TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain or plasmid | Relevant genotype and/or phenotype | Source or reference |
|---|---|---|
| V. cholerae | ||
| 569B | Wild type (O1 classical) | 3 |
| C6709 | Wild type (O1 E1 Tor); Smr | J. J. Mekalanos |
| C6709-R | C6709 Smr Rifr | This study |
| SHK17 | C6709 ΔrelA::kan Smr Kmr | This study |
| E. coli | ||
| DH5α | F′ endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 Δ(argF-lacZYA)U169 (φ80dlacZΔM15) | Promega |
| CF1648 | Wild-type MG1655 | 52 |
| CF1652 | MG1655 relA251 Kmr | 52 |
| CF1693 | MG1655 spoT207 relA251 Kmr Cmr | 52 |
| SM10λpir | thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpir R6K | 36 |
| Plasmids | ||
| pBluescript KS(+) | ColE1; high-copy-number cloning vector; Apr | Stratagene |
| pUC4K | Source of the kanamycin resistance gene cassette; Apr Kmr | Pharmacia |
| pGP704 | Suicide vector; pBR322 derivative with oriR6K mobRP4; Apr | 36 |
| pRELVCH | 3.21-kb PCR-amplified relA gene region of V. cholerae strain 569B in pBluescript KS(+); Apr | This study |
| pSHC4 | 1.7-kb HindIII fragment of pRELVCH cloned into the HindIII site of pBluescript KS(+); Apr | This study |
| pSH99.5 | 418-bp HindIII-PstI fragment of pRELVCH; end filled and cloned into EcoRV site of pBluescript KS(+); Apr | This study |
| pSH99.6 | 1.13-kb PstI-MluI fragment of pRELVCH; end filled and cloned into EcoRV site of pBluescript KS(+); Apr | This study |
| pSHEG17 | 1.24-kb EcoRV-HincII fragment of pRELVCH; cloned into the EcoRV site of pGP704; Apr | This study |
| pSHK17 | 660-bp NsiI fragment of the relA gene of V. cholerae present in pSHEG17 was replaced by 1.24-kb PstI fragment carrying the kanamycin resistance gene cassette of pUC4K; Apr Kmr | This study |
| Oligonucleotides | ||
| VCR-F | 5′-ACGCCACTCATAACGCGAAATT-3′ | This study |
| VCR-R | 5′-CCAAGGCTATCCCAATCAAATC-3′ | This study |
| ctxA-F | 5′-CTCAGACGGGATTTGTTAGGCACG-3′ | This study |
| ctxB-R | 5′-GGTTGCTTCTCATCATCGAACCAC-3′ | This study |
| tcpA-F | 5′-CACGATAAGAAAACCGGTCAAGAG-3′ | This study |
| tcpA-R | 5′-GAAAGGACCTTCTTTCACGTTG-3′ | This study |
| toxT-F | 5′-ACTGTCGACGCAAAGCATATTCAGAGA-3′ | This study |
| toxT-R | 5′-CGCGGATCCATACAATCGAAAATAGGA-3′ | This study |
| toxR-F | 5′-CGGGATCCATGTTCGGATTAGGACAC-3′ | This study |
| toxR-R | 5′-CGGGATCCTACTCACACACTTTGATGGC-3′ | This study |
| PMBEX1 | 5′-ACGAATTCAATGGTTGCGGTACG-3′ | This study |