摘要

大肠杆菌K-12与苯甲酸盐（质子动力（PMF）的部分解偶联剂）的实验进化选择降低抗生素抗性的突变。我们在羰基氰化物间氯苯腙（CCCP）存在下进行了实验演化，CCCP是一种强解偶联剂。将培养物在含有20-150μMCCCP的LBK培养基中每天1：100连续稀释，所述CCCP在pH 6.5或pH 8.0下缓冲。经过1000代，人群可耐受高达150μM的CCCP。序列分离株在mprA（emrR）中具有突变，其下调输出CCCP的EmrAB-TolC泵。 mprA :: kanR缺失在60 CCCP时赋予生长，但不是在进化菌株（150μM）抵抗的较高水平。一些mprA突变菌株也具有影响emrA的点突变，但emrA的缺失消除了CCCP抗性。因此，CCCP进化的分离株包含额外的适应性。一个分离株缺乏emrA或mprA突变，但在cecR（ybiH）中有突变，其产物上调药物泵YbhG和YbhFSR;在上调多药泵mdtEF的gadE中，cecR :: kanR缺失赋予CCCP部分抗性。其他具有突变的多药外排基因包括ybhR和acrAB.acrB分离株对AcrAB底物氯霉素和四环素敏感。 CCCP进化菌株中的其他突变基因包括rng（核糖核酸酶G）和cyaA（腺苷酸环化酶）。总体而言，实验进化揭示了通过EmrA增加CCCP流出的突变的CCCP依赖性适应性优势;对于可能使不存在的药物（cecR，gadE，acrAB，ybhR）的质子驱动泵失活的突变。这些结果与我们先前关于药物敏感性与进化的苯甲酸盐耐受性相关的报告一致。对细菌遗传反应的质子动力（PMF）消耗以及它们对耐药性的影响的了解很少。 PMF驱动许多抗生素的输出，但是当不存在抗生素时，能量成本可能降低适应性。我们的进化实验揭示了适应PMF解偶联剂CCCP的遗传机制，包括选择增加的CCCP流出，但也反对PMF驱动的泵用于不存在的药物的表达。这些结果对我们对肠道微生物组的理解产生了影响，肠道微生物组经历了高水平的有机酸，可以降低PMF。

Experimental evolution of Escherichia coli K-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 cyanide m-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20-150 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 in mprA (emrR), which downregulates the EmrAB-TolC pump that exports CCCP. A mprA::kanR deletion conferred growth at 60 CCCP, though not at the higher levels resisted by evolved strains (150 µM). Some mprA mutant strains also had point mutations affecting emrA, but deletion of emrA abolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lacked emrA or mprA mutations but had mutations in cecR (ybiH) whose product upregulates drug pumps YbhG and YbhFSR; and in gadE, which upregulates multidrug pump mdtEF A cecR::kanR deletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations include ybhR and acrAB The acrB isolate was sensitive to AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains include rng (ribonuclease G) and cyaA (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, ybhR). 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.

https://www.ncbi.nlm.nih.gov/pubmed/30578262