Respiration then runs a futile cycle, as protons continue to be pumped but PMF is not maintained ( 4– 6). The transmembrane proton flux equilibrates both ΔpH and Δψ, and thus depletes PMF ( 1, 2). These cations then shuttle protons across the membrane via protonation/deprotonation ( 1– 3). Uncouplers such as CCCP are generally hydrophobic compounds with an acidic proton that reside in the inner membrane of the cell. The proton motive force (PMF) is diminished or abolished by uncouplers of oxidative phosphorylation such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) ( 1). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate). 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). Other mutant genes in CCCP-evolved strains include adhE (alcohol dehydrogenase), rng (ribonuclease G), and cyaA (adenylate cyclase). The acrB isolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Another isolate showed acrB (MDR efflux pump). A later evolved descendant of the C-B11 population showed mutations in ybhR (MDR efflux). A cecR∷kanR deletion conferred partial resistance to CCCP.

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). Deletion of emrA abolished the CCCP resistance of these strains. 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. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. ABSTRACT Experimental 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).

Cccp project replacement