TABLE 1.
Bacterial strains, plasmids, and PCR primers used in this study
| Strain, plasmid, or primer | Relevant genotype or phenotype or sequence | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | F−endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1(A) Δ(argF-lac)U169 φ80dlacZΔM15 | BRL |
| BL21(DE3) | F−ompT hsdSB (rS− mS−) gal dcm (DE3) | Novagen |
| S. aureus strains | ||
| 8325 | NCTC 8325 (wild type, 11-bp deletion in rsbU) | Laboratory stock |
| RN4220 | NCTC 8325-4-r (restriction mutant, 11-bp deletion in rsbU) | Laboratory stock |
| 8325ΔpknB | pknB deletion in strain 8325 | This study |
| 8325ΔpknB/pRB473pknB | 8325 containing pRB473pknB for ΔpknB complementation | This study |
| COLΔpknB | pknB deletion strain COL | This study |
| Plasmids | ||
| pBT2 | Shuttle vector, Apr in E. coli, Cmr in S. aureus | 6 |
| pBT2ΔpknB | Deletion vector for pknB, ermB fragment flanked by fragments upstream and downstream of pknB in pBT2, Emr and Cmr in S. aureus | This study |
| pGEM-T | Apr | Promega |
| pEC1 | Apr EmrermB fragment in pUC18 | 6 |
| pRB473 | Shuttle vector, Apr Cmr | 6 |
| pRB473pknB | pRB473 containing pknB fragment for ΔpknB complementation | This study |
| pKG31 | Apr Emr EcoRI-PstI fragment of ermB in pGEM-T | This study |
| pET-28a(+) | His6 expression vector, Kanr | Novagen |
| pET-28aPknB | Encodes His6-PknB, cloned in pET28a(+), Kanr | This study |
| pET28aPurA | Encodes His6-PurA, cloned in pET28a(+), Kanr | This study |
| Primers | ||
| pknBEcoRI | 5′-CGGAATTCTATCACCTTCAATAGCCGCG-3′ | |
| pknBHindIII | 5′-CCCAAGCTTGTTTGGTGGTGTGAATGACC-3′ | |
| pknBBamHI | 5′-CGGGATCCAATAAATTCAGTCTCATAGCC-3′ | |
| pknBPstI | 5′-AACTGCAGAGTGACGATATTGATGAGGG-3′ | |
| ermBEcoRI | 5′-CGGAATTCGGTGACATCTCTCTATTGTG-3′ | |
| ermBPstI | 5′-AACTGCAGGGAAGCTGTCAGTAGTATACC-3′ | |
| SA1063-fw | 5′-AGCCATATGATAGGTAAAATAATAAATGAACG-3′ | |
| SA1063-rev | 5′-CCGCTCGAGTTATACATCATCATAGCTGACTTC-3′ | |
| SA0016-fw | 5′-GCTAGCTCATCAATCGTAGTAGTTGGG-3′ | |
| SA0016-rev | 5′-CGCCCTAGGCTACCACAATTCTTTTAATAGG-3′ |