Enterococcus faecalis OG1RF Evolution at Low pH Selects Fusidate-sensitive Mutants in Elongation Factor G and at High pH Selects Defects in Phosphate Transport

Enterococcus bacteria inhabit human and soil environments that show a wide range of pH. Strains include commensals as well as antibiotic-resistant pathogens. We investigated adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution in acid (pH 4.8), neutral pH (pH 7.0), and base (pH 9.0). Serial planktonic culture was performed for 500 generations, and in high-pH biofilm culture for four serial bead transfers. Nearly all mutations led to nonsynonomous codons, indicating adaptive selection. All acid-adapted clones from planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a tradeoff of decreased resistance to fusidic acid (fusidate). All base-adapted clones from planktonic cultures, and some from biofilm-adapted cultures, showed mutations affecting the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). Biofilm culture produced small-size colonies on brain-heart infusion agar; these variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a tradeoff of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones evolved at high pH (but not at low pH) include oppBCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, experimental evolution of E. faecalis showed strong pH dependence, favoring fusidate-sensitive elongation factor G modification at low pH and loss of phosphate transport genes at high pH. IMPORTANCEE. faecalis bacteria are found in dental biofilms where they experience low pH as a result of fermentative metabolism. Thus the effect of pH on antibiotic resistance has clinical importance. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extreme high pH. In other environments such as soil and plant rhizosphere, enterococci experience acidification associated with climate change. Thus the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.