Metabolic Erosion Primarily Through Mutation Accumulation,and Not Tradeoffs,Drives Limited Evolution of Substrate Specificity in Escherichia coli

Evolutionary adaptation to a constant environment is often accompanied by specialization and a reduction of fitness in other environments. We assayed the ability of the Lenski Escherichia coli populations to grow on a range of carbon sources after 50,000 generations of adaptation on glucose. Using direct measurements of growth rates, we demonstrated that declines in performance were much less widespread than suggested by previous results from Biolog assays of cellular respiration. Surprisingly, there were many performance increases on a variety of substrates. In addition to the now famous example of citrate, we observed several other novel gains of function for organic acids that the ancestral strain only marginally utilized. Quantitative growth data also showed that strains with a higher mutation rate exhibited significantly more declines, suggesting that most metabolic erosion was driven by mutation accumulation and not by physiological tradeoffs. These reductions in growth by mutator strains were ameliorated by growth at lower temperature, consistent with the hypothesis that this metabolic erosion is largely caused by destabilizing mutations to the associated enzymes. We further hypothesized that reductions in growth rate would be greatest for substrates used most differently from glucose, and we used flux balance analysis to formulate this question quantitatively. To our surprise, we found no significant relationship between decreases in growth and dissimilarity to glucose metabolism. Taken as a whole, these data suggest that in a single resource environment, specialization does not mainly result as an inevitable consequence of adaptive tradeoffs, but rather due to the gradual accumulation of disabling mutations in unused portions of the genome.     

ASYMMETRIC, BIMODAL TRADE-OFFS DURING ADAPTATION OF METHYLOBACTERIUM TO DISTINCT GROWTH SUBSTRATES

Trade-offs between selected and nonselected environments are often assumed to exist during adaptation. This phenomenon is prevalent in microbial metabolism, where many organisms have come to specialize on a narrow breadth of substrates. One well-studied example is methylotrophic bacteria that can use single-carbon (C₁) compounds as their sole source of carbon and energy, but generally use few, if any, multi-C compounds. Here, we use adaptation of experimental populations of the model methylotroph, Methylobacterium extorquens AM1, to C₁ (methanol) or multi-C (succinate) compounds to investigate specialization and trade-offs between these two metabolic lifestyles. We found a general trend toward trade-offs during adaptation to succinate, but this was neither universal nor showed a quantitative relationship with the extent of adaptation. After 1500 generations, succinate-evolved strains had a remarkably bimodal distribution of fitness values on methanol: either an improvement comparable to the strains adapted on methanol or the complete loss of the ability to grow on C₁ compounds. In contrast, adaptation to methanol resulted in no such trade-offs. Based on the substantial, asymmetric loss of C₁ growth during growth on succinate, we suggest that the long-term maintenance of C₁ metabolism across the genus Methylobacterium requires relatively frequent use of C₁ compounds to prevent rapid loss.     

Evolution of Escherichia coli in different carbon environments for 2,000 generations

Cellular energetics is thought to have played a key role in dictating all major evolutionary transitions in the history of life on Earth. However, how exactly cellular energetics and metabolism come together to shape evolutionary paths is not well understood. In particular, when an organism is evolved in different energy environments, what are the phenomenological differences in the chosen evolutionary trajectories, is a question that is not well understood. In this context, starting from an Escherichia coli K‐12 strain, we evolve the bacterium in five different carbon environments—glucose, arabinose, xylose, rhamnose and a mixture of these four sugars (in a predefined ratio) for approximately 2,000 generations. At the end of the adaptation period, we quantify and compare the growth dynamics of the strains in a variety of environments. The evolved strains show no specialized adaptation towards growth in the carbon medium in which they were evolved. Rather, in all environments, the evolved strains exhibited a reduced lag phase and an increased growth rate. Sequencing results reveal that these dynamical properties are not introduced via mutations in the precise loci associated with utilization of the sugar in which the bacterium evolved. These phenotypic changes are rather likely introduced via mutations elsewhere on the genome. Data from our experiments indicate that evolution in a defined environment does not alter hierarchy in mixed‐sugar utilization in bacteria.     

Simultaneous Growth on Citrate Reduces the Effects of Iron Limitation during Toluene Degradation in Pseudomonas

Rhizoremediation has been suggested as an attractive bioremediation strategy for the effective breakdown of pollutants in soil. The presence of plant root exudates such as organic acids, sugars, and amino acids that may serve as carbon sources or biosynthetic building blocks and the limited bioavailability of iron may influence the degradation of pollutants in the rhizosphere. To test the effect of such compounds on hydrocarbon degradation, trace concentrations of yeast extract or mixtures of organic acids and amino acids were added to continuous cultures of Pseudomonas putida mt2 and P. putida WCS358 (TOL) growing on toluene. By addition of these compounds increased growth yields and higher specific growth rates on toluene were obtained. The effects of iron limitation on the substrate utilization pattern of both strains were tested by growing the strains on a mixture of toluene and the readily degradable carbon source citrate while the iron concentration was varied. Simultaneous use of both substrates under carbon-limited as well as iron-limited conditions was observed. Growth yields were less reduced and iron requirement was lower during iron-limited growth in the toluene + citrate grown cultures compared to cultures in which toluene was used as the sole carbon source. The kinetic properties of the cells for toluene degradation were less hampered by the lack of iron when citrate was used as an additional carbon source. The results indicate that the availability of low concentrations of natural organic compounds, such as produced in the rhizosphere, may positively influence the degradative performance of hydrocarbon-degrading bacteria.     

The Ability of Flux Balance Analysis to Predict Evolution of Central Metabolism Scales with the Initial Distance to the Optimum

The most powerful genome-scale framework to model metabolism, flux balance analysis (FBA), is an evolutionary optimality model. It hypothesizes selection upon a proposed optimality criterion in order to predict the set of internal fluxes that would maximize fitness. Here we present a direct test of the optimality assumption underlying FBA by comparing the central metabolic fluxes predicted by multiple criteria to changes measurable by a ¹³C-labeling method for experimentally-evolved strains. We considered datasets for three Escherichia coli evolution experiments that varied in their length, consistency of environment, and initial optimality. For ten populations that were evolved for 50,000 generations in glucose minimal medium, we observed modest changes in relative fluxes that led to small, but significant decreases in optimality and increased the distance to the predicted optimal flux distribution. In contrast, seven populations evolved on the poor substrate lactate for 900 generations collectively became more optimal and had flux distributions that moved toward predictions. For three pairs of central metabolic knockouts evolved on glucose for 600–800 generations, there was a balance between cases where optimality and flux patterns moved toward or away from FBA predictions. Despite this variation in predictability of changes in central metabolism, two generalities emerged. First, improved growth largely derived from evolved increases in the rate of substrate use. Second, FBA predictions bore out well for the two experiments initiated with ancestors with relatively sub-optimal yield, whereas those begun already quite optimal tended to move somewhat away from predictions. These findings suggest that the tradeoff between rate and yield is surprisingly modest. The observed positive correlation between rate and yield when adaptation initiated further from the optimum resulted in the ability of FBA to use stoichiometric constraints to predict the evolution of metabolism despite selection for rate.     

From nature to the laboratory: the impact of founder effects on adaptation

Most founding events entail a reduction in population size, which in turn leads to genetic drift effects that can deplete alleles. Besides reducing neutral genetic variability, founder effects can in principle shift additive genetic variance for phenotypes that underlie fitness. This could then lead to different rates of adaptation among populations that have undergone a population size bottleneck as well as an environmental change, even when these populations have a common evolutionary history. Thus, theory suggests that there should be an association between observable genetic variability for both neutral markers and phenotypes related to fitness. Here, we test this scenario by monitoring the early evolutionary dynamics of six laboratory foundations derived from founders taken from the same source natural population of Drosophila subobscura. Each foundation was in turn three‐fold replicated. During their first few generations, these six foundations showed an abrupt increase in their genetic differentiation, within and between foundations. The eighteen populations that were monitored also differed in their patterns of phenotypic adaptation according to their immediately ancestral founding sample. Differences in early genetic variability and in effective population size were found to predict differences in the rate of adaptation during the first 21 generations of laboratory evolution. We show that evolution in a novel environment is strongly contingent not only on the initial composition of a newly founded population but also on the stochastic changes that occur during the first generations of colonization. Such effects make laboratory populations poor guides to the evolutionary genetic properties of their ancestral wild populations.     

How phenotypic convergence arises in experimental evolution

Evolutionary convergence is a core issue in the study of adaptive evolution, as well as a highly debated topic at present. Few studies have analyzed this issue using a “real‐time” or evolutionary trajectory approach. Do populations that are initially differentiated converge to a similar adaptive state when experiencing a common novel environment? Drosophila subobscura populations founded from different locations and years showed initial differences and variation in evolutionary rates in several traits during short‐term (～20 generations) laboratory adaptation. Here, we extend that analysis to 40 more generations to analyze (1) how differences in evolutionary dynamics among populations change between shorter and longer time spans, and (2) whether evolutionary convergence occurs after 60 generations of evolution in a common environment. We found substantial variation in longer term evolutionary trajectories and differences between short‐ and longer term evolutionary dynamics. Although we observed pervasive patterns of convergence toward the character values of long‐established populations, populations still remain differentiated for several traits at the final generations analyzed. This pattern might involve transient divergence, as we report in some cases, indicating that more generations should lead to final convergence. These findings highlight the importance of longer term studies for understanding convergent evolution.     

Utilization of Iron Gallate and Other Organic Iron Complexes by Bacteria from Water Supplies

The degradation of four soluble organic iron compounds by bacteria isolated from surface waters and the precipitation of iron from these complexes by the isolates was studied. All eight isolates brought about the precipitation of iron when grown on ferric ammonium citrate agar. Three isolates were able to degrade ferric malonate, and three others degraded ferric malate with iron precipitation. Only three isolates, two strains of Pseudomonas and one of Moraxella, were able to degrade gallic acid when this was supplied as the sole carbon source. One strain of Pseudomonas was found to be active in degrading ferric gallate. Electron microscopy of cells of this bacterium after growth in ferric gallate as the sole carbon source yielded results indicating uniform deposition of the iron on or in the bacterial cells. Seven of the isolates could degrade the iron gallate complex if supplied with additional carbon in the form of yeast extract.     

Experimental adaptation of Salmonella typhimurium to mice

Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization. By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment. Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected into mice. After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11-0.58). Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10(-6)/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations. For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability. During adaptation there was no indication that a high mutation rate was counterselected. These data show that S. typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.     

The adaptive response of a natural microbial population to carbon- and nitrogen-limitation

The majority of experiments investigating the population and community level consequences of altered carbon and nitrogen availability implicitly assume a fixed range of ability to obtain and process these nutrients. This may be fair when considering long‐lived organisms, however, short‐lived organisms (e.g. microbes) will pass through many more generations in the same time period providing scope for evolutionary adaptation. Using isolates of a natural fungal population we demonstrate significant adaptation to carbon‐limitation, but not to nitrogen‐limitation, over a modest number of generations. However, adaptation comes at a cost: those adapted to carbon‐limitation are significantly worse competitors for limited nitrogen when compared with their ancestors. The potential for adaptation (and correlated cost) we demonstrate with our model system highlights the need to consider evolutionary adaptation when interpreting responses of populations, communities and ecosystems to treatments that manipulate, either directly or indirectly, resource availability.     

Rapid evolution of metabolic traits explains thermal adaptation in phytoplankton

Understanding the mechanisms that determine how phytoplankton adapt to warming will substantially improve the realism of models describing ecological and biogeochemical effects of climate change. Here, we quantify the evolution of elevated thermal tolerance in the phytoplankton, Chlorella vulgaris. Initially, population growth was limited at higher temperatures because respiration was more sensitive to temperature than photosynthesis meaning less carbon was available for growth. Tolerance to high temperature evolved after ≈ 100 generations via greater down‐regulation of respiration relative to photosynthesis. By down‐regulating respiration, phytoplankton overcame the metabolic constraint imposed by the greater temperature sensitivity of respiration and more efficiently allocated fixed carbon to growth. Rapid evolution of carbon‐use efficiency provides a potentially general mechanism for thermal adaptation in phytoplankton and implies that evolutionary responses in phytoplankton will modify biogeochemical cycles and hence food web structure and function under warming. Models of climate futures that ignore adaptation would usefully be revisited.     

The Impact of Macroscopic Epistasis on Long-Term Evolutionary Dynamics

Genetic interactions can strongly influence the fitness effects of individual mutations, yet the impact of these epistatic interactions on evolutionary dynamics remains poorly understood. Here we investigate the evolutionary role of epistasis over 50,000 generations in a well-studied laboratory evolution experiment in Escherichia coli. The extensive duration of this experiment provides a unique window into the effects of epistasis during long-term adaptation to a constant environment. Guided by analytical results in the weak-mutation limit, we develop a computational framework to assess the compatibility of a given epistatic model with the observed patterns of fitness gain and mutation accumulation through time. We find that a decelerating fitness trajectory alone provides little power to distinguish between competing models, including those that lack any direct epistatic interactions between mutations. However, when combined with the mutation trajectory, these observables place strong constraints on the set of possible models of epistasis, ruling out many existing explanations of the data. Instead, we find that the data are consistent with a “two-epoch” model of adaptation, in which an initial burst of diminishing-returns epistasis is followed by a steady accumulation of mutations under a constant distribution of fitness effects. Our results highlight the need for additional DNA sequencing of these populations, as well as for more sophisticated models of epistasis that are compatible with all of the experimental data.     

Adaptive evolution in invasive species

Many emerging invasive species display evidence of rapid adaptation. Contemporary genetic studies demonstrate that adaptation to novel environments can occur within 20 generations or less, indicating that evolutionary processes can influence invasiveness. However, the source of genetic or epigenetic variation underlying these changes remains uncharacterised. Here, we review the potential for rapid adaptation from standing genetic variation and from new mutations, and examine four types of evolutionary change that might promote or constrain rapid adaptation during the invasion process. Understanding the source of variation that contributes to adaptive evolution in invasive plants is important for predicting future invasion scenarios, identifying candidate genes involved in invasiveness, and, more generally, for understanding how populations can evolve rapidly in response to novel and changing environments.     

Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments

Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.     

Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation

Microorganisms have been mutating and evolving on Earth for billions of years. Now, a field of research has developed around the idea of using microorganisms to study evolution in action. Controlled and replicated experiments are using viruses, bacteria and yeast to investigate how their genomes and phenotypic properties evolve over hundreds and even thousands of generations. Here, we examine the dynamics of evolutionary adaptation, the genetic bases of adaptation, tradeoffs and the environmental specificity of adaptation, the origin and evolutionary consequences of mutators, and the process of drift decay in very small populations.     

Citric acid as a siderophore of enterococci?

In the pool of 70 enterococcal strains of the genus Enterococcus 61.4% released citrate into the medium. This metabolite has occurred more frequently in E. faecium strains. There was no correlation between hydroxamate siderophores production and citrate releasing. Only nine (10, 3%) of 70 strains have used Fe3+-dicitrate complex as iron sources. Iron restricted condition causing moderate inhibition of growth have not increased citrate releasing. When iron deficiency has caused stronger growth inhibition, E. faecalis strains did not release citrate and E. faecium strains its smaller amounts. The resting cells grown in iron-restricted condition have incorporated 59Fe3+ complexed by citrate more active than cells grown in the medium with excess of iron. So, citrate has not been a siderophore in enterococci.     

Relation of Salmonella typhi and Salmonella gallinarum auxotrophicity to the ability to grow on Simmons' medium

A total of 9 S. typhi strains and 1 S. gallinarum strain isolated from clinical material are auxotrophic with respect to tryptophan. The growth of S. typhi is accelerated in a glucose- and tryptophan-containing minimal medium with cysteine and methionine added. S. typhi strains are incapable of using sodium citrate as the only source of carbon. S. typhi cannot grow on Simmons citrate agar as these organisms are auxotrophic with respect to tryptophan and incapable of using sodium citrate as the only source of carbon, and S. gallinarum cannot grow on this medium as they are auxotrophic with respect to tryptophan.     

The Escherichia coli BarA-UvrY two-component system is needed for efficient switching between glycolytic and gluconeogenic carbon sources

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.     

Gene expression changes in the coccolithophore Emiliania huxleyi after 500 generations of selection to ocean acidification

Coccolithophores are unicellular marine algae that produce biogenic calcite scales and substantially contribute to marine primary production and carbon export to the deep ocean. Ongoing ocean acidification particularly impairs calcifying organisms, mostly resulting in decreased growth and calcification. Recent studies revealed that the immediate physiological response in the coccolithophore Emiliania huxleyi to ocean acidification may be partially compensated by evolutionary adaptation, yet the underlying molecular mechanisms are currently unknown. Here, we report on the expression levels of 10 candidate genes putatively relevant to pH regulation, carbon transport, calcification and photosynthesis in E. huxleyi populations short-term exposed to ocean acidification conditions after acclimation (physiological response) and after 500 generations of high CO₂ adaptation (adaptive response). The physiological response revealed downregulation of candidate genes, well reflecting the concomitant decrease of growth and calcification. In the adaptive response, putative pH regulation and carbon transport genes were up-regulated, matching partial restoration of growth and calcification in high CO₂-adapted populations. Adaptation to ocean acidification in E. huxleyi likely involved improved cellular pH regulation, presumably indirectly affecting calcification. Adaptive evolution may thus have the potential to partially restore cellular pH regulatory capacity and thereby mitigate adverse effects of ocean acidification.