Table 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Reagent | Description | Source or reference |
|---|---|---|
| E. coli | ||
| DH5α | Φ80 dLacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17(rk- mk+) supE44 relA1 deoRΔ(lacZYA-argF)U169 | Promega |
| S. aureus | ||
| RN4220 | rk- mk+; accepts foreign DNA | [20] |
| RN6390 | Prophage-cured wild-type strain | [21] |
| Newman | Wild-type clinical isolate | [22] |
| H803 | Newman sirA::Km; KmR | [30] |
| H1665 | Newman Δsfa::Km; KmR | [9] |
| H1666 | Newman Δsbn::Tet Δsfa::Km; TetR KmR | [9] |
| H1262 | Newman Δhts::Tet; TetR | [9] |
| H1497 | Newman sirA::Km Δhts::Tet; TetR KmR | [9] |
| H2131 | Newman sbnA::Tc ΔsfaABCsfaD::Km | This study |
| H1718 | Newman sbnB::Tc ΔsfaABCsfaD::Km | This study |
| Plasmids | ||
| pACYC184 | E. coli cloning vector; CmR | ATCC |
| pALC2073 | E. coli/S. aureus shuttle vector; ApR CmR | [26] |
| pAUL-A | Temperature-sensitive S. aureus suicide vector; EmR LcR | [25] |
| pDG1514 | pMTL23 derivative carrying tetracycline resistance cassette; ApR | [24] |
| pFB5 | pALC2073 derivative carrying sbnA; CmR | This study |
| pSED52 | pALC2073 derivative carrying sbnB; CmR | This study |
| Oligonucleotides* | ||
| Cloning of sbnA into pBC SK+ | ||
| sbnA5'-SacI | 5' TGAGCTCGATTCTGTAGGGCAAACACC 3' | |
| sbnA3'-KpnI | 5' TTGGTACCTCTAAGTAACGATCGCCTCG 3' | |
| Amplification/cloning of a tetracycline resistance cassette from pDG1513 | ||
| Tet5'-NsiI | 5' TTGTATATGCATACGGATTTTATGACCGATGA 3' | |
| Tet3' | 5' TGTGTGGAATTGTGAGCGGATAAC 3' | |
| Cloning of sbnA into pALC2073 | ||
| sbnA5'-XhoI | 5' TTTCTCGAGATTTTAAATTTGAGGAGGAA 3' | |
| sbnA3'-EcoRI | 5' TTTGAATTCCCACATAAACTTGTGAATGATT 3' | |
| Cloning of sbnB into pACYC184 | ||
| sbnB5'-BamHI | 5' TTGGATCCTAGTTTATTCAGATACATGG 3' | |
| sbnB3'-BamHI | 5' TTGGATCCTGTCCCAATATTTTGTTGTT 3' | |
| Cloning of sbnB into pALC2073 | ||
| sbnB5'-EcoRI | 5' TTGAATTCTCAAGTGATCCATGTAGATG 3' | |
| sbnB3'-EcoRI | 5' TTGAATTCCAATTCCGGCTATATCTTCA 3' | |
* underlined sequences in oligonucleotides denote restriction sites