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

Abstract

Benzoate, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline in Escherichia coli K-12. Genetic mechanisms of increased benzoate tolerance and decreased drug resistance were analyzed in strains isolated from experimental evolution with benzoate. Transcriptomes showed reversal of benzoate-dependent regulation, including the Gad regulon for acid resistance and multi-drug resistance (MDR). Benzoate-evolved strains knocked down the nitrate reductase narHJ and the acid-consuming hydrogenase Hyd-3, but upregulated OmpF and other porins that admit nutrients and antibiotics. Mutations were found affecting many genes of aromatic catabolism such as folD and add. Several candidate genes from benzoate-evolved strains had a role in benzoate tolerance. Growth with benzoate or salicylate was increased by deletion of the Gad activator ariR, or by deletion of the slp-gadX acid fitness island encoding Gad regulators and the MDR pump MdtEF. Benzoate growth was also increased by deletion of MDR component emrA, the RpoS post-transcriptional regulator cspC, or the adenosine deaminase add. Growth in chloramphenicol with benzoate was decreased by a point mutation in the RNA polymerase alpha-subunit rpoA, which appeared in one chloramphenicol-sensitive benzoate-evolved strain. Growth in chloramphenicol was also decreased by deletion of Mar activator rob, or of rfaY (lipopolysaccharide biosynthesis). Hydrogenase Hyd-3 deletion increased benzoate tolerance. Overall, long-term culture in the presence of benzoate or salicylate favors loss of MDR efflux pumps and of hydrogenases that generate a futile cycle of PMF; and favors upregulation of large-hole porins that increase uptake of fermentable nutrients and of antibiotics. IMPORTANCEBenzoate is a common food preservative, and salicylate is the active form of aspirin. In E. coli, benzoate derivatives upregulate multiple regulons that export antibiotics and other toxic products, and downregulate large outer-membrane porins that allow antibiotic influx. But benzoate or salicylate exposure causes energy stress by depleting the proton motive force. In the absence of antibiotics, long-term benzoate exposure selects against energy-spending systems such as multi-drug efflux pumps and the proton-consuming hydrogenase. Selection favors upregulation of porins that admit fermentable substrates but also allow entry of antibiotics. Thus, evolution with benzoate requires a tradeoff for antibiotic sensitivity. Benzoate and salicylate are naturally occurring plant signal molecules that may influence the evolution of microbiomes in plants and in animal digestive tracts. Effects on hydrogenase activity may be relevant to the biotechnology of hydrogen production.