PT - JOURNAL ARTICLE AU - Kaitlin E. Creamer AU - Frederick Ditmars AU - Preston Basting AU - Santiago Acero AU - Karina Kunka AU - Issam Hamdallah AU - Sean P. Bush AU - Zachary Scott AU - Amanda He AU - Stephanie Penix AU - Alexandra Gonzales AU - Elizabeth K. Eder AU - Dominic Camperchioli AU - Adama Berndt AU - Michelle W. Clark AU - Kerry Rouhier AU - Joan L. Slonczewski TI - Benzoate and Salicylate Tolerant Strains Lose Antibiotic Resistance during Laboratory Evolution of <em>Escherichia coli</em> K-12 AID - 10.1101/063271 DP - 2016 Jan 01 TA - bioRxiv PG - 063271 4099 - http://biorxiv.org/content/early/2016/07/11/063271.short 4100 - http://biorxiv.org/content/early/2016/07/11/063271.full AB - Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted laboratory evolution by daily dilution in increasing concentrations of benzoic acid (from 5 to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from the evolving populations showed change in phenotype from benzoate-sensitive to benzoate-tolerant but sensitive to chloramphenicol and tetracycline. Sixteen clones isolated at 2,000 generations grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth profiles were seen in 10 mM salicylate. The strains showed growth profiles indistinguishable from W3110 in the absence of benzoate; in media buffered at pH 4.8, pH 7.0, or pH 9.0; or in 20 mM acetate or sorbate at pH 6.5. The genomes of 16 strains revealed over 100 mutations including SNPs, large deletions, and insertion sequence knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or associated regulators such as rob and cpx, as well as MDR efflux pumps emrA, emrY, and mdtA. Strains also lost or down-regulated the Gad acid fitness regulon. In 5 mM benzoate, or in 2 mM salicylate, most strains showed increased sensitivity to the antibiotic chloramphenicol, some more sensitive than a marA knockout. Thus, the benzoate-evolved strains may reveal additional unknown drug resistance components.