TABLE 2.

Oligonucleotides used in the present study

FunctionPrimerGeneProcedureSequence (5′→3′)b
CloningacrA-FacrACloningaagatatcATGAACAAAAACAGAGGGTTAACG
acrA-RacrACloningaaaagcttTTAAGACTTGGTTTGTTCAGACTG
acrB-FacrBCloningATGCCTAATTTCTTTATCGATCGC
acrB-RaacrBCloningRTGRTGRTCNACNGGRTGRTT
tolC-FtolCCloningATGCAAATGAAGAAA
tolC-RtolCCloningTTAATGACGGAACGGATT
acrR-FacrR from E. aerogenesCloningaaggatccATGGCACGAAAAACCAAACAACAGG
acrR-RacrR from E. aerogenesCloningaagaattcTCAGGCCATGACCTCAGAGC
pctx-Fpromoter CTX-M-14CloningaaaggatccCCATCAATAAAATTGAG
pctx-Rpromoter CTX-M-14CloningaagatatcCTCAAACTCCCAATACG
acrAKm-FacrAKnockoutGGCGTCGTGACGCTCAAATCCGAACCTCTCCAGATGACAACAGAACTACCGGGCCGCACCAAAGCCACGTTGTGTCTCAA
acrAKm-RacrAKnockoutTGAAGCTACTTCCTGAGCCTTGACCTGCGCGCCAGGACGAACTTTTTGCAAACCAGTAATGCGCTGAGGTCTGCCTCGTG
Expression studies (RT-PCR)rpoB-FarpoBRT-PCRCAGCCGCGAYCAGGTTGACTACA
rpoB-RrpoBRT-PCRGACGCACCGCAGGATACCACCTG
ompC-FompCRT-PCRAGGTTAACGATCAACTGACCGG
ompC-RompCRT-PCRAAATTTCAGACCGGCGAACGCC
ompF-FompFRT-PCRAGTGGGAATATAACTTCCAGGG
ompF-RompFRT-PCRTGCGTCACCGAATTTCAGGCC
acrART-FacrART-PCRGATTATGATTCTGCCTTGGCCG
acrART-RacrART-PCRCAATGCGACCGCTGATAGGGG
Operon assemblyaefA-FcaefA from E. cloacaeAmplification of the acrR-acrA intergenic regionGTACATCGCGTTCTGCACGCG
acrA2-RcacrA from E. cloacaeAmplification of the acrR-acrA intergenic regionTGCCTTGACATCACCGCCTTC
acrA3-FdacrA from E. cloacaeAmplification of the acrAB intergenic regionCGACAAACAACAAGCGTCGGC
acrB2-RdacrB from E. cloacaeAmplification of the acrAB intergenic regionATGATGATTATGGCGATCACCC
  • 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.