Characterization of mutations in the PAS domain of the EvgS sensor kinase selected by laboratory evolution for acid resistance in Escherichia coli

Abstract

Laboratory-based evolution and whole-genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two-component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS-dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.

Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution

“…E. coli experimental evolution in moderate acid (pH 5.3) yields an acid-dependent fitness increase, but genome sequence analysis has not been used to identify the underlying genetic changes (31). In another study, populations that evolved for 1,000 generations with 20 rounds of exposure to pH 2.5 showed 20 new mutations, including mutations in evgS alleles that relieve logphase repression of extreme-acid resistance (32). Here we report on the isolation and characterization of eight E. coli strains from 4 of 24 populations cultured at pH 4.8 (for 730 generations) and then at pH 4.6 (2,000 generations in total).…”

Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution

“…Experimental evolution procedures with E. coli have included the adaptation to high temperatures (43), freeze-thaw cycles (44), high ethanol concentrations (45), and acid (46)(47)(48). We developed a microplate dilution cycle in order to generate evolving populations buffered at low pH (49).…”

“…We sequenced genomes of selected isolates and then identified genetic variants using the breseq pipeline (48)(49)(50). The breseq pipeline assembles a referencebased alignment to predict mutations compared to a previously sequenced genome (NCBI GenBank accession number NC_007779.1, E. coli K-12 W3110).…”

Discrimination and Integration of Stress Signals by Pathogenic Bacteria

“…Binding of a signaling molecule to particular amino acids or cofactors in PAS and GAF domains leads to a protein conformational change that converts the sensor-kinase to an active form. Heme groups of the O 2 receptors FixL and DosT are ligated to PAS and GAF domains, respectively, whereas a PAS-like fold in PhoQ and EvgS responds to antimicrobial peptides, divalent cations, or acid pH (Cho et al, 2006; Johnson et al, 2014). PhoQ also responses to acid stress, and the mechanism has been characterized in detail.…”

“…Acid pH can independently activate PhoQ through allosteric interactions with the adaptor protein SafA. Protonation of a histidine residue in the PAS domain in the periplasmic portion of the EvgS sensor kinase stimulates binding of the SafA adaptor protein to the PAS domain of PhoQ (Johnson et al, 2014). HAMP domains are usually involved in transduction of signals generated by upstream domains.…”