High frequency of hotspot mutations in core genes of Escherichia coli due to short-term positive selection

Abstract

Core genes comprising the ubiquitous backbone of bacterial genomes are not subject to frequent horizontal transfer and generally are not thought to contribute to the adaptive evolution of bacterial pathogens. We determined, however, that at least one-third and possibly more than one-half of the core genes in Escherichia coli genomes are targeted by repeated replacement substitutions in the same amino acid positions-hotspot mutations. Occurrence of hotspot mutations is driven by positive selection, as their rate is significantly higher than expected by random chance alone, and neither intragenic recombination nor increased mutability can explain the observed patterns. Also, commensal E. coli strains have a significantly lower frequency of mutated genes and mutations per genome than pathogenic strains. E. coli strains causing extra-intestinal infections accumulate hotspot mutations at the highest rate, whereas the highest total number of mutated genes has been found among Shigella isolates, suggesting the pathoadaptive nature of such mutations. The vast majority of hotspot mutations are of recent evolutionary origin, implying short-term positive selection, where adaptive mutations emerge repeatedly but are not sustained in natural circulation for long. Such pattern of dynamics is consistent with source-sink model of virulence evolution.

Fig. 1.

Phylogram of concatenated sequences of 7 housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA) for 14 E. coli/Shigella genomes analyzed.

Fig. 2.

(A) Frequency distribution of genes (of reference genome CFT073) across 14 genomes. The genes represented by only 1 genome are unique to CFT073, whereas the ones represented by all 14 genomes (shown as gray bar) are termed “core” genes. (B) Allelic diversity of core genes. The genes with at least 4 alleles (shown as gray bars) could be used for the maximum-likelihood based phylogenetic reconstruction and the following ZP-analysis.

Fig. 3.

Pie charts showing significantly (P < 0.05) enriched functional categories of candidate proteins with adaptive mutations (based on presence of recent hotspot mutations) in 4 ecotypes using DAVID (stringency, medium). For each pie chart, the number in parentheses denotes the number of selection-candidate genes included in the enriched categories.

Fig. 4.

Dependence of the number of genes with recent (shown by open points) or long-term (shown by filled points) hotspots in E. coli on the number of genomes analyzed. The best-fit curve based on data sets of 10 (circles), 11 (triangles), 15 (diamonds), 23 (asterisks) clonally unlinked genomes along with smaller randomly selected data sets of 7, 8, and 9 genomes (circles with error bars) was found to be sigmoidal [y = (a + b√x)/(1 + √x)] with R² = 0.97 for recent hotspots and R² = 0.99 for long-term hotspots.