Escherichia coli cultures maintain stable subpopulation structure during long-term evolution

Abstract

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

Keywords: biofilm; complex environment; niche specialization.

Fig. 1.

Illustration of culture methods for long-term evolution. Fifty WT and 50 mutL− (mismatch-repair deficient) E. coli K-12 MG1655 populations were established from a single isolated colony and grown in glass culture tubes containing 10 mL of LB broth. Cultures were propagated by alternating 37 °C incubation for 24 h with 25 °C incubation for 48 h. Further, five different evolution treatments were established by varying the volume of the transfer bottleneck. Populations were thoroughly vortexed before each transfer, and whole communities were subjected to whole-genome sequencing every 6 mo.

Fig. 2.

Muller plots representing relative abundances of haplotypes over 3 y of evolution. Shades of green, red, and blue denote different haplotypes, with variation in shade both within the figure and the mutation legend representing new variants arising within these haplotypes. Mutations in black occurred and fixed within populations before divergence of subpopulations. Relative abundances of haplotypes were inferred via whole-population sequencing performed every 6 mo, combining with whole-genome sequencing of eight individual clones from each population revealed haplotype associations. Numbers in the legend correspond to a particular population, while mutations included in the legend are present for at least two sampling points at a frequency of <0.05 in the population. Datasets S1 and S2 contain a complete list of mutations identified in each isolated clone.

Fig. 3.

Differences in growth phenotypes of clones isolated from population 125. (A) Growth curves (measured by optical density at a wavelength of 600 nm) for clones isolated from population 125 (MMR+) and revived population 125 (125-P) cultured in LB broth for 24 h at 37 °C. Warmer colors (red, orange, and pink) represent clones belonging to haplotype A and cooler colors (blue, green, and purple) represent clones belonging to haplotype B. MG1655 is the progenitor from which the evolved populations originated. (B) Relative maximum growth rates (U_max) for clones isolated from population 125 determined by fitting a modified Gompertz equation to optical density data. For all bar graphs, red bars represent clones belonging to haplotype A, blue bars represent clones belonging to haplotype B, and purple bars (125-P) represent a revived sample of population 125 after 2.5 y. (C) Relative lag time (L) for clones isolated from population 125 determined by fitting a modified Gompertz equation to optical-density data. (D) Competitive fitness of clones and revived sample of population 125 when cocultured with a rifampicin-resistant MG1655 strain and counted through replica plating determined by the equation $\omega =\ln \left(E_i/E_f\right)/\ln \left(A_i/A_f\right)$, where E_i and E_f are the initial and final counts of the evolved clones (population) and A_i and A_f are the initial and final counts of the MG1655 progenitor within the coculture. Rifampicin resistance was used as a selective marker as this phenotype can be conferred without a measurable fitness effect. Dark bars represent fitness in a culture tube (heterogeneous environment) and light bars represent fitness in a flask (homogeneous environment).

Fig. 4.

Uptake/release of free amino acids from single clones in LB broth through an initial 6 h of growth. Amino acid availability is demonstrated as a ratio of the concentration of a particular amino acid at a particular time point (T_x) and that for the same amino acid at 0 h (T₀) as detected by GC-MS. (A) phenylalanine, (B) proline, and (C) valine show no significant difference in net uptake across samples, while (D) threonine, (E) lysine, and (F) cysteine show significant differences in net uptake and net excretion between the evolved clonal isolates and the WT progenitor.