TABLE 2.
PCR primers used in this study
| Primer name | Sequencea (5′–3′) | Purpose |
|---|---|---|
| atpA_p207_KpnI | CGGGGTACCATGCAACTGAATTCCACCGA | Used to construct recombinant plasmid for atpA complementation |
| atpA_p207_HindIII | CCCAAGCTTTTACTTGTCGTCATCGTCTTTGTAGTCCCAGGATTGGGTTGCTTTGA | |
| atpF_p207_EcoRI | CCGGAATTCATGGTGAATCTTAACGCAACAA | Used to construct recombinant plasmid for atpF complementation |
| atpF_p207_SalI | ACGCGTCGACTTACTTGTCGTCATCGTCTTTGTAGTCCAGTTCAGCGACAAGTTTATC | |
| dsbA_p207_EcoRI | CCGGAATTCATGAAAAAGATTTGGCTGG | Used to construct recombinant plasmid for dsbA complementation |
| dsbA_p207_SalI | ACGCGTCGACTTACTTGTCGTCATCGTCTTTGTAGTCTTTTTTCTCGGACAGATATTTC | |
| dsbB_p207_EcoRI | CCGGAATTCATGTTGCGATTTTTGAACCA | Used to construct recombinant plasmid for dsbB complementation |
| dsbB_p207_SalI | ACGCGTCGACTTACTTGTCGTCATCGTCTTTGTAGTCGCGACCGAACAGATCACGTT | |
| ompF_p207_EcoRI | CCGGAATTCATGATGAAGCGCAATATT | Used to construct recombinant plasmid for ompF complementation |
| ompF_p207_SalI | ACGCGTCGACTTACTTGTCGTCATCGTCTTTGTAGTCGAACTGGTAAACGATACC | |
| ompR_p207_EcoRI | CCGGAATTCATGCAAGAGAACTACAAGA | Used to construct recombinant plasmid for ompR complementation |
| ompR_p207_SalI | ACGCGTCGACCTACTTGTCGTCATCGTCTTTGTAGTCTGCTTTAGAGCCGTCCGGTA | |
| ΔatpA_H1_P1 | CTTGCAGACGTCTTGCAGTCTTAAGGGGACTGGAGC | Used to generate atpA knockout strain |
| ΔatpA_H2_P2 | GCCTTGCGGCCTGCCCTAAGGCAAGCCGCCAGACGT | |
| atpA_up | TGCTGCGATGGAAAAACGTC | |
| atpA_down | TTCTGGACGCTTGCGATCTT | |
| ΔatpF_H1_P1 | TTTATTTAAAGAGCAATATCAGAACGTTAACTAAATAGAGGCATTGTGCT | Used to generate atpF knockout strain |
| ΔatpF_H2_P2 | GGCGTAGGGGCGAGCTACCGTAATAAATTCAGACATCAGCCCCTCCCTCC | |
| atpF_up | ATCGCTGTAGGTCTGGGTCT | |
| atpF_down | ATGTCCTGCCAGCGTTCTAC | |
| ΔdsbA_H1_P1 | GTGAATATTCACGGGCTTTATGTAATTTACATTGAA | Used to generate dsbA knockout strain |
| ΔdsbA_H2_P2 | AATTAACACCTATGTATTAATCGGAGAGAGTAGATC | |
| dsbA_up | AGCGGCAGGATGCATTATCA | |
| dsbA_down | GGGAAGATTACTGGCTGCGA | |
| ΔdsbB_H1_P1 | AATAAATATAGCGGCAGGAAAAAAGCGCTCCCGCAGGAGCGCCGAATGGA | Used to generate dsbB knockout strain |
| ΔdsbB_H2_P2 | AATGAATTGGTTTAAACTGCGCACTCTATGCATATTGCAGGGAAATGATT | |
| dsbB_up | CAATGGCAGATGAAGCGAGC | |
| dsbB_down | TGCAAATGGGCTGGATAGCA | |
| ΔompF_H1_P1 | GTTGTCAGAATCGATCTGGTTGATGATGTAGTCAAC | Used to generate ompF knockout strain |
| ΔompF_H2_P2 | GTGATCGTCCCTGCTCTGTTAGTAGCAGGTACTGCA | |
| ompF_up | CGCTATCAGGGTAACGGGAG | |
| ompF_down | AGCACTTTCACGGTAGCGAA | |
| ΔompR_H1_P1 | GGGCAAATGAACTTCGTGGCGAGAAGCGCAATCGCC | Used to generate ompR knockout strain |
| ΔompR_H2_P2 | CTTACAAATTGTTGCGAACCTTTGGGAGTACAAACA | |
| ompR_up | TGTTGCGAACCTTTGGGAGT | |
| ompR_down | AGCAAGGTGACGATGAGCAA | |
| Ec.ompF_ H1_ His_P1 | GACGACACCGTTGCTGTGGGTATCGTTTACCAGTTCCATCATCACCATCACCATTAA | In cis addition of His tag (C terminal) to ompF in E. coli strain BW25113 |
| Ec.ompF_ H2 _P2 | AAGTCCTGTTTTTTCGGCATTTAACAAAGAGGTGTG | |
| Se.ompF_ H1_ His_P1 | GACGATCAGGCGGCTGTCGGTATTACTTACCAGTTCCATCATCACCATCACCATTGA | In cis addition of His tag (C terminal) to ompF in S. enteritidis strain 22577 |
| Se.ompF_ H2_P2 | AGTCCTGTTTTTGAGGCATAAAACAAAGGGGTCTG | |
| Ec1–72_EcoRI | CCGGAATTCATGATGAAGCGCAATATT | Used to construct KΔompF(ompFEc+Se) |
| Ec1-72_R | CGGTGTTAATTTGAGTTTCCCCTTTAAAACCA | |
| Se69-363_F | GGAAACTCAAATTAACACCGATCTGACCGGT | |
| Se69-363_SalI | ACGCGTCGACTCAATGGTGATGGTGATGATGGAACTGGTAAGTAATACCGA | |
| Se1-68_EcoRI | CCGGAATTCATGATGAAGCGCAAAATCCT | Used to construct KΔompF(ompFSe+Ec) |
| Se1-68_R | CGGAATTGATCTGCGTTTCCCCTTTAAA | |
| Ec73-362_ F | GGAAACGCAGATCAATTCCGATCTGACCG | |
| Ec73-362_SalI | ACGCGTCGACTTAATGGTGATGGTGATGATGGAACTGGTAAACGATACC | |
| ΔSYGG-F | AATGGCGACATGACCTATG | Used to construct KΔompF(Ec.ΔSYGG) |
| ΔSYGG-R | GTTTTCACCGTTACCCTTG | |
| ΔKGN-F | GGTGAAAACAGTTACGGTG | Used to construct KΔompF(Ec.ΔKGN) |
| ΔKGN-R | GGAAAAATAATGCAGACCAAC | |
| ΔtolA_H1_P1 | CTTGCTTGAAAGAGAGCGGGTAACAGGCGAACAGTTTTTGGAAACCGAGA | Used to knock out tolA for the ΔtonB knockout strain (Keio collection no. 67A9) |
| ΔtolA_H2_P2 | CCGTGGCAACCGGTGCCTGATGTTGACCGTCCGAACAGTCAACATCGCGA | |
| tolA_up | TAATGACGCAGCCTATCTAA | |
| tolA_down | ATGCCTGCTTCATCATATCT | |
| ΔtolQ_H1_P1 | AAAATGAAGCCTCGTGCGCTTCCCAAGTCTATTGTCGCGGAGTTTAAGCA | Used to knock out tolQ for the ΔtonB knockout strain (Keio collection no. 67A9) |
| ΔtolQ_H2_P2 | ATTTCGGACTTGAGATCGCGACGACCTCGTCCACGCGCTCTGGCCATGGC | |
| tolQ_up | GTCAACGCCGAGAATACT | |
| tolQ_down | ACCAATACCAGACACTTCAA | |
| ΔtolR_H1_P1 | TTCTGCACCGCCAGGCGTTTACCGTTAGCGAGAGCAACAAGGGGTAAGCC | Used to knock out tolR for the ΔexbD knockout strain (Keio collection no. 57A12) |
| ΔtolR_H2_P2 | AACTGTTCGCCTGTTACCCGCTCTCTTTCAAGCAAGGGAAACGCAGATGT | |
| tolR_up | TGGAACTGAATTACGACAAC | |
| tolR_down | CAACTACCGCACCTGAAT | |
| P1 | TGTGTAGGCTGGAGCTGCTTCG | Used to amplify the kanamycin or chloramphenicol resistance genes |
| P2 | CATATGAATATCCTCCTTA | |
| C1 | TTATACGCAAGGCGACAAGG | Used to verify the resistance gene cassette integration at the desired location combined with gene specific primers |
| C2 | GATCTTCCGTCACAGGTAGG | |
| K1 | CAGTCATAGCCGAATAGCCT | |
| K2 | CGGTGCCCTGAATGAACTGC |
a
For gene deletion mutants, the kanamycin or chloramphenicol primers P1 and P2 were added to the 3′ end of homologous extension sequences (H1 and H2) of the E. coli-specific gene. Restriction enzyme sites are underlined. PCRs were performed in a volume of 50 μl containing 45 μl of Platinum PCR supermix (Invitrogen, Carlsbad, CA), 1 U Taq DNA polymerase, 22 mM Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl2, 220 μM each deoxynucleoside triphosphate, and stabilizers. PCR was carried out under the following conditions: 2 min at 94°C for 1 cycle; 30 s at 94°C, 30 s at 55°C, and 2 min at 68°C for 30 cycles; and 10 min at 68°C for 1 cycle.