ABSTRACTExperimental evolution ofEscherichia coliK-12 with benzoate, a partial uncoupler of the proton motive force (PMF), selects for mutations that decrease antibiotic resistance. We conducted experimental evolution in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20 to 150 µM CCCP buffered at pH 6.5 or at pH 8.0. After 1,000 generations, the populations tolerated up to 150 µM CCCP. Sequenced isolates had mutations inmprA(emrR), which downregulates the EmrAB-TolC pump that exports CCCP. AmprA::kanRdeletion conferred growth at 60 μM CCCP, though not at the higher levels resisted by evolved strains (150 µM). SomemprAmutant strains also had point mutations affectingemrA, but deletion ofemrAabolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lackedemrAormprAmutations but had mutations incecR(ybiH), whose product upregulates drug pumps YbhG and YbhFSR, and ingadE, which upregulates the multidrug pump MdtEF. AcecR::kanRdeletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations includedybhRandacrAB. TheacrBisolate was sensitive to the AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains includerng(RNase G) andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP-dependent fitness advantage for mutations increasing CCCP efflux via EmrA and for mutations that may deactivate proton-driven pumps for drugs not present (cecR,gadE,acrAB, andybhR). These results are consistent with our previous report of drug sensitivity associated with evolved benzoate tolerance.IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force (PMF), and their effects on drug resistance, are poorly understood. PMF drives export of many antibiotics, but the energy cost may decrease fitness when antibiotics are absent. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for increased CCCP efflux but also against the expression of PMF-driven pumps for drugs not present. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF.
Compound Efflux in Helicobacter pylori