TABLE 1.
Bacterial strains and plasmids used in this study
| Bacterial strains and plasmids | Relevant featuresa | Source or reference |
|---|---|---|
| Strains | ||
| S. clavuligerus | ||
| NRRL 3585 | Wild type; cephamycin and clavulanic acid producer | NRRL |
| E. coli | ||
| XLI-blue | General cloning host | Stratagene |
| ET12567(pUZ8002) | Methylation deficient; transfer functions from pUZ8002 | 15 |
| Plasmids | ||
| Cloning vectors | ||
| pBluescript II KS(+) | E. coli general cloning vector, Ampr | Stratagene |
| pSL1180 | E. coli general cloning vector, Ampr | Pharmacia |
| pTZ18R | E. coli general cloning vector, Ampr | US Biochemicals |
| pUC119 | E. coli general cloning vector, Ampr | 32 |
| pSET152 | E. coli replicon, Streptomyces φC31 attachment site, aptr | NRRL |
| pCR 2.1-TOPO | E. coli vector for cloning PCR products, Ampr | Invitrogen |
| pJV326 | par-defective, oriT-containing, Streptomyces-E. coli shuttle | 9 |
| pIJ486 | Streptomyces pIJ101 replicon, Tsrr | 33 |
| pMT3226 | pSET152 derivative, xylE controlled by gylP1P2 promoters | 10, 31 |
| Antibiotic resistance cassettes | ||
| pUC120apr | Aprr cassette in pUC120 | 23 |
| pSKNeo | Neor cassette in pBluescript II KS(+) | 20 |
| Intermediate constructs | ||
| K6L2 | E. coli cosmid carrying S. clavuligerus genomic DNA | 2 |
| p667-3PB | middle of orf18 to end of orf19 in pUC119 | This study |
| pCEC001 | Start of orf10 to middle of orf18 in pTZ18R | This study |
| pCEC004 | Middle of orf13 to middle of orf16 in pBluescript II SK(+) | This study |
| pCEC028 | Middle of orf12 to end of orf15 in pBluescript II SK(+) | This study |
| pCEC062 | End of orf17 to middle of orf18 in pBluescript II KS(+) | This study |
| Gene disruption constructs | ||
| pLOG221 | orf11::apr disruption construct | This study |
| pLOG 240 | orf12::apr disruption construct | This study |
| pCEC046 | orf14::apr disruption construct | This study |
| pCEC047 | orf13::apr disruption construct | This study |
| pCEC063 | orf15::apr disruption construct | This study |
| pCEC068 | orf16::apr disruption construct | This study |
| pCEC076 | orf17::apr disruption construct | This study |
| pCEC085 | orf18::neo disruption construct | This study |
| pCEC086 | orf15-Δfs replacement construct | This study |
| pCEC089 | orf19::apr disruption construct | This study |
| pCEC179 | Δorf16 replacement construct | This study |
| Gene insertion construct | ||
| pMT8.34 | pgyl::orf18 construct in pSET152 | This study |
a
Abbreviations: Amp, ampicillin; Apr, apramycin; Neo, neomycin; Tsr, thiostrepton.