TABLE 1.
Bacterial strains and plasmids used
| Bacterial strain or plasmid | Genotype or phenotype | Reference or source |
|---|---|---|
| Strains | ||
| P. putida | ||
| TW3 | Wild type, 4NT+a Pnb+ | 35 |
| PaW340 | Trp− Strr plasmid-free derivative of P. putida mt-2 (PaW1) | 20 |
| E. coli | ||
| DH5α | φ80dlacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17(rK− mK+elA1 deoR Δ(lacZYA-argF)V169 | Promega |
| DH5αPRO | deoR endA1 gyrA96 hsdR17 (rK− mK+) recA1 relA1 supE44 thi-1 Δ(lacZYA-argFV169) φ80dlacZΔM15 F− λ− PN25/tetR PLacIq/LacI Spr | Clontech |
| MHB101 | F′ thi-1 hsdS20 (rB− mB−) supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 rpsL20 (Strr) xyl-5 mtl-1 | Promega |
| Plasmids | ||
| pLAFR5 | IncP Tcr; derivative of pLAFR3 containing a double cos cassette | 22 |
| pRK2013 | IncP Tra Rk2+ ΔrepRk2 repEI+ Kmr | 13 |
| pPROLar.A122 | Plac/ara-1 Kmr p15A origin | Clontech |
| pHN150 | 1.9-kbp XbaI fragment in pUC19 containing protocatechuate 4, 5-dioxygenase genes (ligA and ligB) | 33 |
| pTW3.11 | 6.6-kbp EcoRI clone containing orf1, pnbR, pnbB, and orf2 in pUC18 | This study |
| pTW3.12 | 3.6-kbp EcoRI/SacI subclone of pTW3.11 | This study |
| pTW3.13 | 6-kbp SacI/SmaI clone containing pnbA, orf1, pnbR, pnbB, and orf2 in pLAFR5 | This study |
| pTW3.14 | 4.9-kbp SmaI subclone of pTW3.13 | This study |
| pTW3.15 | 3.4-kbp SalI/PstI subclone of pTW3.14 | This study |
| pTW3.15NX | pTW3.15 with frameshift introduced in XhoI site to create knockout of pnbA | This study |
| pTW3.15NH | Deletion of 366-bp HindIII fragment in pTW3.15 to create knockout of orf1 | This study |
| pTW3.16 | Insertion of PCR fragment carrying pnbB into EcoRI site of pUC18 | This study |
| pPROpnbA | KpnI/BamHI fragment containing pnbA in pPROLar.A122 | This study |
a
4NT, 4-nitrotoluene.