Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism

Abstract

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, 2015, https://doi.org/10.1128/AEM.03494-14). 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 H₂ 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: Escherichia coli; GABA; RNA polymerase; acid; decarboxylase; experimental evolution; fnr; low pH.

FIG 1

pH rise of acid-evolved mutants cultured with lysine or arginine. Bacteria were inoculated in microplate wells containing 200 μl of modified Møller broth medium at pH 6.8 supplemented with 2.5 g/liter l-lysine (A) or at pH 5.5 supplemented with 2.5 g/liter l-arginine (B). The microplates were incubated for 24 h at 37°C. The terminal spectrum ratio for bromocresol purple absorbance (A_570–590/A_400–450) was converted to indicate pH (scale at right) (see Fig. S1). Error bars indicate standard errors of the means (n = 9). One-way ANOVA tests were run with the Tukey comparisons to identify significantly different groups. Asterisks indicate strains for which the results are significantly different from those of the knockout strain (P < 0.05).

FIG 2

Arginine decarboxylase assay. RpoC modulates arginine-dependent pH rise in strain B11-1 but not in strains F9-2 and H9-1. Unique rpo point mutations found in the acid-evolved strains were reverted to represent the original ancestral W3110 sequence for strain B11-1 (A) as well as strains H9-1 and F9-2 (B). The method described in the legend of Fig. 1B was modified for kinetic recording of absorbance ratios. Absorbance readings were taken every 2 h for 48 h. Replicates for each strain are shown as curves of the same color.

FIG 3

GABA production and extreme-acid survival of acid-evolved strains. (A) GABA assays of anaerobic overnight cultures after exposure at pH 2.0 for 2 h, as measured by GC-MS (see Materials and Methods). ANOVA tests identified no significant difference in GABA production levels between strains W3110, B11, and H9. (B) Survival of overnight cultures after dilution and incubation at pH 2.0 for 2 h (see Materials and Methods). The y axis shows the log₂ ratios of the colony counts at the indicated pH values. The axis cutoff (10⁻⁶) represents the lower limit of colony detection. One-way ANOVA tests were run with the Tukey method comparison to identify significantly different groups. Asterisks indicate strains that show significant difference in results from those of the ancestral strain W3110 (P < 0.05).