SupplementaryInformation
Plasmid constructions
All plasmids and oligonucleotides used in this study are listed in Tables S2 and S3, respectively. The bamA coding sequences fromS. Typhimurium LT2, E. cloacae ATCC 13047, D. dadantii 3937 and Y. pseudotuberculosis YPIII were amplified from the appropriate genomic DNAs using primer pairs: bamA-Eco (pZS21)/bamA-Xba (pZS21), bamA-Eco (pZS21)/Ecloa-bamA-Xba, 3937-bamA-Eco/3937-bamA-Xba, and YP-bamA-Bam/YP-bamA-Xba, respectively. The resulting products were digested with EcoRI/XbaI or BamHI/XbaI and ligated to plasmid pZS21.Plasmid pZS21::bamAHA4Ecoliwas created by amplifying E. coli bamA with bamA-Eco (pZS21)/bamA-L4-Sal-rev and bamA-L4-HA-for/bamA-Xba (pZS21). The PCR products were digested with EcoRI/SalI and SalI/XbaI (respectively) and ligated into plasmid pZS21 digested with EcoRI and XbaI. Plasmid pZS21::bamAHA6Ecoli was created by amplifying E. coli bamA with bamA-Eco (pZS21)/bamA-L6-Sal-rev and bamA-L6-HA-for/bamA-Xba (pZS21). The products were digested with EcoRI/SalI and SalI/XbaI (respectively) and ligated into plasmid pZS21 digested with EcoRI and XbaI. Plasmid pZS21::bamAHA7Ecoli was created by amplifying E. coli bamA with bamA-Eco (pZS21)/bamA-L7-Sal-revand bamA-L7-HA-for/bamA-Xba (pZS21). Products were digested with EcoRI/SalI and SalI/XbaI (respectively) and ligated to plasmid pZS21 digested with EcoRI and XbaI.Plasmid pZS21::bamA∆4Ecoliwas created by amplifying E. coli bamA with bamA-Eco (pZS21)/bamA-∆L4-rev and bamA-∆L4-for/bamA-Xba (pZS21). Products were digested with EcoRI/NheI and NheI/XbaI respectively and ligated into pZS21 digested with EcoRI and XbaI. Plasmid pZS21::bamA∆6Ecoliwas created by amplifying E. coli bamA with bamA-Eco (pZS21)/bamA-∆L6-rev and bamA-∆L6-for/bamA-Xba (pZS21). Amplicons were digested with EcoRI/NheI and NheI/XbaI respectively and ligated into pZS21 digested with EcoRI and XbaI.
Plasmid pZS21::bamAEc4Ecloacaewas created by amplifying E. cloacae bamA with bamA-Eco (pZS21) and Ecloa-bamA-L4(Ecoli). The resulting product was used as a megaprimer to amplify E. cloacae bamA with oligonucleotideEcloa-bamA-Xba. The final product was digested with EcoRI and XbaI and ligated to pZS21. Plasmid pZS21::bamAEc6Ecloacaewas created by amplifying E. cloacae bamA with bamA-Eco (pZS21)/Ecloa-bamA-L6(Ecoli)-revand Ecloa-bamA-L6(Ecoli)-for/Ecloa-bamA-Xba. The twoPCR products were digested with EcoRI/NheI and NheI/XbaI and ligated to plasmid pZS21 digested with EcoRI and XbaI. Plasmid pZS21::bamAEc7Ecloacaewas created by amplifying E. cloacae bamA with bamA-Eco (pZS21) and Ecloa-bamA-L7(Ecoli). The resulting product was used with Ecloa-bamA-Xba to amplify E. cloacae bamA. The final PCR product was digested with EcoRI and XbaI and ligated to pZS21. Plasmid pZS21::bamAEc4/7Ecloacaewas generated by amplifying a fragment from plasmid pZS21::bamAEc4Ecloacaewith bamA-Eco (pZS21) and Ecloa-bamA-L7(Ecoli). The resulting product was used with Ecloa-bamA-Xba to amplify E. cloacae bamA. The final product was digested with EcoRI and XbaI and ligated to pZS21. Plasmid pZS21::bamAEc6/7Ecloacaewas created by amplifying pZS21::bamAEc6Ecloacaewith bamA-Eco (pZS21) and Ecloa-bamA-L7(Ecoli). The resulting product was used with Ecloa-bamA-Xba to amplify E. cloacae bamA. The final product was digested with EcoRI and XbaI and ligated to plasmid pZS21. All bamA fragments were also subcloned into plasmid pZS21amp using EcoRI and XbaI restriction sites. The coding sequence for CdiAEC93 residues Val2905 – Lys3132 was amplified from pDAL660∆1-39 with primers EC93-CT-Nco/EC93-CT-Bam and the product digested with NcoI/BamHI for ligation to plasmid pSH21 {Shoji, 2010 #72}. An NcoI/XhoI fragment containing cdiA-CTEC93 was then subcloned into pCH450 to generate plasmid pCH450-cdiA-CTEC93. The pCH450-cdiA-CT/cdiIEC93 construct was generated by amplifying the toxin/immunity gene pair with EC93-CT-Nco/EC93-CdiI-Xho followed by ligation to pCH450.