Benzoate-Tolerant Escherichia coli Strains from Experimental Evolution Lose the Gad Regulon, Multi-Drug Efflux Pumps, and Hydrogenases

Benzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during experimental evolution of Escherichia coli K-12. Transcriptomes of E. coli isolates evolved with benzoate showed reversal of benzoate-dependent regulation, including downregulation of multi-drug efflux pumps; the Gad acid resistance regulon; the nitrate reductase narHJ; and 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 activator ariR, or of the slp-gadX acid fitness island encoding Gad regulators and the multi-drug pump mdtEF. Benzoate tolerance was also increased by deletion of multi-drug component emrA, RpoS post-transcriptional regulator cspC, adenosine deaminase add, hydrogenase hyc (Hyd-3), and the RNA chaperone/DNA-binding regulator hfq. Chloramphenicol resistance was decreased by mutations in global regulators, such as RNA polymerase alpha-subunit rpoA, Mar activator rob, and hfq. Deletion of lipopolysaccharide biosynthetic kinase rfaY decreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism such as aroF (tyrosine biosynthesis) and apt (adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for loss of multi-drug efflux pumps and of hydrogenases that generate a futile cycle of PMF; and upregulates porins that admit fermentable nutrients and antibiotics. IMPORTANCE: Benzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentration, 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 also allow entry of antibiotics. Thus, evolution with benzoate, and related molecules such as salicylates, requires a tradeoff for antibiotic sensitivity--a tradeoff 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.