Abstract
Multidrug transporters (MDTs) are major contributors to microbial drug resistance and are further utilized for improving host phenotypes in biotechnological applications. Therefore, the identification of these MDTs and the understanding of their mechanisms of action in vivo are of great importance. However, their promiscuity and functional redundancy represent a major challenge towards their identification. Here, a multistep tolerance adaptive laboratory evolution (TALE) approach was leveraged to achieve this goal. Specifically, a wild-type E. coli K-12-MG1655 and its cognate knockout individual mutants ΔemrE, ΔtolC, and ΔacrB were evolved separately under increasing concentrations of two lipophilic cations, tetraphenylphosphonium (TPP(+)), and methyltriphenylphosphonium (MTPP(+)). The evolved strains showed a significant increase in MIC values of both cations and an apparent cross-cation resistance. Sequencing of all evolved mutants highlighted diverse mutational mechanisms that affect the activity of nine MDTs including acrB, mdtK, mdfA, acrE, emrD, tolC, acrA, mdtL, and mdtP. Besides regulatory mutations, several structural mutations were recognized in the proximal binding domain of acrB and the permeation pathways of both mdtK and mdfA. These details can aid in the rational design of MDT inhibitors to efficiently combat efflux-based drug resistance. Additionally, the TALE approach can be scaled to different microbes and molecules of medical and biotechnological relevance.