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Key Points The evolve and resequence (E&R) approach is a powerful paradigm for understanding the molecular basis of adaptation. Several E&R systems exist, ranging from in vitro RNA and DNA molecules to microorganisms evolving from an isogenic clone and sexual eukaryotes harbouring standing variation. E&R experiments are producing different results in the different systems. Can observed differences be reconciled with evolutionary theoretical models? The systems differ in: population size, level of standing variation, initial variance in fitness and level of genetic exchange. We argue that when these differences between systems are taken into account many of the apparent differences can be explained. Nevertheless, enigmas remain. Why do ploidy changes and/or large duplications and deletions seem to be more important in asexual microorganisms and sexual eukaryotes? At what point do sexually reproducing organisms need newly arising mutations? In sexually reproducing organisms, does allele frequency change often plateau before fixation? How much can macroscopic epistasis help us to understand evolution in microorganisms, and what is the role of epistasis in sexually reproducing organisms? Combining experimental evolution with next-generation sequencing, the evolve and resequence (E&R) approach is a powerful method for dissecting the genomic changes underlying the adaptation of populations of laboratory organisms or molecules. This Review describes the E&R results from diverse systems and discusses the extent to which various features, including population genetics, experimental setups and reproduction modes, account for the distinct observed outcomes. Evolve and resequence (E&R) experiments use experimental evolution to adapt populations to a novel environment, then next-generation sequencing to analyse genetic changes. They enable molecular evolution to be monitored in real time on a genome-wide scale. Here, we review the field of E&R experiments across diverse systems, ranging from simple non-living RNA to bacteria, yeast and the complex multicellular organism Drosophila melanogaster . We explore how different evolutionary outcomes in these systems are largely consistent with common population genetics principles. Differences in outcomes across systems are largely explained by different starting population sizes, levels of pre-existing genetic variation, recombination rates and adaptive landscapes. We highlight emerging themes and inconsistencies that future experiments must address.

Key Points Escherichia coli is, paradoxically, both the most frequent commensal aero-anaerobic Gram-negative bacillus of the vertebrate gut and one of the main pathogens, being responsible for both intraintestinal and extraintestinal infections. Deciphering the ecological and evolutionary forces that shape the population structure of the commensal strains will help to understand the emergence of virulence in the species. In the past few decades, successive molecular methods have contributed to the refinement of the clonal concept of E. coli , including serotyping and multilocus enzyme electrophoresis, followed by DNA marker analysis and nucleotide sequencing. Recently, whole-genome sequencing has revealed the organization of the genome and solved the contradiction between the occurence of recombination events and the observed clonality of the species, allowing the reconstruction of a robust phylogenetic history. In parallel, population genetics-based epidemiology has shown that in a single individual there are predominant strains and also resident and transient strains. Clones, which are characterised by their phylogenetic group, are distributed according to environmental factors and the diet, gut morphology and body mass of their hosts. Finally, the relationships between commensalism and virulence have been clarified. The coincidental hypothesis proposes that 'virulence factors' and their change in prevalence among hosts may reflect some local adaptation to commensal habitats rather than virulence per se . Likewise, intestinal microbiota has been shown to play an important part in the emergence of antibiotic resistance. In the future, with the arrival of next-generation sequencing technology, the study of complete genomes of numerous isolates will allow the development of 'population genomics', and metagenomics approaches will take into account the vast accompanying intestinal microbiota that has been largely ignored in defining the commensal niche of E. coli . Denamur and colleagues review the population structure of commensal Escherichia coli and discuss how commensal strains can adapt to different niches and how commensalism can evolve into pathogenicity. The primary habitat of Escherichia coli is the vertebrate gut, where it is the predominant aerobic organism, living in symbiosis with its host. Despite the occurrence of recombination events, the population structure is predominantly clonal, allowing the delineation of major phylogenetic groups. The genetic structure of commensal E. coli is shaped by multiple host and environmental factors, and the determinants involved in the virulence of the bacteria may in fact reflect adaptation to commensal habitats. A better characterization of the commensal niche is necessary to understand how a useful commensal can become a harmful pathogen. In this Review we describe the population structure of commensal E. coli , the factors involved in the spread of different strains, how the bacteria can adapt to different niches and how a commensal lifestyle can evolve into a pathogenic one.

Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli . IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS 150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS 150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS 150 -mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability. Insertion sequences (IS) are common mobile genetic elements in bacteria, but their effects on bacterial evolution are not well understood. Here, Consuegra and colleagues investigate the dynamics and fitness consequences of IS elements in E. coli over 50,000 generations.

Mutators represent a successful strategy in rapidly adapting asexual populations, but theory predicts their eventual extinction due to their unsustainably large deleterious load. While antimutator invasions have been documented experimentally, important discrepancies among studies remain currently unexplained. Here we show that a largely neglected factor, the mutational idiosyncrasy displayed by different mutators, can play a major role in this process. Analysing phylogenetically diverse bacteria, we find marked and systematic differences in the protein-disruptive effects of mutations caused by different mutators in species with different GC compositions. Computer simulations show that these differences can account for order-of-magnitude changes in antimutator fitness for a realistic range of parameters. Overall, our results suggest that antimutator dynamics may be highly dependent on the specific genetic, ecological and evolutionary history of a given population. This context-dependency further complicates our understanding of mutators in clinical settings, as well as their role in shaping bacterial genome size and composition. Mutators are expected to re-evolve low mutation rates to reduce deleterious load, but empirical evidence is mixed. Here, the authors show that load can vary across mutators and genetic backgrounds, which their simulations suggest can substantially alter antimutator dynamics.

With the molecular revolution in Biology, a mechanistic understanding of the genotype–phenotype relationship became possible. Recently, advances in DNA synthesis and sequencing have enabled the development of deep mutational scanning assays, capable of scoring comprehensive libraries of genotypes for fitness and a variety of phenotypes in massively parallel fashion. The resulting empirical genotype–fitness maps pave the way to predictive models, potentially accelerating our ability to anticipate the behaviour of pathogen and cancerous cell populations from sequencing data. Besides from cellular fitness, phenotypes of direct application in industry (e.g. enzyme activity) and medicine (e.g. antibody binding) can be quantified and even selected directly by these assays. This review discusses the technological basis of and recent developments in massively parallel genetics, along with the trends it is uncovering in the genotype–phenotype relationship (distribution of mutation effects, epistasis), their possible mechanistic bases and future directions for advancing towards the goal of predictive genetics.

Background Bacterial colonies are dynamic evolutionary microenvironments where spatial heterogeneity and nutrient gradients generate diverse ecological niches. Understanding how such structured environments drive genetic and functional diversification, including the emergence of clinically relevant traits such as antibiotic resistance, remains a major challenge. Methods To dissect adaptive strategies in structured populations, we performed whole-genome sequencing on 24 Escherichia coli isolates recovered from a three-week-old colony. Transcriptomic profiling was conducted on two representative mutants, and targeted competition assays were used to assess fitness relative to the parental strain. Results Genome sequencing uncovered extensive evolutionary diversification, with 34 distinct mutations, mostly insertion-sequence events, affecting transcriptional, stress-response, metabolic, and envelope regulators. Strikingly, half of all isolates carried mutations in the yobF-cspC operon, identifying it as a major adaptive hotspot in structured populations. Transcriptomic analyses revealed a broad regulatory shift in yobF-cspC mutants, with substantial activation of central metabolism and biosynthesis coupled to repression of acid resistance and stress pathways. Functionally, deletion of cspC alone conferred a robust σS-independent fitness advantage and fully restored competitiveness in an rpoS -inactivated background in aging colonies. Beyond this dominant adaptive trajectory, genome analysis revealed isolates carrying mutations that confer gain-of-function phenotypes, including β-lactam and rifamycin resistance, despite the absence of antibiotic pressure, highlighting aging colonies as potential reservoirs of clinically relevant diversity. Conclusions Our results suggest that aging bacterial colonies can serve as incubators of evolutionary innovation, simultaneously generating diverse functional variants while selecting for metabolic specialization. Together, these findings show how spatial structure and nutrient recycling shape bacterial adaptation and diversification, providing a powerful and tractable model to investigate evolutionary processes in natural and pathogenic communities.

To estimate the number and diversity of beneficial mutations, we experimentally evolved 115 populations of Escherichia coli to 42.2°C for 2000 generations and sequenced one genome from each population. We identified 1331 total mutations, affecting more than 600 different sites. Few mutations were shared among replicates, but a strong pattern of convergence emerged at the level of genes, opérons, and functional complexes. Our experiment uncovered a set of primary functional targets of high temperature, but we estimate that many other beneficial mutations could contribute to similar adaptive outcomes. We inferred the pervasive presence of epistasis among beneficial mutations, which shaped adaptive trajectories into at least two distinct pathways involving mutations either in the RNA polymerase complex or the termination factor rho.

Characterizing the effect of mutations is key to understand the evolution of protein sequences and to separate neutral amino-acid changes from deleterious ones. Epistatic interactions between residues can lead to a context dependence of mutation effects. Context dependence constrains the amino-acid changes that can contribute to polymorphism in the short term, and the ones that can accumulate between species in the long term. We use computational approaches to accurately predict the polymorphisms segregating in a panel of 61,157 Escherichia coli genomes from the analysis of distant homologues. By comparing a context-aware Direct-Coupling Analysis modelling to a non-epistatic approach, we show that the genetic context strongly constrains the tolerable amino acids in 30% to 50% of amino-acid sites. The study of more distant species suggests the gradual build-up of genetic context over long evolutionary timescales by the accumulation of small epistatic contributions. Predicting the effects of mutations in a species is a major challenge in genetics. Here, the authors investigate protein sequence landscapes using diverged E. coli sequences, to predict tolerated mutations and capture interactions between mutations.

RNA polymerase (RNAP) is emblematic of complex biological systems that control multiple traits involving trade-offs such as growth versus maintenance. Laboratory evolution has revealed that mutations in RNAP subunits, including RpoB, are frequently selected. However, we lack a systems view of how mutations alter the RNAP molecular functions to promote adaptation. We, therefore, measured the fitness of thousands of mutations within a region of rpoB under multiple conditions and genetic backgrounds, to find that adaptive mutations cluster in two modules. Mutations in one module favor growth over maintenance through a partial loss of an interaction associated with faster elongation. Mutations in the other favor maintenance over growth through a destabilized RNAP-DNA complex. The two molecular handles capture the versatile RNAP-mediated adaptations. Combining both interaction losses simultaneously improved maintenance and growth, challenging the idea that growth-maintenance tradeoff resorts only from limited resources, and revealing how compensatory evolution operates within RNAP. Mutations in an RNA polymerase fragment, frequently found in lab adaptation, cluster in two modules favoring growth or maintenance via loss of interactions. Combining mutations in both modules enhances both traits, promoting compensatory evolution.

Background Beneficial mutations play an essential role in bacterial adaptation, yet little is known about their fitness effects across genetic backgrounds and environments. One prominent example of bacterial adaptation is antibiotic resistance. Until recently, the paradigm has been that antibiotic resistance is selected by the presence of antibiotics because resistant mutations confer fitness costs in antibiotic free environments. In this study we show that it is not always the case, documenting the selection and fixation of resistant mutations in populations of Escherichia coli B that had never been exposed to antibiotics but instead evolved for 2000 generations at high temperature (42.2°C). Results We found parallel mutations within the rpoB gene encoding the beta subunit of RNA polymerase. These amino acid substitutions conferred different levels of rifampicin resistance. The resistant mutations typically appeared, and were fixed, early in the evolution experiment. We confirmed the high advantage of these mutations at 42.2°C in glucose-limited medium. However, the rpoB mutations had different fitness effects across three genetic backgrounds and six environments. Conclusions We describe resistance mutations that are not necessarily costly in the absence of antibiotics or compensatory mutations but are highly beneficial at high temperature and low glucose. Their fitness effects depend on the environment and the genetic background, providing glimpses into the prevalence of epistasis and pleiotropy.