TABLE 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristics or sequence (5′ to 3′)a | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| EC100λpir | supE44 ΔlacU169 (ϕ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 (λpirR6K) | Epicentre |
| SM10λpir | thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::mu Kmr (λpirR6K) | Lab collection |
| V. cholerae | ||
| JB58 | O1 El Tor strain N16961 ΔlacZ Smr | Lab collection |
| XBV222 | JB58 ΔleuO | 23 |
| DT733 | JB58 ΔtoxRS | 23 |
| SS4 | JB58 toxRΔppd | This study |
| Plasmids | ||
| pCM10 | luxCDABE reporter plasmid, Kmr | 51 |
| pJB906 | pCM10 containing leuO promoter | 23 |
| pTL61T | lacZ transcriptional reporter plasmid, Cbr | 52 |
| pXB266 | pTL61T containing leuO promoter region with two ToxR binding sites | |
| pVA258 | pTL61T containing leuO promoter region with one ToxR binding site | This study |
| pVA261 | pTL61T containing leuO promoter region with no ToxR binding sites | This study |
| pXB233 | pTL61T containing vexRAB promoter | 53 |
| pXB228 | pTL61T containing vexEF promoter | 54 |
| pXB229 | pTL61T containing vexGH promoter | 54 |
| pXB230 | pTL61T containing vexIJK promoter | 54 |
| pXB231 | pTL61T containing vexCD promoter | 54 |
| pXB232 | pTL61T containing vexLM promoter | 54 |
| pBAD18 | Arabinose-regulated expression plasmid, Cbr | 55 |
| pBAD33 | Arabinose-regulated expression plasmid, Cmr | 55 |
| pXB298 | pBAD18Km expressing leuO | 23 |
| pVA94 | pBAD18 expressing leuO | This study |
| pXB289 | pBAD18 expressing toxRS | 23 |
| pXB286 | pBAD18 expressing toxRΔppdS | 23 |
| pXB302 | pBAD33 expressing toxRS | This study |
| pDT1391 | pBAD33 expressing toxRΔppdS | This study |
| pWM91 | Suicide plasmid vector used for allelic exchange | 41 |
| pWM91::ΔtoxRppd | pWM91 containing a fragment of toxR harboring a deletion of periplasmic domain | This study |
| Oligonucleotides | ||
| PleuO1-F | CGCCCGGGAAATGCATTTTTATAGATTTTT | |
| PleuO2-F | CGCCCGGGAATCGTATTGATTATTAAGGCT | |
| PleuO-R | GGGGATCCGCGTCTTTTTTATCTAACATTTGCATGCCT | |
| toxRΔppd-F1 | GGGAGCTCGGTCCTCAAAAGAGATAT | |
| toxRΔppd-F2 | CTGCTCACTAACTAGGATCTTGCTAT | |
| toxRΔppd-R1 | AACCCGGGCATGCCGCTCAGTCAGG | |
| toxRΔppd-R2 | AGCAAGATCCTAGTTAGTGAGCAGTA | |
| 5′BIO | GCGGGAGTCGGCAGCG | |
| leuO-F-EMSA | GCGGGAGTCGGCAGCGGTTAAAACATTTTTGACGTGAATATTAGTG | |
| leuO-R-EMSA | GCGGGAGTCGGCAGCGCGTCACTAGCGATAAATATGCATAAATC | |
| ompU-F-EMSA | GCGGGAGTCGGCAGCGCAATTAGATTGCGTGCATTT | |
| ompU-R-EMSA | GCGGGAGTCGGCAGCGTTTTTTTACTCCCAAAGTTC | |
| vexR-F-EMSA | GCGGGAGTCGGCAGCGTGCAAAACAGGGGGTATTAG | |
| vexR-R-EMSA | GCGGGAGTCGGCAGCGGCCGTACACTATTTCAGACA | |
| leuO-qPCR-F | GACCACTTCGCCACAAATCACCA | |
| leuO-qPCR-R | CGTTGGATGGCGGAAAATGCG | |
| ompU-qPCR-F | ACACCGTATAGGCTGTCATTG | |
| ompU-qPCR-R | GTGCTGAAGCTCGCCTATCTC | |
| gyrA-qPCR-F | CAATGCCGGTACACTGGTACG | |
| gyrA-qPCR-R | AAGTACGGATCAGGGTCACG |
a
Kmr, kanamycin resistant; Cbr, carbenicillin resistant; Cmr, chloramphenicol resistant.