TABLE 2.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| Strains | ||
| Nocardia interforma ATCC 21072 | Wild-type FOR-A producer | ATCC |
| S. kaniharaensis ATCC 21070 | Wild-type FOR-A producer | ATCC |
| S. resistomycificus NRRL 2290 | New FOR-A producer | NRRL |
| Salinispora arenicola CNS-205 | Potential FOR-A producer | 29 |
| S. candidus NRRL 3601 | Wild-type PRF-A producer | NRRL |
| Nocardia interforma PC1 | ATCC 21072 derivative with deletion of a 1.8-kb region (containing focI, foc1, and foc2) | This study |
| S. kaniharaensis ΔforT | ATCC 21070 derivative with in-frame deletion of forT | This study |
| S. candidus ΔprfT | NRRL 3601 derivative with in-frame deletion of prfT | This study |
| E. coli DH10B | F− mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara leu)7697 galU galK λ− rpsL nupG | Gibco-BRL |
| E. coli Rosetta(DE3)/pLysS | F− ompT hsdSB(rB− mB−) gal dcm λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) pLysS(Cmlr) | Novagen |
| E. coli ET12567(pUZ8002) | ET12567 containing helper plasmid pUZ8002 | 28 |
| Plasmids | ||
| pEASY-Blunt | pUCori lacZ f1 ori; Kan Amp | TransGen Biotech |
| pET28a | neo reppMB1; T7 promoter | Novagen |
| pET28a/forT | pET28a derivative carrying an NdeI-EcoRI fragment containing forT | This study |
| pET28a/forK | pET28a derivative carrying an NdeI-EcoRI fragment containing forK | This study |
| pET28a/prfT | pET28a derivative carrying an NdeI-EcoRI fragment containing prfT | This study |
| pET28a/prfK | pET28a derivative carrying an NdeI-EcoRI fragment containing prfK | This study |
| pET28a/nbtG | pET28a derivative carrying an NdeI-EcoRI fragment containing nbtG | This study |
| pCHW351 | pOJ446 derivative carrying a 2.8-kb XbaI-BglII left arm, a 2.9-kb BglII blunt right arm, and tsr for the replacement of focI, foc1, and foc2 | This study |
| pCHW352 | pCRISPR-Cas9 derivative carrying an XbaI-NcoI gRNA, a 2.0-kb left arm, and a 1.8-kb right arm for in-frame deletion of forT | This study |
| pCHW353 | pCRISPR-Cas9 derivative carrying an XbaI-NcoI gRNA, a 1.8-kb left arm, and a 2.2-kb right arm for in-frame deletion of prfT | This study |