Deletion of AcrS Results in Increased Expression of acrE and Confers an Increase in Kanamycin Resistance in Escherichia coli BW25113

Escherichia coli bacteria contain numerous efflux pumps that are responsible for intrinsic resistance to antimicrobial compounds. Previous studies have shown that the efflux pumps AcrAB and AcrEF are similar in structure and may be regulated with enzymes that have homologous functions. AcrR is a known repressor of the acrAB operon. It has been suggested that regulatory proteins AcrR and AcrS have analogous functions due to their encoding sequences being located upstream of acrAB and acrEF, respectively. Therefore, we hypothesized that deletion of the predicted regulatory gene acrS in E. coli BW25113 would result in increased expression of efflux pump protein acrE, and increased intrinsic kanamycin resistance. To test this hypothesis, we used a ΔacrEΔkan and a ΔacrSΔkan deletion mutant. Using an MIC assay, we found that the wild-type strain (BW25113), ΔacrSΔkan and ΔacrEΔkan had kanamycin MICs of 3.1 μg/ml, 12.5 μg/ml, and 3.1 μg/ml, respectively. Consistent with our MIC assay results, we showed using a quantitative polymerase chain reaction assay that the expression of acrE in ΔacrSΔkan increased when cultured in the presence of sub-inhibitory concentrations of kanamycin. This effect was almost doubled when 6.3 μg/ml kanamycin was used, which may indicate that acrE expression increases in response to higher concentrations of antibiotic. In contrast to our expectations, acrA expression levels remained unchanged in all three of the strains, indicating that the AcrAB efflux pump may not be involved in acquiring kanamycin resistance in E. coli. These results support that AcrS may negatively regulate acrEF, and AcrEF participates in the intrinsic kanamycin resistance in E. coli BW25113.