TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| JM109 | recA1 supE44 endA1 hsdR7 gyrA96 relA1 thi | 21 |
| BL21(DE3) | F− ompT hsdSB(rB− mB−) dcm gal (DE3) | Novagen |
| H. mediterranei | ||
| DF50 | pyrF deletion mutant of H. mediterranei ATCC 33500 | 22 |
| ΔphaP mutant | phaP deletion mutant of H. mediterranei DF50 | 20 |
| ΔphaR mutant | phaR deletion mutant of H. mediterranei DF50 | This study |
| ΔphaRP mutant | phaRP deletion mutant of H. mediterranei DF50 | This study |
| ΔphaEC mutant | phaEC deletion mutant of H. mediterranei DF50 | This study |
| ΔphaPEC mutant | phaPEC deletion mutant of H. mediterranei DF50 | This study |
| ΔphaRPEC mutant | phaRPEC deletion mutant of H. mediterranei DF50 | This study |
| E24A | DF50 strain with PhaR carrying E24A mutation | This study |
| Q28A | DF50 strain with PhaR carrying Q28A mutation | This study |
| Q30A | DF50 strain with PhaR carrying Q30A mutation | This study |
| R32A | DF50 strain with PhaR carrying R32A mutation | This study |
| K67A | DF50 strain with PhaR carrying K67A mutation | This study |
| R75A | DF50 strain with PhaR carrying R75A mutation | This study |
| E82A | DF50 strain with PhaR carrying E82A mutation | This study |
| R83A | DF50 strain with PhaR carrying R83A mutation | This study |
| Plasmids | ||
| pHFX | 4.0-kb integration vector containing pyrF and its native promoter, Ampr | 22 |
| pWL502 | 7.8-kb expression vector containing pyrF and its native promoter, Ampr | 20 |
| pSCM307 | 8.2-kb shuttle vector containing promoter of hsp5 of Halobacterium sp. strain NRC-1, Ampr | 26 |
| pJAM1020 | 10.7-kb expression plasmid containing smRSGFP gene, Ampr | 24 |
| pM1915 | 8.8-kb expression vector pWL502 containing smRSGFP gene and mutated promoter of PTS | 28 |
| pGEM-T Easy | 3.0-kb cloning vector, Ampr | Promega |
| pET-28a | 5.4-kb IPTG-inducible expression vector with His6 tag | Novagen |
| pDR | 5.6-kb integration vector of pHFX for knockout of phaR | This study |
| pDRP | 5.6-kb integration vector of pHFX for knockout of phaRP | This study |
| pDEC | 5.3-kb integration vector of pHFX for knockout of phaEC | This study |
| pDPEC | 5.4-kb integration vector of pHFX for knockout of phaPEC | This study |
| pDRPEC | 5.4-kb integration vector of pHFX for knockout of phaRPEC | This study |
| pR-IN | 5.2-kb integration vector of pHFX for knock-in of phaR | This study |
| pRF | 8.7-kb expression vector pWL502 containing smRSGFP gene and the phaRP promoter (−151 to +17) | This study |
| pEF | 8.7-kb expression vector pWL502 containing smRSGFP gene and phaEC promoter | This study |
| pWLR | 8.4-kb expression vector pWL502 containing phaR and phaRP promoter | This study |
| pWLP | 8.5-kb expression vector pWL502 containing phaP and phaRP promoter | 20 |
| pWLRP | 8.8-kb expression vector pWL502 containing phaRP and phaRP promoter | This study |
| pHP | 8.4-kb expression vector pWL502 containing phaP and hsp5 promoter | This study |
| pHRRF | 9.2-kb pRF-derived vector for additional expression of phaR under hsp5 promoter | This study |
| pRmyc | 8.3-kb pWL502 derived vector, expressing phaR-myc under mutated PTS promoter from pM1915 | This study |
| pT-Rpro | 2.9-kb pGEM-T Easy-derived cloning vector of phaRP promoter | This study |
| pM1 to pM16 | 8.7-kb pRF-derived vectors with mutations introduced into phaRP promoter | This study |
| pD41 | 8.6-kb pRF-derived vector with phaRP promoter truncated (−41 to +17) | This study |
| pD86 | 8.6-kb pRF-derived vector with phaRP promoter truncated (−86 to +17) | This study |
| pET-28aR | 5.7-kb pET-28a-derived vector, expressing phaR-His6 | This study |
| pET-28aP | 5.8-kb pET-28a derived vector, expressing phaP-His6 | This study |
| pR-IN-E24A | 5.2-kb integration vector of pHFX for knock-in of phaR with E24A mutation | This study |
| pR-IN-Q28A | 5.2-kb integration vector of pHFX for knock-in of phaR with Q28A mutation | This study |
| pR-IN-Q30A | 5.2-kb integration vector of pHFX for knock-in of phaR with Q30A mutation | This study |
| pR-IN-R32A | 5.2-kb integration vector of pHFX for knock-in of phaR with R32A mutation | This study |
| pR-IN-K67A | 5.2-kb integration vector of pHFX for knock-in of phaR with K67A mutation | This study |
| pR-IN-R75A | 5.2-kb integration vector of pHFX for knock-in of phaR with R75A mutation | This study |
| pR-IN-E82A | 5.2-kb integration vector of pHFX for knock-in of phaR with E82A mutation | This study |
| pR-IN-R83A | 5.2-kb integration vector of pHFX for knock-in of phaR with R83A mutation | This study |