TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s)a | Source or reference |
|---|---|---|
| Comamonas sp. | ||
| E6 | Wild type, TPA+ | 30 |
| EME009 | E6 derivative, ΔtphRItphCI | This study |
| EME010 | EME009 derivative, ΔtphRII | This study |
| EME011 | EME009 derivative, ΔtphA2IA3IBIA1ItphA2II | This study |
| E. coli | ||
| DH5α | supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 12 |
| XL2-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tcr) Amy Cmr] | Stratagene |
| BL21(DE3) | F−ompT hsdSB(rB− mB−) gal dcm (DE3), T7 RNA polymerase gene under control of the lacUV promoter | 35 |
| Plasmids | ||
| pBluescript II SK(+) | Cloning vector, Apr | 34 |
| pT7Blue | Cloning vector, Apr T7 promoter | Novagen |
| pK19mobsacB | oriT sacB Kmr | 31 |
| pKT230MC | IncQ broad-host-range cloning vector, Kmr | 25 |
| pJB866 | RK2 broad-host-range expression vector, Tcr PmxylS | 2 |
| pET16b | Expression vector, N-terminal His10 tag, Apr T7 promoter | Novagen |
| pPR9TZ | Broad-host-range promoter probe vector, RK2 ori Apr Cmr | 17 |
| pSS92R | pBluescript II SK(+) with a 4.0-kb SalI fragment carrying tphRI and tphCI | This study |
| pK19RCI | pK19mobsacB with a 2.5-kb EcoRV-HindIII fragment of pSS92R | This study |
| pDTRCI | pK19RCI with a deletion of a 0.5-kb XbaI-SmaI fragment carrying the tphRI-tphCI intergenic region | This study |
| pEJ89 | pJB866 with a 8.9-kb EcoRI fragment carrying tphRIICIIA2IIA3IIBIIA1II | 30 |
| pEJ84 | pEJ89 with a deletion of a 0.6-kb MunI-Tth111I fragment carrying part of tphRII | 30 |
| pDTRII | pK19mobsacB with a 2.3-kb EcoRI-HindIII fragment of pEJ84 | This study |
| pK19A2II | pK19mobsacB with a 2.0-kb HindIII-XhoI fragment carrying tphA2II | This study |
| pDTA2II | pK19A2II with a deletion of a 0.5-kb SphI-PstI fragment carrying part of tphA2II | This study |
| pK19ABIF | pK19mobsacB with a 4.3-kb HindIII fragment carrying tphA2IA3IBIA1I | This study |
| pDTABI | pK19ABIF with a deletion of a 1.9-kb PstI fragment carrying tphA2IA3IBIA1I | This study |
| pKTRII | pKT230MC with a 1.8-kb KpnI fragment carrying tphRII | This study |
| pKTtphII | pKT230MC with a 8.9-kb EcoRI fragment carrying tphRIICIIA2IIA3IIBIIA1II | This study |
| pTRII | pT7Blue with a 779-bp PCR fragment generated by the TphRII-F and TphRII-R primer pair | This study |
| pETtprII | pET16b with a 772-bp NdeI-XhoI fragment of pTRII | This study |
| pTTC | pT7Blue with a 393-bp PCR fragment carrying the tphRII-tphCII intergenic region | This study |
| pZTC | pPR9TZ with a 381-bp HindIII-KpnI fragment of pTTC | This study |
| pTTR | pT7Blue with a 500-bp PCR fragment carrying the tphRII-tphCII intergenic region | This study |
| pZTR | pPR9TZ with a 379-bp HindIII-KpnI fragment of pTTR | This study |
| pTTCT5C | Site-directed mutant of pTTC generated by the SMT5C-F and SMT5C-R primer pair | This study |
| pTTCG6A | Site-directed mutant of pTTC generated by the RM315-F and RM315-R primer pair | This study |
| pTTCC7T | Site-directed mutant of pTTC generated by the SMC7T-F and SMC7T-R primer pair | This study |
| pTTCG8A | Site-directed mutant of pTTC generated by the SMG8A-F and SMG8A-R primer pair | This study |
| pT5C | pPR9TZ with a 381-bp HindIII-KpnI fragment of pTTCT5C | This study |
| pG6A | pPR9TZ with a 381-bp HindIII-KpnI fragment of pTTCG6A | This study |
| pC7T | pPR9TZ with a 381-bp HindIII-KpnI fragment of pTTCC7T | This study |
| pG8A | pPR9TZ with a 381-bp HindIII-KpnI fragment of pTTCG8A | This study |
a
Abbreviations: TPA+, able to grow on TPA as the sole carbon source; Apr, Kmr, Tcr, and Cmr, resistance to ampicillin, kanamycin, tetracycline, and chloramphenicol, respectively.