TABLE 2.
Oligonucleotides used in the present study
| Function | Primer | Gene | Procedure | Sequence (5′→3′)b |
|---|---|---|---|---|
| Cloning | acrA-F | acrA | Cloning | aagatatcATGAACAAAAACAGAGGGTTAACG |
| acrA-R | acrA | Cloning | aaaagcttTTAAGACTTGGTTTGTTCAGACTG | |
| acrB-F | acrB | Cloning | ATGCCTAATTTCTTTATCGATCGC | |
| acrB-Ra | acrB | Cloning | RTGRTGRTCNACNGGRTGRTT | |
| tolC-F | tolC | Cloning | ATGCAAATGAAGAAA | |
| tolC-R | tolC | Cloning | TTAATGACGGAACGGATT | |
| acrR-F | acrR from E. aerogenes | Cloning | aaggatccATGGCACGAAAAACCAAACAACAGG | |
| acrR-R | acrR from E. aerogenes | Cloning | aagaattcTCAGGCCATGACCTCAGAGC | |
| pctx-F | promoter CTX-M-14 | Cloning | aaaggatccCCATCAATAAAATTGAG | |
| pctx-R | promoter CTX-M-14 | Cloning | aagatatcCTCAAACTCCCAATACG | |
| acrAKm-F | acrA | Knockout | GGCGTCGTGACGCTCAAATCCGAACCTCTCCAGATGACAACAGAACTACCGGGCCGCACCAAAGCCACGTTGTGTCTCAA | |
| acrAKm-R | acrA | Knockout | TGAAGCTACTTCCTGAGCCTTGACCTGCGCGCCAGGACGAACTTTTTGCAAACCAGTAATGCGCTGAGGTCTGCCTCGTG | |
| Expression studies (RT-PCR) | rpoB-Fa | rpoB | RT-PCR | CAGCCGCGAYCAGGTTGACTACA |
| rpoB-R | rpoB | RT-PCR | GACGCACCGCAGGATACCACCTG | |
| ompC-F | ompC | RT-PCR | AGGTTAACGATCAACTGACCGG | |
| ompC-R | ompC | RT-PCR | AAATTTCAGACCGGCGAACGCC | |
| ompF-F | ompF | RT-PCR | AGTGGGAATATAACTTCCAGGG | |
| ompF-R | ompF | RT-PCR | TGCGTCACCGAATTTCAGGCC | |
| acrART-F | acrA | RT-PCR | GATTATGATTCTGCCTTGGCCG | |
| acrART-R | acrA | RT-PCR | CAATGCGACCGCTGATAGGGG | |
| Operon assembly | aefA-Fc | aefA from E. cloacae | Amplification of the acrR-acrA intergenic region | GTACATCGCGTTCTGCACGCG |
| acrA2-Rc | acrA from E. cloacae | Amplification of the acrR-acrA intergenic region | TGCCTTGACATCACCGCCTTC | |
| acrA3-Fd | acrA from E. cloacae | Amplification of the acrAB intergenic region | CGACAAACAACAAGCGTCGGC | |
| acrB2-Rd | acrB from E. cloacae | Amplification of the acrAB intergenic region | ATGATGATTATGGCGATCACCC |
a
Oligonucleotide degenerated where R is A or G; N is A, C, G, or T; and Y is C or T.
b
Lowercase and italic nucleotides indicate restriction sites for cloning.
c
Oligonucleotides used for amplification, cloning, and sequencing of the acrR gene and the acrR-acrA intergenic region of the operon from E. cloacae EcDC64. F, forward; R, reverse.
d
Oligonucleotides used for amplification, cloning, and sequencing of the acrAB intergenic region of the operon from E. cloacae EcDC64. F, forward; R, reverse.