TABLE 1.
Strains and plasmids used in this study
| Strains/Plasmids | Descriptions | References |
|---|---|---|
| E. coli strains | ||
| DH5αλpir | F‐ Δ(lacZYA‐argF)U169 recA1 endA1 hsdR17 supE44 thi‐1 gyrA96 relA1 λ::pir | Hanahan, (1983) |
| SM10λpir | thi thr leu tonA lacY supE recA::RPA‐2‐Te::Mu λpirR6K, Kmr | Miller and Mekalanos (1988) |
| XL1‐Blue | F ‐ ::Tn10 proA+B+ lacq Δ(lacZ)M151 recA1 endA1 gyrA46 (Nalr) thi hsdR17 (rK−mK+) supE44 relA1 lac | Bullock et al. (1987) |
| BL21 (DE3) | fhuA2 [lon] ompTgal (λ DE3) [dcm] ∆hsdS λ DE3=λsBamHIo ∆EcoRI‐B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 | NEB |
| W3110 ΔcyaA | F‐ λ‐ rpoS(Am) rph‐1 Inv(rrnD‐rrnE) ΔcyaA::scar | Herbst et al. (2018) |
| V. cholerae strains | ||
| WT | P27459‐S, O1 Inaba, El Tor, clinical isolate, Bangladesh 1976, spontaneous Smr | Pearson et al. (1993) |
| ΔtoxR | P27459‐S with deletion in toxR, Smr | Fengler et al. (2012) |
| ΔtoxRS | P27459‐S with deletion in toxR and toxS, Smr | Fengler et al. (2012) |
| toxRCC | P27459‐S with toxR replaced by FLAG‐toxRC236SC293S, Smr | Fengler et al. (2012) |
| toxRC236S | P27459‐S with toxR replaced by FLAG‐toxRC236S, Smr | This study |
| toxRC293S | P27459‐S with toxR replaced by FLAG‐toxRC293S, Smr | This study |
| toxRC293W76RS | P27459‐S with toxR replaced by FLAG‐toxRC293SW76R, Smr | This study |
| ΔdsbA | P27459‐S with dsbA replaced by km cassette, Smr, Kmr | Fengler et al. (2012) |
| dsbC::pGP | P27459‐with dsbC inserted by pGP704, Smr, Apr | Fengler et al., (2012) |
| ΔdsbA toxRC293S | P27459‐S ΔdsbA with toxR replaced by FLAG‐toxRC293S, Smr , Kmr | This study |
| dsbC::pGP toxRC293S | P27459‐S dsbC::pGP with toxR replaced by FLAG‐toxRC293S, Smr , Apr | This study |
| ΔdegP | P27459‐S with degP replaced by cat cassette, Smr, Cmr | This study |
| ΔdegP ompU::phoA | P27459‐S ΔdegP with insertion of pGP704phoA downstream of ompU, Smr , Cmr, Apr | This study |
| ΔtoxR ompU::phoA | P27459‐S with deletion in toxR and insertion of pGP704phoA downstream of ompU, Smr , Cmr, Apr | This study |
| ΔdegP toxRCC ompU::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC236SC293S and insertion of pGP704phoA downstream of ompU, Smr, Cmr, Apr | This study |
| ΔdegP toxRC236S ompU::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC236S and insertion of pGP704phoA downstream of ompU, Smr , Cmr, Apr | This study |
| ΔdegP toxRC293S ompU::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC293S and insertion of pGP704phoA downstream of ompU, Smr , Cmr, Apr | This study |
| ΔdegP ompT::phoA | P27459‐S ΔdegP with insertion of pGP704phoA downstream of ompT, Smr , Cmr, Apr | This study |
| ΔtoxR ompT::phoA | P27459‐S with deletion in toxR and insertion of pGP704phoA downstream of ompT, Smr , Cmr, Apr | This study |
| ΔdegP toxRCC ompT::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC236SC293S and insertion of pGP704phoA downstream of ompT, Smr , Cmr, Apr | This study |
| ΔdegP toxRC236S ompT::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC236S and insertion of pGP704phoA downstream of ompT, Smr , Cmr, Apr | This study |
| ΔdegP toxRC293S ompT::phoA | P27459‐S ΔdegP with toxR replaced by FLAG‐toxRC293S and insertion of pGP704phoA downstream of ompT, Smr , Cmr, Apr | This study |
| Plasmids | ||
| pKEK229 | OriR6K, mobRP4, sacB, Apr | Correa et al. (2000) |
| pCVD442 | OriR6K, mobRP4, sacB, Apr | Donnenberg and Kaper (1991) |
| pGP704 | OriR6K, mobRP4, Apr | Miller and Mekalanos (1988) |
| pBAD18‐Kan | Expression vector, oriColE1, arabinose Inducible, Kmr | Guzman et al. (1995) |
| pBAD18 | Expression vector, oriColE1, arabinose Inducible, Apr | Guzman et al. (1995) |
| pACYC184 | Cloning vector, orip15A, Tetr, Cmr | Rose (1988) |
| pFLAG‐MACTM | Expression vector with N‐terminal FLAG‐Tag, IPTG inducible, Apr | Sigma‐Aldrich |
| pKT25 | Expression vector, encodes for the first 224 AA of CyaA (T25 fragment, B. pertussis), C‐terminal heterologous protein fusion, IPTG inducible, Kmr | Karimova et al. (1998) |
| pUT18C | Expression vector, encodes for AA 225 to 399 of CyaA (T18 fragment, B. pertussis), C‐terminal heterologous protein fusion, IPTG inducible, Apr | Karimova et al. (1998) |
| pKEK229dsbA::km | pKEK229 carrying up and down fragments, Apr, Kmr | Fengler et al. (2012) |
| pCVD442degP::cat | pCVD442 carrying up and down fragment of degP flanking a cat cassette, Apr, Cmr | Vorkapic et al. (2019) |
| pCVD442toxR | pCVD442 carrying up and down fragment of toxR, Apr | Fengler et al. (2012) |
| pCVD442FLAGtoxRCC | pCVD442 carrying up and down fragment of FLAGtoxRC236C293S, Apr | Fengler et al. (2012) |
| pCVD442FLAGtoxRC236S | pCVD442 carrying up and down fragment of FLAGtoxRC236S, Apr | This study |
| pCVD442FLAGtoxRC293S | pCVD442 carrying up and down fragment of FLAGtoxRC293S, Apr | This study |
| pCVD442FLAGtoxRC293SW76R | pCVD442 carrying up and down fragment of FLAGtoxRC293SW76R, Apr | This study |
| pFLAGtoxR | toxR of P27495‐S in pFLAG‐MACTM, Apr | Fengler et al. (2012) |
| pFLAGtoxRS | toxR and toxS of P27495‐S in pFLAG‐MACTM, Apr | Fengler et al. (2012) |
| pFLAGtoxRCC | toxRC236SC293S point mutant of P27495‐S in pFLAG‐MACTM, Apr | Fengler et al. (2012) |
| pFLAGtoxRCCtoxS | toxRC236SC293S point mutant and toxS of P27495‐S in pFLAG‐MACTM, Apr | Fengler et al. (2012) |
| pFLAGtoxRC236S | toxRC236S point mutant of P27495‐S in pFLAG‐MACTM, Apr | This study |
| pFLAGtoxRC293S | toxRC293S point mutant of P27495‐S in pFLAG‐MACTM, Apr | This study |
| pFLAGtoxRW76RS | toxRW76RS of point mutant of P27495‐S in pFLAG‐MACTM, Apr | This study |
| pBAD18‐KanFLAGtoxRS | FLAG‐toxRS of P27495‐S in pBAD18‐Kan, Kmr | This study |
| pBAD18‐FLAGtoxRS | FLAG‐toxRS of P27495‐S in pBAD18, Apr | This study |
| pGP704dsbC | pGP704 carrying internal fragment of dsbC, Apr | Fengler et al. (2012) |
| pGP704phoAompU | pGP704phoA with ompU gene fragment, Apr | Lembke et al. (2018) |
| pGP704phoAompT | pGP704phoA with ompT gene fragment, Apr | Lembke et al. (2018) |
| pKT25‐zip | BACTH positive control, leucine zipper of GCN4 (yeast) fused to the T25 fragment, Kmr | Karimova et al. (1998) |
| pUT18C‐zip | BACTH positive control, leucine zipper of GCN4 (yeast) fused to the T18 fragment, Apr | Karimova et al. (1998) |
| pKT25toxR | toxR of P27495‐S in pKT25, Kmr | This study |
| pKT25toxRS | toxR and toxS of P27495‐S in pKT25, Kmr | This study |
| pKT25toxRW76R | toxRW76R of P27495‐S in pKT25, Kmr | This study |
| pKT25toxRW76RS | toxRW76R and toxS of P27495‐S in pKT25, Kmr | This study |
| pUT18CtoxR | toxR of P27495‐S in pUT18C, Apr | This study |
| pUT18CtoxRS | toxR and toxS of P27495‐S in pUT18C, Apr | This study |
| pUT18CtoxRW76R | toxRW76R of P27495‐S in pUT18C, Apr | This study |
| pUT18CtoxRW76RS | toxRW76R and toxS of P27495‐S in pUT18C, Apr | This study |
| pUT18CtoxSFLAG | toxS‐FLAG of P27495‐S in pUT18C, Apr | This study |
| pUT18CopompU | ompU operators O123 of P27495‐S in pUT18C, Apr | This study |
| pUT18CopompU toxR | toxR of P27495‐S in pUT18CopompU, Apr | This study |
| pUT18CopompU toxRS | toxR and toxS of P27495‐S in pUT18CopompU, Apr | This study |
| pUT18CopompU toxRW76R | toxRW76R of P27495‐S in pUT18CopompU, Apr | This study |
| pUT18CopompU toxRW76RS | toxRW76R and toxS of P27495‐S in pUT18CopompU, Apr | This study |