TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant characteristics | Source or reference |
|---|---|---|
| E. coli strains | ||
| XL1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB+lacIqZΔM15::Tn10(Tetr)] | Stratagene |
| M15(pREP4) | NaIs Strs Rifs Δlac-ara-gal-mtl F−recA uvr (pREP4 lacI Kanr) | QIAGEN |
| Cloning vectors | ||
| pBluescript II SK(−) | E. coli phagemid cloning vector (Ampr) | Stratagene |
| pQE30 | E. coli expression vector with six-His tag 5′ of the polylinker (Ampr) | QIAGEN |
| pGEX-2T | E. coli expression vector with glutathione S-transferase tag 5′ of the polylinker (Ampr) | GE Biosciences |
| Plasmids | ||
| pCNF24 | Wild-type cnf1 gene (nucleotides 1 to 3045) amplified from pHLK102 and cloned into BamHI/KpnI sites of pQE30 | 21 |
| p2CNF | Coding region of cnf2 cloned into SacI/KpnI sites of pQE30 | This study |
| pEOSW30 | 3.3-kb BanI/HaeII fragment containing the cnf2 gene cloned into the SmaI site of pK184 | 23 |
| pΔN63 | pCNF24 with deletion of 187 nucleotides from the 5′ end of the cnf1 gene | 21 |
| pΔN75 | 2.8-kb BamHI/KpnI cnf1 fragment (nucleotides 226 to 3045) cloned into pQE30 | 21 |
| pΔN134 | 2.6- kb BamHI/KpnI cnf1 fragment (nucleotides 403 to 3045) cloned into pQE30 | 21 |
| pΔN272 | 2.2-kb BamHI/KpnI cnf1 fragment (nucleotides 817 to 3045) cloned into pQE30 | 21 |
| pΔN545 | 1.4-kb PstI cnf1 fragment (nucleotides 1637 to 3045) cloned into pQE30 | 21 |
| pΔC469 | 1.6-kb BamHI/PstI cnf1 fragment (nucleotides 1 to 1637) cloned into pQE30 | 21 |
| pΔ442 | pCNF24 with an internal in-frame BclI deletion (nucleotides 1115 to 2348) | 21 |
| pGEX-2T-LRP | Full-length lrp gene cloned into BamHI/EcoRI sites of pGEX-2T | 5 |
| pHisLRP | Full-length lrp gene subcloned into BamHI/XmaI sites of pQE30 | This study |