Table 1.
Bacterial strains and plasmids used in this studya
| Strain or plasmid | Description | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | F−endA1 hsdR17 (rK− mK+) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacZYA argF)U169 φ80dlacZΔM15 | 38 |
| BL21(DE3) | hsdS gal (λcIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1) | 42 |
| V. cholerae strains | ||
| 569BSR | Classical Inaba, Smr, wild-type V. cholerae | 47 |
| WK3 | ΔphoBR mutant of 569BSR; Smr Kmr | 47 |
| Plasmids | ||
| pGEX4T-3 | High-copy vector containing an MCS preceded by GST coding region; Apr | GE Healthcare |
| pGEXphoWT | 0.7-kbp 569BSR phoB ORF inserted into BamHI/XhoI pGEX4T-3 restriction sites, fused 5′ to GST coding region; Apr | This work |
| pIC552 | Low-copy vector containing lacZ preceded by an MCS to insertion of a promoter region; Apr | 23 |
| pIC234 | pIC552 containing a region of 234 bp upstream of phoBR putative transcription start site regulating lacZ; Apr | 10 |
| pIC86 | pIC552 containing a region of 86 bp upstream of phoBR putative transcription start site regulating lacZ; Apr | This work |
| pIC62 | pIC552 containing a region of 62 bp upstream of phoBR putative transcription start site regulating lacZ; Apr | This work |
| pIC234mut | pIC552 containing the mutated region of 234 bp upstream of phoBR putative transcription start site regulating lacZ; Apr | This work |
| pIC86mut | pIC552 containing the mutated region of 86 bp upstream of phoBR putative transcription start site regulating lacZ; Apr | This work |
| pWKS30 | Low-copy vector; Apr | 49 |
| pGEM-T Easy | High-copy easy cloning vector; Apr | Promega |
| pWK5 | High-copy plasmid; contains phoBR operon, including entire regulatory region; Apr | 47 |
| pWK20 | 3 kbp pWK5 PstI fragment inserted into PstI pWKS30 restriction site; contains phoBR coding region plus 234-bp promoter; Apr | This work |
| pCG7 | pWK5 containing a SacII restriction site in the center of phoBR regulatory region; Apr | This work |
| pCG8 | 3-kbp pCG7 PstI fragment inserted into PstI pWKS30 restriction site; contains phoBR coding region plus 234-bp mutated promoter; Apr | This work |
| pLC1 | pWK5 reverse amplified, digested with BglII, and linked; contains phoBR plus 62-bp promoter; Apr | This work |
| pLC2 | pWK5 reverse amplified, digested with BglII, and linked; contains phoBR plus 86-bp promoter; Apr | This work |
| pLC3 | 2.3-kbp pLC1 BglII/PstI fragment, inserted into BamHI/PstI pWKS30 restriction sites; contains phoBR plus 62-bp promoter; Apr | This work |
| pLC4 | 2.3-kbp pLC2 BglII/PstI fragment, inserted into BamHI/PstI pWKS30 restriction sites; contains phoBR plus 86-bp promoter; Apr | This work |
| pLC5 | 2.3-kbp fragment of pCG7 amplified and cloned into vector pGEM-T Easy; contains phoBR plus 86-bp mutated promoter; Apr | This work |
| pLC6 | 2.3-kbp pLC5 NotI fragment, inserted into NotI pWKS30 restriction site; contains phoBR plus 86-bp mutated promoter; Apr | This work |
a
Sm, streptomycin; Km, kanamycin; Ap, ampicillin; Cm, chloramphenicol; MCS, multiple-cloning site; ORF, open reading frame.