CCCP experimental evolution of Escherichia coli K-12 selects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

ABSTRACTExperimental evolution was conducted with Escherichia coli K-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 M CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations in emrAB or in its negative repressor mprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion of emrA abolished the CCCP resistance of these strains. One CCCP-evolved isolate lacked emrA or mprA mutations; this strain (C-B11-1) showed mutations in drug efflux regulators cecR (ybiH) (upregulates drug pumps YbhG and YbhFSR) and gadE (upregulates drug pump mdtEF). A cecR::kanR deletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations in ybhR (MDR efflux). Another isolate showed acrB (MDR efflux pump). The acrB isolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains include adhE (alcohol dehydrogenase), rng (ribonuclease G), and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).\n\nIMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.