TABLE 1.
Bacterial strains and plasmids used
| Bacterial strains or plasmids | Relevant characteristicsa | Reference or source |
|---|---|---|
| Bacterial strains | ||
| E. coli C-5641 | Prototrophic, pnp::Tn5-Km | 35 |
| P. fluorescens N3 | Naf+ Sal+/pN3 | 5 |
| P. aeruginosa PAO11 | tfp-54 nal-19 fon-1 | B. W. Holloway Collection |
| P. putida TMB | Tmb+ Tol+/pGB | 3 |
| P. putida KT2440 | hsdR hdsM+ Ben+ Rifs | 2 |
| PPM101 | P. putida TMB pnp-1 (INS pKRF7A), Kanr Strr Sucs | This study |
| PPM103 | P. putida KT2440 pnp-1 (INS pKRF7A), Kanr Strr Sucs | This study |
| Plasmids | ||
| pGEM-4Z | Cloning vector | Promega |
| pGEM-4Z-Km1 | pHP45Ω-Km 2.2-kb BamHI fragment containing the Ω-Km interposon cloned in BamHI-digested pGEM-4Z | This study |
| pGeRF1 | P. putida pnp EcoRI (698)/XhoI (1268) fragment from pRF17 cloned in EcoRI/SalI-digested pGEM-4Z, 5′ end of pnp | This study |
| pGZ119EH (HE) | Cloning vector, Cmr | 29 |
| pHP45Ω-Km | pBR322 derivative carrying the Ω-Kmr interposon | 15 |
| pJB3Pnp2 | 2.5-kb SphI/SacI fragment from pRF17, containing the P. putida pnp gene, cloned in SphI/SacI-digested pJB3Tc19 | This study |
| pJB3Tc19 | Broad-host-range cloning vector, Apr/Tcr, RK2 oriT oriV | 6 |
| pKNG101 | Suicide cloning vector, R6K ori, RK2 (RP4) mob, strA strB (Srr) sacB (sucrose sensitivity) | 27 |
| pKRF7A | pMA9 3.8-kb BamHI/HindIII fragment, carrying the central part of P. putida pnp gene interrupted by Ω-Km interposon, cloned in SmaI-digested pKNG101 | This study |
| pMA5 | P. putida pnp fragment 889-2452 obtained by PCR with P. putida genomic DNA by using the FG218 and FG219 primers, cloned in HincII-digested pGEM-4Z | This study |
| pMA9 | Klenow-fragment-treated HindIII fragment of pGEM-4Z-Km1 containing the Ω-Km interposon cloned in HincII fragment of pMA5 interrupting the central fragment of pnp | This study |
| pRF14 | P. putida pnp fragment 1089-2888 obtained by PCR with P. putida genomic DNA by using the FG231 and FG393 primers, cloned in SmaI-digested pGZ119-EH; contains the 3′ end of pnp | This study |
| pRF16 | P. putida pnp fragment 396-2243 obtained by PCR with P. putida genomic DNA by using the FG390 and FG172 primers, cloned in SmaI-digested pGZ119-EH; contains the 5′ end of pnp | This study |
| pRF17 | 4.8-kb HindIII-EcoRI (partial digestion) fragment of pRF16 ligated to the 1.5-kb EcoRI-HindIII fragment of pRF14; contains the P. putida pnp gene (396-2857) reconstructed in pGZ119EH | This study |
| pURF10 | 215-bp P. putida pnp fragment amplified with degenerate primers FG143 and FG144, cloned in pUC18 SmaI | This study |
a
Numbers are coordinates. See Table 2 for oligonucleotides.