The evolution of robustness and fragility during long-term bacterial adaptation

Abstract

Theory predicts that well-adapted populations may evolve mechanisms to counteract the inevitable influx of deleterious mutations. While mutational robustness can be directly selected in the laboratory, evidence for its spontaneous evolution during general adaptation is mixed. Moreover, whether robustness evolves to include pleiotropic effects remains largely unexplored. Here, we studied the effects of point mutations in the RNA polymerase of Escherichia coli over a 15,000-generation adaptive trajectory. Fitness effects of both beneficial and deleterious mutations were attenuated in fitter backgrounds. In contrast, pleiotropic effects became more severe and widespread with greater adaptation. These results show that trade-offs between robustness and fragility can evolve in regulatory networks, regardless of whether driven by adaptive or nonadaptive processes. More broadly, they illustrate how adaptation can generate hidden variability, with unpredictable evolutionary consequences in new environments.

Keywords: experimental evolution; genetic robustness; pleiotropy.

Fig. 1.

Fitness effects of RNA polymerase mutations in the LTEE environment. (A) Average fitness increases over time in the LTEE. We examined fitness effects in the ancestor and two evolved clones with fitness gains of ~25% (2 K) and ~50% (15 K). (B) Using lambda-red recombineering, we introduced the same 10 rpoB mutations, clustered within 171-bp, into each background. (C) Insets show mutant-to-wildtype ratios during bulk competitions for the Anc, 2 K, and 15 K backgrounds (Left to Right). Bar plot shows fitness effects with SE (Anc, black; 2 K, red; 15 K, blue). (D) Difference in fitness effects in evolved strains (2 K, red; 15 K, blue) relative to the ancestor. Both deleterious and beneficial effects are mitigated in evolved strains. The gray rectangle indicates 95% CI from two replicates. The vertical line marks the transition from deleterious to beneficial effects in the ancestor.

Fig. 2.

Pleiotropic effects of RNA polymerase mutations. (A) Mutations tend to be more deleterious, with ranks changing in LB compared to DM. (B) Fitness effects in LB correlate strongly across backgrounds, with a few exceptions. Symbols as in Fig. 1C. (C) Evolved strains show no directional trend in robustness. (D) Heatmaps show alterations in virulence-related traits caused by the 10 mutations across backgrounds (scale in E, number of stressor levels by which mutations shift the point of growth inhibition, see SI Appendix). Traits include biofilm formation (BIO) and resistance to bile salts (BS), oxidative (OX), thermal (THE), iron starvation (IR), acidic (AC), and alkaline (ALK) stresses. (E) Alterations increase progressively with adaptation to the LTEE environment, indicating a robustness–fragility trade-off. (F) Susceptibility profiles of the evolved backgrounds (Top). Fragility correlates strongly with each mutation’s median fitness effect across backgrounds and media (Bottom).