Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli

Abstract

Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit(+)) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit(+) variants achieve a much higher population density in this environment. All Cit(+) individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit(+) phenotype on its own. To determine which of the other >70 mutations in early Cit(+) clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit(+) from an evolved strain. We discovered a mutation that increased expression of the dctA C4-dicarboxylate transporter greatly enhanced the Cit(+) phenotype after it evolved. Surprisingly, strains containing just the citT and dctA mutations fully use citrate, indicating that earlier mutations thought to have potentiated the initial evolution of Cit(+) are not required for expression of the refined version of this trait. Instead, this metabolic innovation may be contingent on a genetic background, and possibly ecological context, that enabled citT mutants to persist among competitors long enough to obtain dctA or equivalent mutations that conferred an overwhelming advantage. More generally, refinement of an emergent trait from a rudimentary form may be crucial to its evolutionary success.

Keywords: epistatic network; experimental evolution; genetic basis of adaptation.

Fig. 1.

REGRES. (1) Suicide F plasmids are transferred to the donor strain by conjugation, and multiple F plasmid integrant Hfr strains are selected. (2) Isolated Hfr strains are mated with an F^– recipient strain, permitting genome transfer and homologous recombination with the genomes of recipient cells. (3) A suitable selection or screening procedure for individual transconjugants displaying the phenotype of interest is applied. (4) Isolated strains are genotyped against a panel of known alleles. (5) Strains of interest, usually with the fewest donor alleles, are selected for whole-genome sequencing and can be used as donors for another round of REGRES, if desired.

Fig. 2.

Evolved mutations present in Cit⁺ REGRES strains. Whole-genome sequencing was used to determine the CZB154 alleles and other genetic changes present in seven REGRES clones chosen on the basis of genotyping results (Figs. S1–S4). Selected alleles are shown here, ordered by position in the ancestral chromosome and named for the genes they impact (e.g., yaaH), for the flanking genes if an allele is intergenic (e.g., dctA/yhjK) or for a range of genes for alleles that represent large chromosomal duplications or deletions (e.g., rbsD–yieO). Full results are shown in Fig. S5, and complete details for each allele are provided in Table S1.

Fig. 3.

Evolved dctA* mutation is sufficient for Cit⁺⁺ in conjunction with CitT activation, increases dctA mRNA expression, and enables utilization of succinate. (A) Average growth curves of strains with either the ancestral (REL607) or evolved dctA allele [REL607(dctA*) and CZB154] grown in DM25 media as in the evolution experiment. Certain strains also carry a multicopy plasmid with the activated rnk–citT promoter configuration (pCitT). DM25 media contains 0.0025% (wt/vol) glucose and 0.032% (wt/vol) citrate. Error bars are the SD of at least three replicates. (B) Average growth curves in DM25 of strains containing either the ancestral or evolved dctA allele or with a reversion of the evolved allele to the ancestral state (dctA^wt). Error bars are the SD of at least three replicates. (C) mRNA expression of dctA gene determined by qRT-PCR for strains containing either the ancestral or evolved dctA allele. Transcript levels are shown relative to strain REL607. Error bars are the SEM for biological triplicate samples. (D) Average growth curves of strains containing either the ancestral or evolved dctA allele in DM media with no citrate or glucose and containing 0.01% (wt/vol) succinate as the sole carbon source. Error bars are the SD of at least three replicates. Similar growth was observed for these strains on other C₄-dicarboxylates such as fumarate and aspartate (Fig. S7).

Fig. 4.

Model for the evolution of citrate utilization in the E. coli long-term evolution experiment. (A) There were three major epochs in the evolution of this metabolic innovation: potentiation, actualization, and refinement. Weakly Cit⁺ cells were first isolated from the population after the rnk–citG actualizing mutation that amplified and activated the CitT transporter (shown as the citT allele). There was not an appreciable increase in the final cell density at the end of each growth cycle in the evolution experiment at this point (Top). After the dctA* refinement mutation, there was a substantial increase in population size because cells were able to fully use citrate, which we distinguish from the rudimentary Cit⁺ phenotype as the strong Cit⁺⁺ phenotype (Middle). The evolution of Cit⁺⁺ is statistically more likely from certain Cit^– genetic backgrounds that arose later in this population, presumably because they accumulated one or more potentiating mutations relative to the ancestor (3, 10). Key mutations are shown with their approximate timings relative to these evolutionary epochs and one another (Bottom). (B) Cit⁺⁺ phenotype is not the product of all-or-none epistasis with potentiating mutations. The progression of citrate utilization phenotypes as they evolved in the LTEE in strains that contained key mutations in the context of earlier evolved alleles (Upper) and qualitative phenotypes of reconstructed strains containing only key evolved alleles in the ancestral genetic background (Bottom) are shown. The evolved citT mutation alone is sufficient for detectable but extremely limited citrate utilization, as observed in early Cit⁺ isolates (3). The citT and dctA* mutations together are sufficient for full citrate utilization characteristic of the Cit⁺⁺ phenotype, even in the absence of potentiating mutations. (C) Mechanism of Cit⁺⁺ refinement. When both the CitT and DctA transporters are expressed, due to the citT and dctA* mutations, their activities can be coupled so that the proton-motive force (H⁺) powers reuptake of succinate or other C₄-dicarboxylate substrates for continued citrate import, yielding the Cit⁺⁺ phenotype. It is possible that the unknown potentiating mutations make sufficient succinate available from glucose metabolism to power limited citrate import through CitT but that this does not result in a sustainable cycle.