Biology subjects

Lenski, R. E.

Publications and source records attributed to Lenski, R. E..

18 recordsLinked to original sources

bioRxiv2026

The historical domestication of a Clostridium botulinum strain used for the industrial production of botulinum neurotoxin

Laboratory production of botulinum neurotoxin (BoNT) began more than 90 years ago for medical, military, and later pharmaceutical applications, creating one of the longest-running examples of microbial cultivation under sustained human control. A single Clostridium botulinum Group I lineage, Army Hall A (AHA), was established by the U.S. military in 1942 and later gave rise to Hall A-hyper (HAH), the strain widely used for pharmaceutical BoNT production. We analyzed more than 1,000 C. botulinum genomes to identify AHAs closest relatives and to infer the most recent common ancestor (MRCA) shared with its nearest wild lineage. Relative to this MRCA, AHA accumulated nearly 100 genetic changes, including 46 single-nucleotide substitutions, 44 small insertions or deletions, and several large deletions and structural variants that led to the loss of more than 80 genes. A nonsense mutation in mutS generated a hypermutator phenotype that accelerated mutation rates and increased genetic diversity. This event occurred early in the lineages domestication and appears to have facilitated laboratory-adaptive traits, including the lack of sporulation and increased BoNT yield. Competition experiments under standard growth conditions confirmed substantial laboratory adaptation, with AHA exhibiting a strong fitness advantage over a close wild relative. Together, these results reconstruct the genomic trajectory of a bacterium evolving under prolonged human-mediated selection and provide a genome-resolved example of microbial domestication. The findings show how laboratory conditions, industrial selection, and changing mutation rates can jointly shape bacterial evolution, and they offer a general framework for understanding domestication and laboratory adaptation across microbial systems. SignificanceHuman-mediated species domestication has been central to the development of civilization, with well-known examples in plants and animals. Microbes have also been domesticated, often unintentionally and before the advent of modern microbiology. The military and pharmaceutical applications of botulinum neurotoxin led to the sustained cultivation and eventual domestication of a high toxin-producing Clostridium botulinum strain that remains widely used today. We reconstruct the genomic changes along this trajectory and identify a key early mutation that generated a hypermutator phenotype, increasing genetic variation available for human-directed selection. These findings reveal fundamental evolutionary processes shaping bacterial genomes under long-term human control. This genome-resolved example of microbial domestication offers a general framework for understanding laboratory adaptation in both evolutionary and applied microbiology.

microbiology↗

bioRxiv2025

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

Most mutations affecting fitness are harmful, and their inevitable occurrence reduces mean population fitness. Theory predicts that well-adapted populations may evolve mechanisms to minimize this deleterious load. Direct selection to increase mutational robustness can be achieved in the laboratory. However, its spontaneous evolution during general adaptation remains uncertain, with mixed evidence across model systems. Here, we studied the effects of highly pleiotropic point mutations in Escherichia coli over a 15,000-generation adaptive trajectory. The fitness effects of both beneficial and deleterious mutations were attenuated with increased adaptation over time. In contrast, pleiotropic effects in new environments became more severe and widespread with greater adaptation. These results show that trade-offs between robustness and fragility can rapidly evolve in regulatory networks, regardless of whether driven by adaptive or non-adaptive processes. More broadly, these results show that adaptation can generate a hidden potential for phenotypic diversity, unpredictably shaping evolutionary prospects in new environments.

evolutionary biology↗

bioRxiv2024

Reduced selection during sweeps lead to adaptive momentum on rugged landscapes

AbstractEvolutionary theory seeks to explain the remarkable diversity and adaptability of life on Earth. Current theory offers substantial explanatory power, but it overlooks important transient dynamics that are prominent only when populations are outside equilibrium, such as during selective sweeps. We identify a dynamic that we call "adaptive momentum" whereby lineages with a selective advantage can temporarily sustain more deleterious mutations. This reduction in the strength of purifying selection allows populations to explore fitness valleys that are usually too costly to enter, potentially leading to the discovery of otherwise inaccessible fitness peaks. Using mathematical and agent-based simulations, we demonstrate adaptive momentum and show how periods of disequilibrium become windows of enhanced adaptation. Genetic exploration can occur during these windows without requiring mechanisms such as changing environments or complex landscapes. Adaptive momentum provides a simple potential explanation for bursts of rapid evolution observed in nature, including in pathogens such as SARS-CoV-2 and cancers. (152 words)

evolutionary biology↗

bioRxiv2024

Parasite contingency loci and the evolution of host specificity: Simple sequence repeats mediate Bartonella adaptation to a wild rodent host

Parasites can adapt to better exploit their hosts on many scales, ranging from within an infection of a single individual to series of infections spanning multiple host species. However, little is known about how the genomes of parasites in natural communities evolve when they face diverse hosts. We investigated how Bartonella bacteria that circulate in rodent communities in the dunes of the Negev Desert in Israel adapt to different species of rodent hosts. We propagated 15 Bartonella populations through infections of either a single host species (Gerbillus andersoni or Gerbillus pyramidum) or alternating between the two. After 20 rodent passages, strains with de novo mutations replaced the ancestor in most populations. Mutations in two mononucleotide simple sequence repeats (SSRs) that caused frameshifts in the same adhesin gene dominated the evolutionary dynamics. They appeared exclusively in populations that encountered G. andersoni and altered the dynamics of infections of this host. Similar SSRs in other genes are conserved and exhibit ON/OFF variation in Bartonella isolates from the Negev Desert dunes. Our results suggest that SSR-based contingency loci could be important not only for rapidly and reversibly generating antigenic variation to escape immune responses but that they may also mediate the evolution of host specificity.

evolutionary biology↗

bioRxiv2022

Experimental Test of the Contributions of Initial Variation and New Mutations to Adaptive Evolution in a Novel Environment

Experimental evolution is an approach that allows researchers to study organisms as they evolve in controlled environments. Despite the growing popularity of this approach, there are conceptual gaps among projects that use different experimental designs. One such gap concerns the contributions to adaptation of genetic variation present at the start of an experiment and that of new mutations that arise during an experiment. The primary source of genetic variation has historically depended largely on the study organisms. In the long-term evolution experiment (LTEE) using Escherichia coli, for example, each population started from a single haploid cell, and therefore adaptation depended entirely on new mutations. Most other microbial evolution experiments have followed the same strategy. By contrast, evolution experiments using multicellular, sexually-reproducing organisms typically start with pre-existing variation that fuels the response to selection. New mutations may also come into play in later generations of these experiments, but it is generally difficult to quantify their contribution in these studies. Here, we performed an experiment using E. coli to compare the contributions of initial genetic variation and new mutations to adaptation in a new environment. Our experiment had four treatments that varied in their starting diversity, with 18 populations in each treatment. One treatment depended entirely on new mutations, while the other three began with mixtures of clones, whole-population samples, or mixtures of whole-population samples from the LTEE. By tracking genetic markers associated with particular founders in two of our treatments, we could document the impact of the initial variation during the early generations of our experiment. However, there were no differences in fitness among the treatments after 500 or 2000 generations in the new environment, despite the variation in fitness among the founders. These results indicate that new mutations quickly overcame, and eventually contributed more to adaptation, than did the initial variation. Our study thus shows that pre-existing genetic variation can have a strong impact on early evolution in a new environment, but new beneficial mutations may contribute more to later evolution and can even drive some initially beneficial variants to extinction.

evolutionary biology↗

bioRxiv2022

Parallel changes in gene essentiality over 50,000 generations of evolution

As evolving populations accumulate mutations, the benefits and costs of subsequent mutations change. As fitness increases, the relative benefit of new mutations typically decreases. However, the question remains whether deleterious mutations tend to have larger or smaller costs as a population adapts; theory and experiments provide support for both conflicting hypotheses. To address this question, we compared the effects of insertion mutations in every gene in Escherichia coli between ancestral and 12 independently derived strains after 50,000 generations in a uniform environment. We found both increases and decreases in the fitness costs of mutations, leaving the overall distribution of effects largely unchanged. However, at the extreme, more genes became essential over evolution than vice versa. Both changes in fitness effects and essentiality evolved in parallel across the independent populations, and most changes were not explained by structural variation or altered gene expression. Thus, the macroscopic features of the local fitness landscape remained largely unchanged, even as access to particular evolutionary trajectories changed consistently during adaptation to the experimental environment. One Sentence SummaryLimdi et al. report parallel changes in the cost of mutations in replicate lineages of a decades-long E. coli evolution experiment.

evolutionary biology↗

bioRxiv2022

The evolution of fitness effects during long-term adaptation in bacteria

The distribution of fitness effects of new mutations is central to predicting adaptive evolution, but observing how it changes as organisms adapt is challenging. Here we use saturated, genome-wide insertion libraries to quantify how the fitness effects of new mutations changed in two E. coli populations that adapted to a constant environment for 15,000 generations. The proportions of neutral and deleterious mutations remained constant, despite large fitness gains. In contrast, the beneficial fraction declined rapidly, approximating an exponential distribution, with strong epistasis profoundly changing the genetic identity of adaptive mutations. Despite this volatility, many important targets of selection were predictable from the ancestral distribution. This predictability occurs because genetic target size contributed to the fixation of beneficial mutations as much as or more than their effect sizes. Overall, our results demonstrate that short-term adaptation can be idiosyncratic but empirically predictable, and that long-term dynamics can be described by simple statistical principles. One-Sentence SummaryCouce et al. demonstrate that short-term bacterial adaptation is predictable at the scale of individual genes, while long-term adaptation is predictable at a global scale.

evolutionary biology↗

bioRxiv2022

Idiosyncratic fitness costs of ampicillin-resistant mutants derived from a long-term experiment with Escherichia coli

Antibiotic resistance is a growing concern that has prompted a renewed focus on drug discovery, stewardship, and evolutionary studies of the patterns and processes that underlie this phenomenon. A resistant strains competitive fitness relative to its sensitive counterparts in the absence of drug can impact its spread and persistence in both clinical and community settings. In a prior study, we examined the fitness of tetracycline-resistant clones that evolved from five different Escherichia coli genotypes, which had diverged during a long-term evolution experiment. In this study, we build on that work to examine whether ampicillin-resistant mutants are also less fit in the absence of the drug than their sensitive parents, and whether the cost of resistance is constant or variable among independently derived lines. Like the tetracycline-resistant lines, the ampicillin-resistant mutants were often less fit than their sensitive parents, with significant variation in the fitness costs among the mutants. This variation was not associated with the level of resistance conferred by the mutations, nor did it vary across the different parental backgrounds. In our earlier study, some of the variation in fitness costs associated with tetracycline resistance was explained by the effects of different mutations affecting the same cellular pathway and even the same gene. In contrast, the variance among the ampicillin-resistant mutants was associated with different sets of target genes. About half of the resistant clones suffered large fitness deficits, and their mutations impacted major outer-membrane proteins or subunits of RNA polymerases. The other mutants experienced little or no fitness costs and with, one exception, they had mutations affecting other genes and functions. Our findings underscore the importance of comparative studies on the evolution of antibiotic resistance, and they highlight the nuanced processes that shape these phenotypes.

evolutionary biology↗

bioRxiv2021

Effects of periodic bottlenecks on the dynamics of adaptive evolution in microbial populations

Population bottlenecks are common in nature, and they can impact the rate of adaptation in evolving populations. On the one hand, each bottleneck reduces the genetic variation that fuels adaptation. On the other hand, each founder that survives a bottleneck can undergo more generations and leave more descendants in a resource-limited environment, which allows surviving beneficial mutations to spread more quickly. A theoretical model predicted that the rate of fitness gains should be maximized using [~]8-fold dilutions. Here we investigate the impact of repeated bottlenecks on the dynamics of adaptation using numerical simulations and experimental populations of Escherichia coli. Our simulations confirm the models prediction when populations evolve in a regime where beneficial mutations are rare and waiting times between successful mutations are long. However, more extreme dilutions maximize fitness gains in simulations when beneficial mutations are common and clonal interference prevents most of them from fixing. To examine the simulations predictions, we propagated 48 E. coli populations with 2-, 8-, 100-, and 1000-fold dilutions for 150 days. Adaptation began earlier and fitness gains were greater with 100- and 1000-fold dilutions than with 8-fold dilutions, consistent with the simulations when beneficial mutations are common. However, the selection pressures in the 2-fold treatment were qualitatively different from the other treatments, violating a critical assumption of the model and simulations. Thus, varying the dilution factor during periodic bottlenecks can have multiple effects on the dynamics of adaptation caused by differential losses of diversity, different numbers of generations, and altered selection. SignificanceMany microorganisms experience population bottlenecks during transmission between hosts or when propagated in the laboratory. These bottlenecks reduce genetic diversity, potentially impeding natural selection. However, bottlenecks can also increase the number of generations over which selection acts, potentially accelerating adaptation. We explored this tension by performing simulations that reflect these opposing factors, and by evolving bacterial populations under several dilution treatments. The simulations show that the dilution factor that maximizes the rate of adaptation depends critically on the rate of beneficial mutations. On balance, the simulations agree well with our experimental results, which imply a high rate of beneficial mutation that generates intense competition between mutant lineages.

evolutionary biology↗

bioRxiv2021

Evolution with private resources reverses some changes from long-term evolution with public resources

A population under selection to improve one trait may evolve a sub-optimal state for another trait due to tradeoffs and other evolutionary constraints. How this evolution affects the capacity of a population to adapt when conditions change to favor the second trait is an open question. We investigated this question using isolates from a lineage spanning 60,000 generations of the Long-Term Evolution Experiment (LTEE) with Escherichia coli, where cells have access to a shared pool of resources, and have evolved increased competitive ability and a concomitant reduction in numerical yield. Using media-in oil emulsions we shifted the focus of selection to numerical yield, where cells grew in isolated patches with private resources. We found that the time spent evolving under shared resources did not affect the ability to re-evolve toward higher numerical yield. The evolution of numerical yield commonly occurred through mutations in the phosphoenolpyruvate phosphotransferase system. These mutants exhibit slower uptake of glucose, making them poorer competitors for public resources, and produce smaller cells that release less carbon as overflow metabolites. Our results demonstrate that mutations that were not part of adaptation under one selective regime may enable access to ancestral phenotypes when selection changes to favor evolutionary reversion.

evolutionary biology↗

bioRxiv2021

How the footprint of history shapes the evolution of digital organisms

Goulds thought experiment of "replaying lifes tape" provides a conceptual framework for experiments that quantify the contributions of adaptation, chance, and history to evolutionary outcomes. For example, we can empirically measure how varying the depth of history in one environment influences subsequent evolution in a new environment. Can this "footprint of history"--the genomic legacy of prior adaptation--grow too deep to overcome? Can it constrain adaptation, even with intense selection in the new environment? We investigated these questions using digital organisms. Specifically, we evolved ten populations from one ancestor under identical conditions. We then replayed evolution from three time points in each populations history (corresponding to shallow, intermediate, and deep history) in two new environments (one similar and one dissimilar to the prior environment). We measured the contributions of adaptation, chance, and history to the among-lineage variation in fitness and genome length in both new environments. In both environments, variation in genome length depended largely on history and chance, not adaptation, indicating weak selection. By contrast, adaptation, chance, and history all contributed to variation in fitness. Crucially, whether the depth of history affected adaptation depended on the environment. When the ancestral and new environments overlapped, history was as important as adaptation to the fitness achieved in the new environment for the populations with the deepest history. However, when the ancestral and novel environments favored different traits, adaptation overwhelmed even deep history. This experimental design for assessing the influence of the depth of history is promising for both biological and digital systems.

evolutionary biology↗

bioRxiv2021

Sustained coevolution of phage Lambda and Escherichia coli involves inner as well as outer membrane defenses and counter-defenses

Bacteria often evolve resistance to phage through the loss or modification of cell-surface receptors. In Escherichia coli and phage {lambda}, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyze another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair {lambda}s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that {lambda} evolved an additional counter-defense that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral {lambda}, but some evolved phage grew well on the deletion mutants, indicating they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfills the model of an inverse-gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated. IMPACT STATEMENTLaboratory studies of coevolution help us understand how host defenses and pathogen counter-defenses change over time, which is often essential for predicting the future dynamics of host-pathogen interactions. One particular model, termed "inverse-gene-for-gene" coevolution, predicts that coevolution proceeds through alternating steps, whereby hosts lose the features exploited by pathogens, and pathogens evolve to exploit alternative features. Using a classic model system in molecular biology, we describe the nature and timing of a previously overlooked step in the coevolution of E. coli and bacteriophage lambda. Our work demonstrates that this mode of coevolution can profoundly re-shape the interactions between bacteria and phage.

microbiology↗

bioRxiv2020

Idiosyncratic variation in the fitness costs of tetracycline-resistance mutations in Escherichia coli

A bacteriums fitness relative to its competitors, both in the presence and absence of antibiotics, plays a key role in its ecological success and clinical impact. In this study, we examine whether tetracycline-resistant mutants are less fit in the absence of the drug than their sensitive parents, and whether the fitness cost of resistance is constant or variable across independently derived lines. Tetracycline-resistant lines suffered, on average, a reduction in fitness of almost 8%. There was substantial among-line variation in the fitness cost. This variation was not associated with the level of phenotypic resistance conferred by the mutations, nor did it vary significantly across several different genetic backgrounds. The two resistant lines with the most extreme fitness costs involved functionally unrelated mutations on different genetic backgrounds. However, there was also significant variation in the fitness costs for mutations affecting the same pathway and even different alleles of the same gene. Our findings demonstrate that the fitness costs of antibiotic resistance do not always correlate with the phenotypic level of resistance or the underlying genetic changes. Instead, these costs reflect the idiosyncratic effects of particular resistance mutations and the genetic backgrounds in which they occur.

evolutionary biology↗

bioRxiv2020

Genomic evolution of antibiotic resistance is contingent on genetic background following a long-term experiment with Escherichia coli

Antibiotic resistance is a growing health concern. Efforts to control resistance would benefit from an improved ability to forecast when and how it will evolve. Epistatic interactions between mutations can promote divergent evolutionary trajectories, which complicates our ability to predict evolution. We recently showed that differences between genetic backgrounds can lead to idiosyncratic responses in the evolvability of phenotypic resistance, even among closely related Escherichia coli strains. In this study, we examined whether a strains genetic background also influences the genotypic evolution of resistance. Do lineages founded by different genotypes take parallel or divergent mutational paths to achieve their evolved resistance states? We addressed this question by sequencing the complete genomes of antibiotic-resistant clones that evolved from several different genetic starting points during our earlier experiments. We first validated our statistical approach by quantifying the specificity of genomic evolution with respect to antibiotic treatment. As expected, mutations in particular genes were strongly associated with each drug. Then, we determined that replicate lines evolved from the same founding genotypes had more parallel mutations at the gene level than lines evolved from different founding genotypes, although these effects were more subtle than those showing antibiotic specificity. Taken together with our previous work, we conclude that historical contingency can alter both genotypic and phenotypic pathways to antibiotic resistance. SignificanceA fundamental question in evolution is the repeatability of adaptation. Will independently evolving populations respond similarly when facing the same environmental challenge? This question also has important public-health implications related to the growing problem of antibiotic resistance. For example, efforts to control resistance might benefit from accurately predicting mutational paths to resistance. However, this goal is complicated when a lineages prior history alters its subsequent evolution. We recently found that differences between genetic backgrounds can lead to unpredictable responses in phenotypic resistance. Here, we report that genetic background can similarly alter genotypic paths to resistance. This historical contingency underscores the importance of accounting for stochasticity, in the past as well as at present, when designing evolutionarily informed treatment strategies.

evolutionary biology↗

bioRxiv2020

Maintenance of metabolic plasticity despite relaxed selection in a long-term evolution experiment with Escherichia coli

AO_SCPLOWBSTRACTC_SCPLOWTraits that are unused in a given environment are subject to processes that tend to erode them, leading to reduced fitness in other environments. Although this general tendency is clear, we know much less about why some traits are lost while others are retained, and about the roles of mutation and selection in generating different responses. We addressed these issues by examining populations of a facultative anaerobe, Escherichia coli, that have evolved for >30 years in the presence of oxygen, with relaxed selection for anaerobic growth and the associated metabolic plasticity. We asked whether evolution led to the loss, improvement, or maintenance of anaerobic growth, and we analyzed gene expression and mutational datasets to understand the outcomes. We identified genomic signatures of both positive and purifying selection on aerobic-specific genes, while anaerobic-specific genes showed clear evidence of relaxed selection. We also found parallel evolution at two interacting loci that regulate anaerobic growth. We competed the ancestor and evolved clones from each population in an anoxic environment, and we found that anaerobic fitness had not decayed, despite relaxed selection. In summary, relaxed section does not necessarily reduce an organisms fitness in other environments. Instead, the genetic architecture of the traits under relaxed selection and their correlations with traits under positive and purifying selection may sometimes determine evolutionary outcomes.

evolutionary biology↗

bioRxiv2020

Genomic and phenotypic evolution of Escherichia coli in a novel citrate-only resource environment

Evolutionary innovations allow populations to colonize new, previously inaccessible ecological niches. We previously reported that aerobic growth on citrate (Cit+) evolved in a population of Escherichia coli during adaptation to a minimal glucose medium containing citrate (DM25). Cit+ can grow in citrate-only medium (DM0), which is a novel environment for E. coli. To study adaptation to this new niche, we evolved one set of Cit+ populations for 2,500 generations in DM0 and a control set in DM25. We identified numerous parallel mutations, many mediated by transposable elements. Several lineages evolved multi-copy amplifications containing the maeA gene, constituting up to [~]15% of the genome. We also found substantial cell death in ancestral and evolved clones. Our results demonstrate the importance of copy-number variation and transposable elements in the refinement of the Cit+ trait. However, the observed mortality suggests a persistent evolutionary mismatch between E. coli physiology and a citrate-only environment.

evolutionary biology↗

bioRxiv2019

Historical contingency in the evolution of antibiotic resistance after decades of relaxed selection

Populations often encounter changed environments that remove selection for the maintenance of particular phenotypic traits. The resulting genetic decay of those traits under relaxed selection reduces an organisms fitness in its prior environment. However, whether and how such decay alters the subsequent evolvability of a population upon restoration of selection for a previously diminished trait is not well understood. We addressed this question using Escherichia coli strains from the long-term evolution experiment (LTEE) that independently evolved for multiple decades in the absence of antibiotics. We first confirmed that these derived strains are typically more sensitive to various antibiotics than their common ancestor. We then subjected the ancestral and derived strains to various concentrations of these drugs to examine their potential to evolve increased resistance. We found that evolvability was idiosyncratic with respect to initial genotype; that is, the derived strains did not generally compensate for their greater susceptibility by \"catching up\" to the resistance level of the ancestor. Instead, the capacity to evolve increased resistance was constrained in some backgrounds, implying that evolvability depended upon prior mutations in a historically contingent fashion. We further subjected a time-series of clones from one LTEE population to tetracycline and determined that an evolutionary constraint arose early in that population, corroborating the role of contingency. In summary, relaxed selection not only can drive populations to increased antibiotic susceptibility, but it can also affect the subsequent evolvability of antibiotic resistance in an unpredictable manner. This conclusion has potential implications for public health, and it underscores the need to consider the genetic context of pathogens when designing drug-treatment strategies.

evolutionary biology↗

bioRxiv2017

Analysis of bacterial genomes from an evolution experiment with horizontal gene transfer shows that recombination can sometimes overwhelm selection

We analyzed genomes from an experiment in which Escherichia coli K-12 Hfr donors were periodically introduced into 12 evolving populations of E. coli B. Previous work showed that recombination did not increase adaptation, despite increasing variation relative to asexual controls. The effects of recombination were highly variable: one lineage was mostly derived from the donors, while another acquired almost no donor DNA. In most lineages, some regions showed repeated introgression and others almost none. Regions with high introgression tended to be near the donors origin of transfer sites. To determine whether introgressed alleles imposed a genetic load, we extended the experiment for 200 generations without recombination and sequenced whole-population samples. Beneficial alleles in the recipient populations were occasionally driven extinct by maladaptive donor-derived alleles. On balance, our analyses indicate that the plasmid-mediated recombination was sufficiently frequent to drive donor alleles to fixation without providing much, if any, selective advantage.

evolutionary biology↗