Table 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Description | Source or reference |
|---|---|---|
| Strains | ||
| S. flexneri | ||
| 2457T | S. flexneri serotype 2a | 41 |
| AWY3 | 2457T virB::Tn5 | 14 |
| E. coli | ||
| MC4100 | E. coli strain K-12 with araD and lacZ deletion | 42 |
| MC4100 hns | MC4100 hns::Kn. The first 37 amino acids of H-NS are expressed, giving rise to a trans-dominant negative effect over other H-NS-like proteins in the cell. | 43 |
| Plasmids | ||
| pHJW20 | PicsP-lacZ reporter plasmid derived from pACYC184. Carries 1,259 bp upstream and 48 bp downstream of the icsP gene on a PstI-XbaI DNA fragment. Restriction with SalI and XbaI completely removes icsP promoter and gene sequences. | 15 |
| pDB05 | pHJW20 carrying 1,613 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site in the SalI and XbaI sites. Also known as pospZ_3′t(+32). | This work |
| pospZ_3′t(+11) | pHJW20 carrying 1,613 bp of wild-type sequence upstream and 11 bp downstream of the ospZ translation start site in the SalI and XbaI sites | This work |
| pospZ_3′t(−14) | pHJW20 carrying wild-type sequences located between 1613 bp and 14 bp upstream of the ospZ translation start site in the SalI and XbaI sites | This work |
| pospZ_3′t(−51) | pHJW20 carrying wild-type sequences located between 1613 bp and 51 bp upstream of the ospZ translation start site in the SalI and XbaI sites | This work |
| pospZ_3′t(−189) | pHJW20 carrying wild-type sequences located between 1613 bp and 189 bp upstream of the ospZ translation start site in the SalI and XbaI sites | This work |
| pAFW04 | Identical to pHJW20, but the lambda oop terminator is cloned immediately upstream of the icsP promoter fragment to prevent possible transcriptional read-through into the promoter region. Restriction with SalI and XbaI completely removes icsP promoter sequences but leaves the lambda oop terminator. | This work |
| pospZ_5′t(−1613) | pAFW04 carrying 1,613 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site on a SalI-XbaI DNA fragment. Identical to pDB05, but the lambda oop terminator is cloned immediately upstream of the ospZ promoter fragment to prevent possible transcriptional read-through into the promoter region. | This work |
| pospZ_5′t(−1203) | pAFW04 carrying 1,203 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site on a SalI-XbaI DNA fragment | This work |
| pospZ_5′t(−731) | pAFW04 carrying 731 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site on a SalI-XbaI DNA fragment | This work |
| pospZ_5′t(−495) | pAFW04 carrying 495 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site on a SalI-XbaI DNA fragment | This work |
| pospZ_5′t(−412) | pAFW04 carrying 412 bp of wild-type sequence upstream and 32 bp downstream of the ospZ translation start site on a SalI-XbaI DNA fragment | This work |
| pospZ_Δ45 | pAFW04 with a 45-bp deletion between −270 and −225 relative to the ospZ translation start site | This work |
| pMAP07 | pAFW04 lacking all promoter sequences | This work |
| pMIC16 | pBluescript II KS(+) carrying 809 bp upstream and 331 bp downstream of the ospZ translation start site with 14-bp substitutions in the VirB binding sites | 15 |
| pDB15 | pospZ_5′t(−1613) carrying 14-bp substitutions in the VirB binding sites, so that the original sequence ATTTCAGtATGAAAT was altered to GCCCAGCtCGACCCGa | This work |
| pMIC21 | pHJW20 lacking all promoter sequences | 15 |
| pBAD-virB | pATM324 derived from pBAD18; Ampr | 44 |
| pBAD18 | Arabinose-inducible pBAD expression vector, pBR ori; Ampr | 45 |
a
Uppercase letters represent sites found to be essential for VirB-dependent regulation of the icsP promoter (16) or the corresponding mutated sites. Note that the original sites are organized as a near-perfect inverted repeat, separated by a single nucleotide (lowercase).