TABLE 1.
Bacterial strains used in this study
| Strain or plasmid | Relevant characteristic(s) or description and/or construction | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1 | 9 |
| DH5α/λpir | λpir φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1 | 16 |
| BW20767 | RP4-2-Tc::Mu-1kan::Tn7 integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA(ΔMluI)::pir+ | 13 |
| A. faecalis strains | ||
| WM2072 | Wild type; Hpt+ Pt+ isolate from pig lagoon | This study |
| WM3425 | ΔptxD; integration and segregation with pMW12 | This study |
| WM3544 | ΔhtxA; integration and segregation with pMW14 | This study |
| WM3627 | ΔhtxBCD; integration and segregation with pMW15 | This study |
| WM3652 | ΔptxE; integration and segregation with pMW18 | This study |
| WM3818 | Δhtx/ptx; integration and segregation with pMW19 | This study |
| This study | ||
| Plasmids | ||
| pMAL-c2X | Apr; malE fusion protein expression vector | New England Biolabs |
| PBluescript SK(+) | Apr cloning vector | Stratagene |
| pRL27 | Tn5-RL27 (Kmr-oriR6K) delivery vector | 11 |
| pWM405 | 11 | |
| pRL47 | Recircularized SstI-cut pWM405 after treatment with T4 DNA polymerase and deoxynucleoside triphosphates | This study |
| pRL17 | Kmr; circularized Tn5-RL27 | 11 |
| pRL48 | aph-oriR6K containing KpnI-XbaI fragment of pRL17 inserted into KpnI-XbaI-cut pRL47 | This study |
| p34S-Cm | Apr/Cmr; carries cat gene cassette | |
| pRL50 | aph-containing SstI fragment of pRL48 replaced with the cat-containing SstI fragment of p34S-Cm | This study |
| pRL10 | AprtetAp-tnp oriT | 11 |
| pRL55 | Apr Tn5-RL55 (Cmr-oriR6K) delivery vector; BamHI-cut PCR fragment from pRL10 (primers pRL10-1 and pRL10-2) ligated to BglII-cut PCR fragment from pRL50 (primer Tn5-OE1) | This study |
| pAW19 | Kmr/AprsacB suicide vector | 24 |
| pMW6 | HindIII-digested PCR of ptxD inserted into HindIII and XmnI sites of pMAL-c2X | This study |
| pMW12 | NotI- and EcoRI-digested PCR of ptxD upstream region (primers AptxDupF and AptxDupR) and SpeI- and EcoRI-digested PCR of ptxD downstream region (primers AptxDDNF and AptxDDNR) inserted into NotI- and SpeI-digested pAW19 | This study |
| pMW14 | NotI- and EcoRI-digested PCR of htxA upstream region (primers AhtxAdelY and AhtxARUP) and SacI- and EcoRI-digested PCR of htxA downstream region (primers AhtxAdelX and AhtxAFDN) inserted into NotI- and SacI-digested pAW19 | This study |
| pMW15 | NotI- and EcoRI-digested PCR of htxBCD upstream region (primers htxBDUPF and htxBDUPR) and SacI- and EcoRI-digested PCR of htxBCD downstream region (primers htxBDDNF and htxBDDNR) inserted into NotI- and SacI-digested pAW19 | This study |
| pMW18 | NotI- and EcoRI-digested PCR of ptxE upstream region (primers delptxEUPF and delptxEUPR) and SpeI- and EcoRI-digested PCR of ptxE downstream region (primers delptxEDNF and delptxEDNR) inserted into NotI- and SpeI-digested pAW19 | This study |
| pMW19 | NotI- and EcoRI-digested PCR of htxA upstream region (primers AhtxAdelY and AhtxAUPR) and SpeI- and EcoRI-digested PCR of ptxE downstream region (primers delptxEDNF and delptxEDNR) inserted into NotI- and SpeI-digested pAW19 | This study |
| pMW20 | BamHI-digested PCR of WM2072 16S rRNA gene inserted into BamHI-digested pBluescript SK(+) | This study |