TABLE 1.
Bacterial strains, plasmids, and cosmids used in this study
| Bacterial strain, plasmid, or cosmid | Relevant genotype and/or phenotype | Source or reference |
|---|---|---|
| Strains | ||
| S. viridochromogenes | ||
| Tü494 | PTT-producing wild type | 4 |
| MphsA | Replacement of 1.7 kb of phsA; apr; no PTT production | This study |
| MtheA | Gene interruption of theA; apr; reduced PTT production | This study |
| MtheB | Gene interruption of theB; apr; reduced PTT production | This study |
| Tü494(ΔtheA/theB::aprP) | Replacement of theA and theB; apr; reduced PTT production | This study |
| E. coli | ||
| XL1 Blue | recA1 end A1 gyrA96 thi-1 hsdR17 supE44 relA1lac[F′ proAB lac1qZΔ M15 Tn10(tetr)] | 8 |
| Cosmids | ||
| pPtcos2 | Carries a part of the PTT biosynthetic gene cluster | 1 |
| Plasmids | ||
| pK18/19 | pUC derivative; aphII; lacZ′ complementation system | 29 |
| pRSETB | T7 promoter expression system, 6× His tag, bla | Invitrogen |
| pJOE890 | bla, two multiple cloning sites, two ter genes | 2 |
| pGEM-T-Easy | pUC-derivative, lacZ′ bla | Promega |
| pDRIVE | lacZ′ complementation system, ampicillin and kanamycin resistance, multiple cloning site | Qiagen |
| pGM190 | Streptomyces-E. coli shuttle vector, aphII tsr, inducible tipA promoter, medium copy number | G. Muth, personal communication |
| pWHM3 | Streptomyces-E. coli shuttle vector, trs bla | 47 |
| pDS1 | pUC18 derivative carrying the phsA gene | 35 |
| pDS3 | pQE30 derivative carrying the mutated phsA gene phsA2(Δ1-45), in which the first 135 bp are deleted | 34 |
| pEH13 | pUC21 derivative carrying the aprP resistance cassette | 17 |
| pEH15 | pK18 derivative carrying constitutive ermEp | 17 |
| pSE1 | pJOE890 derivative carrying mphsA1, the fragment upstream of phsA | This study |
| pSE2 | pJOE890 derivative carrying mphsA2, the fragment downstream of phsA | This study |
| pSE3 | pK18 derivative carrying mphsA1 | This study |
| pSE4 | pSE3 derivative carrying mphsA2 behind mphsA1 | This study |
| pSE5 | pSE4 derivative, carrying aprP inserted between mphsA1 and mphsA2 | This study |
| pSE6 | pK18 derivative carrying the 622-bp PstI fragment (phsA′) of the 5′ end of phsA | This study |
| pSE6* | pSE6 derivative carrying phsA1′(S16A) | This study |
| pSE7 | pDS1 derivative carrying phsA1(S16A) | This study |
| pSE10 | pGEM-T-Easy derivative carrying the fragment mtheA | This study |
| pSE11 | pJOE980 derivative carrying the fragment mtheB | This study |
| pSE12 | pK19 derivative carrying the fragment mtheA | This study |
| pSE13 | pK18 derivative carrying the fragment mtheB | This study |
| pSE14 | pSE12 derivative carrying aprP inserted into mtheA | This study |
| pSE15 | pSE13 derivative carrying aprP inserted into mtheB | This study |
| pSE16 | pGEM-T-Easy derivative carrying the theA′ gene | This study |
| pSE17 | pGEM-T-Easy derivative carrying the theB′ gene | This study |
| pSE18 | pRSETB derivative carrying the theA′ gene | This study |
| pSE19 | pRSETB derivative carrying the theB′ gene | This study |
| pSE20 | pEH15 derivative carrying the RBS of pRSETB followed by the theA′ gene | This study |
| pSE21 | pEH15 derivative carrying the RBS of pRSETB followed by the theB′ gene | This study |
| pSE22 | pWHM3 derivative carrying ermEp theA′ | This study |
| pSE23 | pWHM3 derivative carrying ermEp theB′ | This study |
| pSE31 | pK18 derivative carrying the mutated phsA gene phsA2(Δ1-45), in which the first 135 bp are deleted | This study |
| pSE32 | pRSETB derivative carrying phsA2(Δ1-45) | This study |
| pSE34 | pGM190 derivative carrying the mutated phsA gene phsA2(Δ1-45) | This study |
| pSE40 | pDRIVE derivative carrying the phsA gene | This study |
| pSE41 | pGM190 derivative carrying the native phsA gene | This study |
| pSE42 | pDRIVE derivative carrying phsA1(S16A) | This study |
| pSE43 | pGM190 derivative carrying the point-mutated phsA1(S16A) gene | This study |
| pSE44 | pDRIVE derivative carrying the fragment mΔA/B1 | This study |
| pSE45 | pDRIVE derivative carrying the fragment mΔA/B2 | This study |
| pSE46 | pK18 derivative carrying mΔA/B1 | This study |
| pSE47 | pSE46 derivative carrying mΔA/B2 behind mΔA/B1 | This study |
| pSE48 | pSE47 derivative carrying aprP inserted between mΔA/B1 and mΔA/B2 | This study |