Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, https://doi.org/10.1128/AEM. . Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY. The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.KEYWORDS acid, Escherichia coli, experimental evolution, GABA, low pH, RNA polymerase, decarboxylase, fnr E nteric bacteria need to survive a wide range of pH values throughout the human intestinal tract. The pH of the intestinal tract ranges from 1.5 to 3.5 in the stomach, increases to 4 to 7 in the duodenum, reaches 7 to 9 in the jejunum, and decreases to pH 5 to 6 in the cecum (1). On a microscopic scale, extreme gradients of pH occur across the intestinal epithelium where enterobacteria adhere. To cope with such conditions, Escherichia coli maintains a cytoplasmic pH homeostasis...

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 experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up 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 evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like 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. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA. Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

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Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabelling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of LTR retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.

Experimental evolution of Escherichia coli K-12 with benzoate, a partial uncoupler of the proton motive force (PMF), selects for mutations that decrease antibiotic resistance. We conducted experimental evolution in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20 to 150 µM CCCP buffered at pH 6.5 or at pH 8.0. After 1,000 generations, the populations tolerated up to 150 µM CCCP. Sequenced isolates had mutations in mprA (emrR), which downregulates the EmrAB-TolC pump that exports CCCP. A mprA::kanR deletion conferred growth at 60 μM CCCP, though not at the higher levels resisted by evolved strains (150 µM). Some mprA mutant strains also had point mutations affecting emrA, but deletion of emrA abolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lacked emrA or mprA mutations but had mutations in cecR (ybiH), whose product upregulates drug pumps YbhG and YbhFSR, and in gadE, which upregulates the multidrug pump MdtEF. A cecR::kanR deletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations included ybhR and acrAB. The acrB isolate was sensitive to the AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains include rng (RNase G) and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP-dependent fitness advantage for mutations increasing CCCP efflux via EmrA and for mutations that may deactivate proton-driven pumps for drugs not present (cecR, gadE, acrAB, and ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved benzoate tolerance. IMPORTANCE The genetic responses of bacteria to depletion of proton motive force (PMF), and their effects on drug resistance, are poorly understood. PMF drives export of many antibiotics, but the energy cost may decrease fitness when antibiotics are absent. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for increased CCCP efflux but also against the expression of PMF-driven pumps for drugs not present. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF.