TABLE 1.
Bacterial strains and plasmids used in this study
| Strain, plasmid, or cosmid | Descriptiona | Source reference |
|---|---|---|
| Strains | ||
| Bacillus sp. strain ATCC 27860 | Indicator strain for alanyl clavam bioassay | 36 |
| E. coli ESS | Indicator strain for cephamycin C bioassay | A. L. Demain, Drew University, Madison, N.J. |
| E. coli BW25113/pIJ790 | Recombination host for Redirect PCR targeting system | 16 |
| K. pneumoniae ATCC 15380 | Indicator strain for clavulanic acid bioassay | 37 |
| S. clavuligerus NRRL3585 | Wild type | Northern Regional Research Laboratory, Peoria, Ill. |
| S. clavuligerus 4B and 4B-C | ceaS2 disruption mutant (ceaS2::apr) | 20 |
| S. clavuligerus O2FS | ceaS2 frameshift mutant | This study |
| S. clavuligerus orf3::apr | bls disruption mutant | 20 |
| S. lividans TK24 | Plasmid-less cloning host (SLP2− SLP3−), str-6 | D. A. Hopwood, John Innes Institute, Norwich, United Kingdom |
| Plasmids and cosmids | ||
| 6G9 | pWE15-derived cosmid carrying DNA fragment encoding ceaS1, bls1, pah1, and oat1 and flanking sequences | 22a |
| 14E10 | pWE15-derived cosmid carrying DNA fragment encoding ceaS1, bls1, pah1, and oat1 and flanking sequences | 22a |
| p2.8-18 | pUC18 containing 2.8-kb EcoRI fragment carrying part of ceaS1 | This study |
| p5K-6 | pUC118 containing 4.3-kb NcoI fragment carrying pah1, oat1, and upstream sequences but with an internal KpnI fragment deleted to remove pah1 | This study |
| p5.7 | pUC118 containing 5.7-kb EcoRI fragment carrying the 3′ end of ceaS1, all of bls1, pah1, and oat1, and flanking sequences | This study |
| p5.7-T | p5.7 containing tsr from pTSR#8 inserted at FseI site within bls1 | This study |
| p5.7-TH | E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 | This study |
| pApOrf6 | Disruption construct carrying oat2 disrupted with apr | 20 |
| pCAD2-3 | pUC120 carrying ceaS2 and bls2 | 20 |
| pCAD2-3(L1-5) | pCAD2-3 but with a frameshift mutation in ceaS2 | This study |
| pCAD2-3(L1-5)486 | E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 | This study |
| pFDNeo-S | pUC18 carrying neo from Tn5 | 13 |
| pIJ486 | Streptomyces plasmid vector, Tsrr | D. A. Hopwood |
| pIJ773 | Template plasmid for PCR targeting; acc(3)IV plus oriT | 16 |
| pJOE829 | Streptomyces plasmid vector; Hygr | J. Altenbucher, University of Stuttgart, Stuttgart, Germany |
| pNEO5K-6A | p5K-6 containing neo from pFDNeo-S inserted at RsrII site in oat1 | This study |
| pTSR#8 | pUC118 containing tsr gene from pIJ702 | 1 |
| pUC18 | E. coli cloning vector; Ampr | Stratagene |
| pUC118 | E. coli phagemid; Ampr | 44 |
| pUC120Apr | pUC120 containing the apr gene flanked by NcoI sites | 34 |
a
Hygr, hygromycin resistance; Ampr, ampicillin resistance.