Since the MAR phenotype agrees with the substrate profile of AcrAB [15], [19], sequencing of the regulatory loci (acrR, soxRS, marRAB and ramR) reported to regulate AcrAB expression, as well as their promoters, was performed in order to detect any possible mutation that could justify the MAR phenotype (Figure 1).
Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis.
This sequence was inserted at 220 bp downstream from the initiation codon of the acrR gene, accompanied by the doubling of the tetramer CTAG.
Linear DNA fragments comprising the rpsL-neo cassette for the introduction of the acrR and/or marR region were obtained by PCR using the rpsL-neo template DNA (GeneBridges) as the template.
Of human ciprofloxacin-resistant S. enterica serotype Choleraesuis, all were associated with two-base substitutions in the QRDR of the gyrA gene at codon 83 (Ser83Phe) and 87 (ASer80Ile, Ser80ine in the QRDR of the parC gene, and Gln78Stp in the QRDR of the acrR gene (Table 3).
The nucleotide sequences of entire acrR, marR, and soxR genes in organic solvent-tolerant mutants were determined by DNA sequencing to identify mutations in these genes.
The sequences of acrR, marR, and soxR genes in strain JA300 were identical to those present in the E. coli K-12 strain MG1655 genome deposited in GenBank (accession number NC_000913.2).
We also tested the solvent tolerance of the acrR-deleted mutant in which the expression of the acrAB operon increased due to the lack of repressor AcrR, but the acrR mutant showed no enhanced tolerance (Table 2).
The sequences of the primers for the polyermase chain reaction (PCR) amplification of gryA, gyrB, parC, parE, and acrR have been previously described (27 – 30 ).
