TABLE 1.
Bacterial strains, plasmids, and primers used in this study
The following abbreviations are used: ApR, ampicillin resistance; CmR, chloramphenicol resistance; EryR, erythromycin resistance; KmR, kanamycin resistance; TetR, tetracycline resistance; AUAP, abridged universal anchor primer; UAP, universal anchor primer.
| Bacterial strains, plasmids, oligonucleotides | Description | Source or Ref. |
|---|---|---|
| E. coli | ||
| DH5α | F-Ф80 dLacZΔM15 recA1 endA1 nupG gyrA96 glnV44 thi-1 hsdR17 (rk− mk+) λ−supE44 relA1 deoR Δ (lacZYA-argF)U169 | Promega |
| BL21(DE3) | F− ompT gal dcm lon hsdSB (rB− mB+)λ (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) | Novagen |
| S. aureus | ||
| RN4220 | rk−mk+ accepts foreign DNA | 47 |
| RN6390 | Prophage-cured wild-type strain | 48 |
| H306 | RN6390 sirA::Km; KmR | 25 |
| H984 | RN6390 sbnI::Tet; TetR | This study |
| H1312 | H984/pSbnI; sbnI mutant complemented with sbnI | This study |
| H1313 | H187 containing pCN51 | This study |
| H1317 | H984 containing pCN51 | This study |
| H1324 | RN6390 sbn; TetR | 23 |
| H1448 | RN6390 hts::Tet; TetR | 23 |
| Plasmids | ||
| pET28a(+) | Overexpression vector for His6-tagged proteins; KmR | Novagen |
| pCN51 | Cadmium-inducible expression vector; EryR | 49 |
| psbnI (pAB1) | pCN51-sbnI; pCN51 vector for expression of sbnI; EryR | This study |
| pGylux | Promoterless E. coli/S. aureus shuttle vector for luminescence, ApR, CmR | 33 |
| pCM326 | 1479-bp sbnA/B cloned into pGylux; ApR, CmR (fragment A) | This study |
| pCM328 | 2322-bp sbnA/B/C cloned into pGylux; ApR, CmR (fragment B) | This study |
| pCM330 | 2389-bp sbnC/D cloned into pGylux; ApR, CmR (fragment C) | This study |
| pCM332 | 2395-bp sbnD/E cloned into pGylux; ApR, CmR (fragment D) | This study |
| Oligonucleotides | ||
| Cloning of sbnI from S. aureus for pET28a(+) | GC AGC CAT ATG AAT CAT ATT GCT GAA CAT TTA A (forward) | |
| TTG TGT CTC GAG TCA TCA TAT TTC CCT CAA CAT (reverse) | ||
| Cloning of sbnI from S. aureus for complementing vector (psbnI) | TTG AGC GGA TCC CTT AAG CCA TCC ACA TCC TG (forward) | |
| TTG CGC GAA TTC TCG CTA TCA ATA CCG TAA ATC (reverse) | ||
| Cloning of fragment A | GCC TCC CGGGTCAATAAAATATTTATGATTTACATGC (forward) | |
| GACTCCCGGGAACTTGCTTCCATAACTGCAATT (reverse) | ||
| Cloning of fragment B | GACTCCCGGGTAATTTAGGCATTGCGTTG (forward) | |
| GACTCCCGGGCTGCAACCATTAAAGG (reverse) | ||
| Cloning of fragment C | GACTCCCGGGTGTCTGAACATTGCAG (forward) | |
| GACTCCCGGGAAATGAACGGCGAATAC (reverse) | ||
| Cloning of fragment D | GACTCCCGGGACTGACAGTACTTGT (forward) | |
| GACTCCCGGGCCTGTGTTACTAAAC (reverse) | ||
| RT-PCR: sbnA | ACTTCTGGTAATTTAGGCATTGCG (forward) | |
| TGCATCAGGTTCTTCAACCATTTC (reverse) | ||
| RT-PCR: sbnB | CCTGAAAATGGACACATCGC (forward) | |
| CAAAATAATGACGCCACTTGC (reverse) | ||
| RT-PCR: sbnC | ATGAATGGGAAGTGGCTCG (forward) | |
| GCTAATGGATGAAATGGACGAT (reverse) | ||
| RT-PCR: sbnD | CGTAGAAATACAGTTGTGGAGTGG (forward) | |
| AAATAAGCATACCGCCAAACC (reverse) | ||
| RT-PCR: sbnE | CGAATCCATTAGGGCAAACAG (forward) | |
| CTTAACATCGGTGAATCAGGC (reverse) | ||
| RT-PCR: sbnF | TTTTATGCGATGGAAGGG (forward) | |
| CGTCGTTTCTACTTTATCTTTGTCG (reverse) | ||
| RT-PCR: sbnG | TCATTAAAGTGTTGGATATGGGTGC (forward) | |
| TTAGCCATCTCCATTGCATCAAG (reverse) | ||
| RT-PCR: sbnH | CGCTCGCAATGCCAAAGATTC (forward) | |
| TAACGCCTATGCCACCACCAAG (reverse) | ||
| RT-PCR: rpoB | AGAGAAAGACGGCACTGAAAACAC (forward) | |
| ATAACGACCCACGCTTGCTAAG (reverse) | ||
| GSP1 | GAAAGCTGATGTGCTGTTAG | |
| GSP2 | GAGCCCGGGTCTAGACGCATACTTGAAGACGACAG | |
| AUAP | GATACCCGGGCCACGCGTCGACTAGTAC | |
| UAP | GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG | |