TABLE 1.
Strains and plasmids
| Strain or plasmid | Description | Reference or source |
|---|---|---|
| Strains | ||
| E. coli S17-1λpir | F− recA hsdR RP4-2 (Tc::Mu) (Km::Tn7) lysogenized with λpir | 23 |
| V. cholerae | ||
| C7258 | Wild-type O1, El Tor biotype, Ogawa | Peru, 1991 |
| C7258ΔlacZ | C7258 lacZ deletion mutant | 48 |
| AJB50 | C7258 ΔrpoS | 52 |
| AJB51 | C7258 ΔhapR | 52 |
| AJB51ΔlacZ | AJB51 ΔlacZ deletion mutant | 48 |
| HX11 | C7258 ΔlacZ ΔrpoS::rpoS-lacZ | This study |
| HX12 | C7258 ΔlacZ ΔhapR ΔrpoS::rpoS-lacZ | This study |
| HX13 | C7258 ΔvpsR | This study |
| HX14 | C7258 ΔhapR ΔvpsR | This study |
| HX15 | C7258 ΔlacZ ΔvpsR ΔrpoS::rpoS-lacZ | This study |
| HX16 | C7258 ΔlacZ ΔhapR ΔvpsR ΔrpoS::rpoS-lacZ | This study |
| HX17 | C7258 ΔvpsT | This study |
| HX18 | C7258 ΔvpsR ΔvpsT | This study |
| HX19 | C7258 ΔlacZ ΔvpsT ΔrpoS::rpoS-lacZ | This study |
| HX20 | C7258 ΔlacZ ΔhapR ΔvpsT ΔrpoS::rpoS-lacZ | This study |
| HX21 | C7258 ΔlacZ ΔvpsR ΔvpsT ΔrpoS::rpoS-lacZ | This study |
| HX03 | C7258 ΔhapR ΔvpsT | 22 |
| HX22 | C7258 vpsT::pCVDVpsT-FLAG | This study |
| HX23 | C7258 ΔvpsR vpsT::pCVDVpsT-FLAG | This study |
| Plasmids | ||
| pVIK111 | Suicide vector with promoterless lacZ; oriR6K Kmr | 27 |
| pCR-RpoS | 0.34-kb DNA fragment containing the rpoS promoter in pCR2.1 | This study |
| pRpoS-Up | 0.8-kb SmaI-SalI DNA fragment encoding the rpoS promoter and the N-terminal 223 amino acids of RpoS in pUC19 | This study |
| pRpoS-Down | 0.45-kb SphI-XbaI DNA fragment containing the 3′ terminus of the rpoS ORF in pUC19 | This study |
| pLacZ | 3.2-kb SalI-SphI DNA fragment encoding the lacZ ORF from pVIK111 in pUC19 | This study |
| pRpoS-Up-lacZ | 3.2-kb SalI-SphI DNA fragment from pLacZ in pRpoS-Up | This study |
| pCVDRpoS-Up-lacZ | 4-kb SmaI-SphI DNA fragment from pRpoS-Up-lacZ in pCVD442 | This study |
| pCVDΔRpoS-lacZ | 0.45-kb SphI-XbaI DNA fragment from pRpoS-Down in pCVDRpoS-Up-lacZ | This study |
| pUC-VpsR-UP | 0.5-kb XbaI-SalI DNA fragment containing the 5′ terminus of vpsR in pUC19 | This study |
| pUC-VpsR-Down | 0.5-kb SalI-SphI DNA fragment containing the 3′ terminus of vpsR in pUC19 | This study |
| pUCΔvpsR | XbaI-SalI and SalI-SphI DNA fragments flanking the vpsR ORF sequentially cloned into pUC19 | This study |
| pCVDΔvpsR | XbaI-SphI fragment harboring a vpsR deletion in pCVD442 | This study |
| pAT1662 | DGC VCA0956 cloned into pBAD33 | 25 |
| pCR2.1 | Bacterial expression vector for TA cloning (Ampr) | Invitrogen |
| pCR-VpsT1 | 0.67-kb XhoI-KpnI DNA fragment encoding the vpsT ORF in pCR2.1 | This study |
| pCR-VpsT2 | 0.67-kb NdeI-SapI PCR fragment encoding the vpsT ORF in pCR2.1 | This study |
| pTXB1 | Expression vector for construction of in-frame fusions with the intein/chitin binding domain | New England BioLabs |
| pTXB1-VpsT | Vector pTXB1 containing a VpsT-intein fusion | This study |
| pFLAG-CTC | Vector for cytoplasmic expression of C-terminal FLAG fusions | Sigma-Aldrich |
| pFLAG-CTC* | pFLAG-CTC with modified multicloning site lacking the SalI restriction site | This study |
| pTT3 | rrnB T1T2 transcription terminator in pUC19 | 28 |
| pTT7 | 0.43-kb SalI-BamHI DNA fragment encoding the rrnB T1T2 transcription terminator in pUC19 | This study |
| pTT7VpsT-FLAG | 0.73-kb BamHI-SmaI DNA fragment containing the vpsT ORF and FLAG epitope coding region in pTT7 | This study |
| pCVDVpsT-FLAG | SalI-SmaI vpsT-FLAG and rrnB T1T2 fragment in pCVD442 | This study |
| pRpoSLac5 | An SphI-HindIII DNA fragment containing the rpoS promoter region cloned between the rrnB T1T2 transcription terminator and a promoterless lacZ gene in pKRZ1a | 29 |
| pACTluz-RpoS | rrnB T1T2-rpoS promoter fragment from pRpoSLac5, promoterless luxCDABE operon, and OriT in pACYC184 | This study |
| pCVDΔvpsT-Km | pCVD442-based vector with vpsT deletion and Km insertion | 22 |
| pCMW75 | V. harveyi IPTG-inducible DGC QrgB expression vector | 20 |
a
See reference 53.