Generation of an acrAacrE double-knockout in Escherichia coli and its role in kanamycin resistance

Abstract

AcrAB and AcrEF are multidrug efflux pumps found in Escherichia coli that are thought to mediate the export of kanamycin, and therefore contribute to kanamycin resistance. Previous studies found that expression of acrE increased in response to increasing concentrations of kanamycin. Another study found that the loss of acrA or acrE was shown to have no effect on kanamycin resistance, indicating that AcrAB and AcrEF may have compensatory effects on each other. The goal of this study was to generate an acrAacrE double-knockout in E. coli and determine if this double-knockout conferred any differences in kanamycin resistance compared to the single-knockouts and wild-type. We hypothesized that by knocking out both the acrA and acrE genes, no compensatory effects would occur to support kanamycin resistance. To test this, we first generated an acrAacrE double-knockout by using lambda Red (λ-Red) recombination to remove the acrA gene from an acrE single-knockout strain. To determine differences in kanamycin resistance, MIC assays were performed on all the strains. The acrAacrE double-knockout was successfully generated and Sanger sequencing confirmed the deletion of acrA. However, MIC results were not conclusive as they did not show any difference in kanamycin resistance between strains. Based on these results, it is still unclear whether AcrAB and AcrEF have compensatory effects on each other, since there was no significant difference in MIC between the strains.