Innovation in an E. coli evolution experiment is contingent on maintaining adaptive potential until competition subsides

Abstract

Key innovations are disruptive evolutionary events that enable a species to escape constraints and rapidly diversify. After 15 years of the Lenski long-term evolution experiment with Escherichia coli, cells in one of the twelve populations evolved the ability to utilize citrate, an abundant but previously untapped carbon source in the environment. Descendants of these cells became dominant in the population and subsequently diversified as a consequence of invading this vacant niche. Mutations responsible for the appearance of rudimentary citrate utilization and for refining this ability have been characterized. However, the complete nature of the genetic and/or ecological events that set the stage for this key innovation is unknown. In particular, it is unclear why it took so long for citrate utilization to evolve and why it still has evolved in only one of the twelve E. coli populations after 30 years of the Lenski experiment. In this study, we recapitulated the initial mutation needed to evolve citrate utilization in strains isolated from throughout the first 31,500 generations of the history of this population. We found that there was already a slight fitness benefit for this mutation in the original ancestor of the evolution experiment and in other early isolates. However, evolution of citrate utilization was blocked at this point due to competition with other mutations that improved fitness in the original niche. Subsequently, an anti-potentiated genetic background evolved in which it was deleterious to evolve rudimentary citrate utilization. Only later, after further mutations accumulated that restored the benefit of this first-step mutation and the overall rate of adaptation in the population slowed, was citrate utilization likely to evolve. Thus, intense competition and the types of mutations that it favors can lead to short-sighted evolutionary trajectories that hide a stepping stone needed to access a key innovation from many future generations.

Fig 1. Evolution of rudimentary citrate utilization by activating citT expression is slightly beneficial in the genetic background in which it evolved and in the LTEE ancestor.

(a) The rnk-citG duplication that evolved in the LTEE creates a genomic configuration in which a novel mRNA encoding the CitT transporter is expressed from the rnk promoter (P_rnk) (right). This mutation alone is sufficient for weak citrate utilization (Cit⁺ phenotype). It is the ‘actualizing mutation’ in the evolution of this key innovation. Strain ZDB564 is the earliest Cit⁺ isolate from the LTEE. In order to measure the effect that this mutation had on competitive fitness when it evolved, a spontaneous Cit⁻ revertant of ZDB564 in which the duplication collapsed back to the ancestral state was isolated (left). (b) Competitive fitness of Cit⁺ versus Cit⁻ strain variants. The ZDB564 versus ZDB706 competitions measure the fitness effect of the rnk-citG duplication when it evolved. The ZDB706 and REL606 competitions test the effect of adding one copy of the evolved P_rnk-citT module into a strain (+) versus adding an empty version of the same cassette (Ø), as pictured in c. An additional ZDB706 competition (in population) was conducted with the two strains together mixed at a 1:99 ratio with the evolved LTEE population from at 31,000 generations to determine if the mutation had a different effect on fitness when rare in the population. Starred strains (*) have a change to the Ara⁺ marker state to allow competition with the corresponding Ara⁻ strain as illustrated in S1 and S2 Figs. The marker change had no effect on competitive fitness in each case (S3 Fig). Error bars are 95% confidence intervals. (c) Schematic of the gene cassettes used in the P_rnk-citT knock-in assay showing how they were integrated into the E. coli chromosome in a way that replaces the native lac locus. (d) citT mRNA expression levels measured relative to the REL606 LTEE ancestor in the evolved Cit⁺ isolate from the LTEE (ZDB564) and strains with the P_rnk-citT and corresponding empty control cassettes integrated into their chromosomes. Error bars are 95% confidence intervals.

Fig 2. Fitness consequences of evolving Cit⁺ in different evolved genetic backgrounds.

(a) Results of the P_rnk-citT knock-in assay on 23 pre-Cit⁺ evolved strains. The clones are ordered by the generation from which they were isolated. Error bars are 95% confidence intervals. Strain construction details and how the results of competition assays were combined into these fitness estimates are described in the Methods and S1–S4 Figs. (b) Increased lag phase upon addition of the P_rnk-citT module in anti-potentiated strains. Growth curves for the ancestor, REL606, and two anti-potentiated strains, ZDB483 and ZDB14, are shown. Error bars are standard deviations of four replicate cultures.

Fig 3. Potential for evolving Cit⁺ mapped onto phylogeny.

Phylogeny of isolates from the LTEE population including 20 new clones sequenced for this study to provide better resolution of the timing of mutations on the lineage leading to Cit⁺ (names in italics). In order to identify changes in the degree of potentiation due to mutations, we mapped the results of the P_rnk-citT knock-in assay onto this phylogenetic tree. Colored symbols reflect the Cit⁺ to Cit⁻ relative fitness measured for those strains. The ancestor and 61 evolved isolates were used to construct this phylogenetic tree (S1 Table). Two clones isolated at 50,000 generations are not shown. Two strains that evolved citrate utilization in replay experiments under the LTEE conditions in a previous study [7] are marked with plus signs (++), and three strains that had evolved alleles added or removed during strain construction as described in S2 Table are starred (*).

Fig 4. Changes in the potential for innovation along the lineage leading to Cit⁺ due to genetic and population factors.

We clustered phylogenetically adjacent strains in which activating citT expression had a similar effect on fitness to reconstruct when major changes in potentiation occurred due to new mutations accumulating in the evolved strain (the genetic background). Each cluster is represented by a different color and symbol in the simplified phylogenetic tree (upper panel) and the graph showing the group-wise fitness estimate over a time period (horizontal lines) in which these strains were representative of the main pre-Cit⁺ lineage (lower panel). Error bars are 95% confidence intervals. Two models of the rate of adaptation of the LTEE populations at different generations are superimposed on the lower panel. Model 1 estimates the fitness effects of winning cohorts of beneficial mutations sequentially sweeping through the population at each generation according to modelling from Wiser et al. [15]. Model 2 estimates the fitness effects of each consecutive beneficial mutation accrued by the winning lineage over time using additional information from Tenaillon et al. [13] (see Methods). These curves represent competing beneficial mutations that can suppress the evolution of Cit⁺ (the population context). If the fitness effect of activating citT is above these curves, as it is by 29,000 generations, then it is predicted to be among the most beneficial new mutations that could appear at that time in the LTEE. This means that rudimentary citrate utilization (Cit⁺) can persist in the population long enough to be refined by further mutations to full citrate utilization (Cit⁺⁺); the metabolic innovation can be achieved.