Table 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristics | Reference or source |
|---|---|---|
| Strains | ||
| E. coli DH5α | λ− φ80dlacZΔM15 Δ(lacZYA-argF)U169recA1endA1 hsdR17(rK− mK−) supE44thi-1gyrArelA1 | Invitrogen |
| E. coli BL21 | F−ompThsdS (rB− mB−) gal | Amersham |
| E. coli M15 | Nals Sms Rifs Thi− Lac− Ara+ Gal+ Mtl− F− RecA+ Uvr+ Lon+ | Qiagen |
| P. putida S12 | Wild-type; srpABC+srpR+srpS+ | 14 |
| P. putida JK1 | srpB::TnMod-KmO | 19 |
| P. putida S12 lacZ | srpABC+srpS+srpR+lacZ, in a single-copy transcriptional fusion behind PsrpA | This study |
| P. putida SrpS−lacZ | srpABC+srpS mutant srpR+lacZ, in a single-copy transcriptional fusion behind PsrpA | This study |
| P. putida SrpR−lacZ | srpABC+srpS+srpR mutant lacZ, in a single-copy transcriptional fusion behind PsrpA | This study |
| Plasmids | ||
| pCR2.1-TOPO | TOPO TA cloning vector for direct insertion of PCR products, blue/white screening; Apr Kmr | Invitrogen |
| pJD101 | Derived from a BamHI digestion of P. putida JK1 chromosome; contains srpR through a partial open reading frame of TnMod-KmO plasposon-mutated srpB | 19 |
| pJD102 | Derived from a PstI digestion of P. putida JK1 chromosome; contains a partial open reading frame of TnMod-KmO plasposon-mutated srpB through srpC | 19 |
| pJD203 | Constructed by digestion (EcoRV and BamHI) and ligation of pJD101 and pJD102; contains a partial open reading frame of TnMod-KmO plasposon-mutated srpB through srpC | This study |
| pJD500 | Apr Kmr; promoterless trp-lacZ fusion downstream of the srpA promoter, for the construction of single-copy chromosomal lacZ reporter fusions | This study |
| pGEX-4T-1 | Apr; cloning vector for overexpression of N-terminal GST fusion proteins | Amersham |
| pGEX-srpR | Entire coding region of srpR cloned into pGEX-4T-1 (BamHI-EcoRI) | This study |
| pQE31 | Apr; cloning vector for overexpression of N-terminal His6 fusion proteins | Qiagen |
| pQE31-srpS | Entire coding region of srpS cloned into pQE31 (BamHI-KpnI) | This study |
| pQE31-arpR | Entire coding region of arpR cloned into pQE31 (SstI-PstI) | This study |
| pREP4 | Kmr; introduced into E. coli M15; constitutively expresses LacI | Qiagen |