted and replaced by the Ωkm-2 kanamycin cassette; Kmr | This study |
| SH1000 ΔnrdH | Φ11/RN4220 ΔnrdH × SH1000b; Kmr | This study |
| Phage φ11 | Transducing phage that can infect S. aureus 8325-4 | Laboratory stock |
| Plasmids | ||
| pGEM-T Easy | EcoRV-linearized pGEM vector with added 3′-terminal T bases; Apr | Promega |
| pBR322::Ωkm-2 | pBR322 carrying the omega-Km2 cassette; Apr Kmr | 15 |
| pMUTIN-4 | pUC18-based suicidal vector for Gram-positive bacteria; Pspac-lacZ PpenP-lacI Apr Emr | 47 |
| pUP | PacI-BamHI-digested pMUTIN-4 containing ∼1.2 kb PCR amplified upstream of the nrdH region; Apr Emr | This study |
| pUP-DW | BamHI-Bsp120I-digested pUP and ∼1.2-kb PCR-amplified 3′ end downstream of the nrdH region; Apr Emr | This study |
| pUP_Km_DW (pKm1) | BamHI-digested pUP-DW containing the BamHI-flanked omega-Km2 cassette; Apr Emr Kmr | This study |
| pET-28a(+) and pET-30a(+) | T7-based protein expression vectors carrying a hexahistidine tag (His tag); Kmr | Novagen |
| pET-nrdH | pET-30a(+)::nrdH (C-terminal His tag); Kmr | This study |
| pET-nrdE | pET-28a(+)::nrdE (N-terminal His tag); Kmr | This study |
| pET-nrdF | pET-28a(+)::nrdF (N-terminal His tag); Kmr | This study |
| pOUA | pET-14b(+)::trxA (N-terminal His tag); Apr | O. Uziel et al., unpublished data |
| pOUB | pET-14b(+)::trxB (N-terminal His tag); Apr | Uziel et al., unpublished |
a
Apr, Emr, Kmr, and Tcr, resistance to ampicillin, erythromycin-lincomycin, kanamycin, and tetracycline, respectively.
b
Transduction with phage φ11 from donor × recipient.