Laboratory Experimental Evolution of Escherichia coli K-12 under Aromatic Acid Stress

The intestinal bacterium Escherichia coli K-12 can survive and protect its cell contents from a wide range of acidic and basic conditions ranging from low pH in the stomach to higher pH levels in the small intestine. We initiated an adaptive laboratory evolution procedure that consisted of daily dilution in increasing concentrations of benzoic acid for 2,000 generations. Fitness increased step-wise over subsequent generations up to the final 2,000th generation. Sixteen unique clones from 12 of the 24-permeant aromatic acid-adapted populations grew significantly better in a lethal concentration of benzoate and salicylate compared to the ancestral strain. The genomes of the 16 isolates were whole genome sequenced and analyzed to find over 100 mutations including SNPs, large deletions, and transposable mobile elements. Some isolates acquired large deletions in the benzoate-induced multiple antibiotic resistance (mar) regulon and thus showed increased sensitivity to the antibiotic chloramphenicol. All isolates grew similarly to the ancestral wild type strain in various external pH conditions and in the presence of permeant aliphatic acids. However, the isolates showed profiles of adaption in salicylate that resembled the effects of benzoate, thus suggesting that the isolates had increased fitness specifically to permeant aromatic acids such as benzoate and salicylate. We predict that evolution in benzoic acid appears to downregulate regulons induced by aromatic acids and this project likely uncovered novel genes and their products that contribute to fitness, resistance, and growth in the presence of benzoate, the aspirin salicylate, and the antibiotics chloramphenicol and streptomycin.