TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant genotype or characteristic(s) | Reference or source |
|---|---|---|
| Burkholderia sp. strains | ||
| SJ98 | PNP and 2C4NP utilizer, wild type | 13 |
| SJ98ΔpnpA | SJ98 mutant with pnpA gene deleted | This study |
| SJ98ΔpnpA1 | SJ98 mutant with pnpA1 gene deleted | This study |
| SJ98ΔpnpA(pRK415-pnpA) | pnpA gene complemented by pRK415-pnpA in SJ98ΔpnpA | This study |
| Pseudomonas sp. strain WBC-3 | PNP degrader, wild type | 6 |
| E. coli strains | ||
| DH5α | supE44 lacU169 (φ80lacZΔM15) recA1 endA1 hsdR17 thi-1 gyrA96 relA1 | Novagen |
| Rosetta(DE3)pLysS | F− ompT hsdS (rB− mB+) gal dcm lacY1(DE3)/pLysSRARE (Cmr) | Novagen |
| WM3064 | Donor strain for conjugation, 2,6-diaminopimelic acid auxotroph, thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341::(erm pir [wild type]) | 30 |
| Plasmids | ||
| pET-28a | Expression vector, Kanr | Novagen |
| pTnMod-Okm | Source of kanamycin resistance gene | 28 |
| pEX18Tc | Gene knockout vector, oriT+ sacB+ Tcr | 29 |
| pRK415 | Broad host range vector, Tcr | 32 |
| pET-pnpA | NdeI-XhoI fragment containing pnpA inserted into pET-28a | This study |
| pZWJJ009 | NdeI-XhoI fragment containing pnpA from WBC-3 inserted into pET-28a | 6 |
| pET-pnpA1 | NdeI-XhoI fragment containing pnpA1 inserted into pET-28a | This study |
| pET-pnpB | NdeI-XhoI fragment containing pnpB inserted into pET-28a | This study |
| pET-pnpCD | NdeI-XhoI fragment containing pnpCD inserted into pET-28a | This study |
| pET-pnpE | EcoRI-XhoI fragment containing pnpE inserted into pET-28a | This study |
| pET-pnpF | NdeI-XhoI fragment containing pnpF inserted into pET-28a | This study |
| pEX18Tc-pnpA | pnpA gene knockout vector containing two DNA fragments homologous to upstream and downstream regions of pnpA and kanamycin resistance gene | This study |
| pEX18Tc-pnpA1 | pnpA1 gene knockout vector containing two DNA fragments homologous to upstream and downstream regions of pnpA1 and kanamycin resistance gene | This study |
| pRK415-pnpA | Vector for pnpA gene complementation made by fusion of pnpA into pRK415 at KpnI/EcoRI restriction sites | This study |