ABSTRACTBenzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution ofEscherichia coliK-12. Transcriptomes ofE. coliisolates evolved with benzoate showed the reversal of benzoate-dependent regulation, including the downregulation of multidrug efflux pump genes, the gene for the Gad acid resistance regulon, the nitrate reductase genesnarHJ, and the gene for the acid-consuming hydrogenase Hyd-3. However, the benzoate-evolved strains had increased expression of OmpF and other large-hole porins that admit fermentable substrates and antibiotics. Candidate genes identified from benzoate-evolved strains were tested for their roles in benzoate tolerance and in chloramphenicol sensitivity. Benzoate or salicylate tolerance was increased by deletion of the Gad activatorariRor of the acid fitness island fromslpto the end of thegadXgene encoding Gad regulators and the multidrug pump genesmdtEF. Benzoate tolerance was also increased by deletion of multidrug component geneemrA, RpoS posttranscriptional regulator genecspC, adenosine deaminase geneadd, hydrogenase genehyc(Hyd-3), and the RNA chaperone/DNA-binding regulator genehfq. Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit generpoA, the Mar activator generob, andhfq. Deletion of lipopolysaccharide biosynthetic kinase generfaYdecreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such asaroF(encoding tyrosine biosynthesis) andapt(encoding adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for the loss of multidrug efflux pumps and of hydrogenases that generate a futile cycle of PMF and upregulates porins that admit fermentable nutrients and antibiotics.IMPORTANCEBenzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentrations, benzoic acid conducts a proton across the membrane, depleting the proton motive force. In the absence of antibiotics, benzoate exposure selects against proton-driven multidrug efflux pumps and upregulates porins that admit fermentable substrates but that also allow the entry of antibiotics. Thus, evolution with benzoate and related molecules, such as salicylates, requires a trade-off for antibiotic sensitivity, a trade-off that could help define a stable gut microbiome. Benzoate and salicylate are naturally occurring plant signal molecules that may modulate the microbiomes of plants and animal digestive tracts so as to favor fermenters and exclude drug-resistant pathogens.
Cellular Response of Campylobacter jejuni to Trisodium Phosphate