Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

SIGN EPISTASIS LIMITS EVOLUTIONARY TRADE‐OFFS AT THE CONFLUENCE OF SINGLE‐ AND MULTI‐CARBON METABOLISM IN METHYLOBACTERIUM EXTORQUENS AM1

Adaptation of one set of traits is often accompanied by attenuation of traits important in other selective environments, leading to fitness trade‐offs. The mechanisms that either promote or prevent the emergence of trade‐offs remain largely unknown, and are difficult to discern in most systems. Here, we investigate the basis of trade‐offs that emerged during experimental evolution of Methylobacterium extorquens AM1 to distinct growth substrates. After 1500 generations of adaptation to a multi‐carbon substrate, succinate (S), many lineages had lost the ability to use one‐carbon compounds such as methanol (M), generating a mixture of M⁺ and M^? evolved phenotypes. We show that trade‐offs in M^? strains consistently arise via antagonistic pleiotropy through recurrent selection for loss‐of‐function mutations to ftfL (formate‐tetrahydrofolate ligase), which improved growth on S while simultaneously eliminating growth on M. But if loss of FtfL was beneficial, why were M trade‐offs not found in all populations? We discovered that eliminating FtfL was not universally beneficial on S, as it was neutral or even deleterious in certain evolved lineages that remained M⁺. This suggests that sign epistasis with earlier arising mutations prevented the emergence of mutations that drove trade‐offs through antagonistic pleiotropy, limiting the evolution of metabolic specialists in some populations.     

Multiple long-term, experimentally-evolved populations of Escherichia coli acquire dependence upon citrate as an iron chelator for optimal growth on glucose

ABSTRACT: BACKGROUND: Specialization for ecological niches is a balance of evolutionary adaptation and its accompanying tradeoffs. Here we focus on the Lenski Long-Term Evolution Experiment, which has maintained cultures of Escherichia coli in the same, defined seasonal environment for 50,000 generations. Over this time, much adaptation and specialization to the environment has occurred. The presence of citrate in the growth media selected one lineage to gain the novel ability to utilize citrate as a carbon source after 31,000 generations. Here we test whether other strains have specialized to rely on citrate after 50,000 generations. RESULTS: We show that in addition to the citrate-catabolizing strain, three other lineages evolving in parallel have acquired a dependence on citrate for optimal growth on glucose. None of these strains were stimulated indirectly by the sodium present in disodium citrate, nor exhibited even partial utilization of citrate as a carbon source. Instead, all three of these citrate-stimulated populations appear to rely on it as a chelator of iron. CONCLUSIONS: The strains we examine here have evolved specialization to their environment through apparent loss of function. Our results are most consistent with the accumulation of mutations in iron transport genes that were obviated by abundant citrate. The results present another example where a subtle decision in the design of an evolution experiment led to unexpected evolutionary outcomes.     

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.     

Negative frequency‐dependent selection maintains coexisting genotypes during fluctuating selection

Natural environments are rarely static; rather selection can fluctuate on timescales ranging from hours to centuries. However, it is unclear how adaptation to fluctuating environments differs from adaptation to constant environments at the genetic level. For bacteria, one key axis of environmental variation is selection for planktonic or biofilm modes of growth. We conducted an evolution experiment with Burkholderia cenocepacia, comparing the evolutionary dynamics of populations evolving under constant selection for either biofilm formation or planktonic growth with populations in which selection fluctuated between the two environments on a weekly basis. Populations evolved in the fluctuating environment shared many of the same genetic targets of selection as those evolved in constant biofilm selection, but were genetically distinct from the constant planktonic populations. In the fluctuating environment, mutations in the biofilm‐regulating genes wspA and rpfR rose to high frequency in all replicate populations. A mutation in wspA first rose rapidly and nearly fixed during the initial biofilm phase but was subsequently displaced by a collection of rpfR mutants upon the shift to the planktonic phase. The wspA and rpfR genotypes coexisted via negative frequency‐dependent selection around an equilibrium frequency that shifted between the environments. The maintenance of coexisting genotypes in the fluctuating environment was unexpected. Under temporally fluctuating environments, coexistence of two genotypes is only predicted under a narrow range of conditions, but the frequency‐dependent interactions we observed provide a mechanism that can increase the likelihood of coexistence in fluctuating environments.     

High-resolution mutation mapping reveals parallel experimental evolution in yeast

Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2–24 kb (0.1–10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene.     

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.     

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Pantoea ananatis is a Gram‐negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non‐pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4‐formamido‐4,6‐dideoxy‐d ‐glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6‐dehydratase, an aminotransferase, and an N‐formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N‐formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis–Menten kinetics with dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as the substrate and N¹⁰‐formyltetrahydrofolate as the carbon source. In addition, the X‐ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N‐formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N‐formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non‐pathogenic apparently do not contain these genes.     

Experimental adaptive radiation in Pseudomonas

We studied the importance of selection and constraint in determining the limits of adaptive radiation and the consequences of adaptive radiation in an experimental system. We propagated four replicate lines of the bacterium Pseudomonas fluorescens derived from a single ancestral clone in 95 environments, where growth was limited by the availability of a single carbon source for 1,000 generations. We then assayed the growth of the ancestral clone and the evolved lines in all 95 environments. Evolved lines increased their performance in almost every selection environment and invaded 70% of the novel environments as a direct response to selection. Direct responses tended to be larger in environments where growth was initially poor. Although evolved lines lost the ability to grow on about three substrates that their ancestor could readily grow on, the correlated response to selection was, on average, positive. The correlated response allowed all of our evolved populations to expand their niches and to occupy collectively the remaining novel habitats. This is inconsistent with classical theories of niche evolution. In the most extreme cases, adaptation occurred through "roundabout selection": lineages became adapted to an environment through selection in another environment but not through selection in the environment itself. Our results indicate that mutation accumulation by neutral drift was responsible for generating the majority of costs of adaptation.     

The genetics of adaptation to discrete heterogeneous environments: frequent mutation or large‐effect alleles can allow range expansion

Range expansions are complex evolutionary and ecological processes. From an evolutionary standpoint, a populations' adaptive capacity can determine the success or failure of expansion. Using individual‐based simulations, we model range expansion over a two‐dimensional, approximately continuous landscape. We investigate the ability of populations to adapt across patchy environmental gradients and examine how the effect sizes of mutations influence the ability to adapt to novel environments during range expansion. We find that genetic architecture and landscape patchiness both have the ability to change the outcome of adaptation and expansion over the landscape. Adaptation to new environments succeeds via many mutations of small effect or few of large effect, but not via the intermediate between these cases. Higher genetic variance contributes to increased ability to adapt, but an alternative route of successful adaptation can proceed from low genetic variance scenarios with alleles of sufficiently large effect. Steeper environmental gradients can prevent adaptation and range expansion on both linear and patchy landscapes. When the landscape is partitioned into local patches with sharp changes in phenotypic optimum, the local magnitude of change between subsequent patches in the environment determines the success of adaptation to new patches during expansion.     

Dynamics of molecular evolution in RNA virus populations depend on sudden versus gradual environmental change

Understanding the dynamics of molecular adaptation is a fundamental goal of evolutionary biology. While adaptation to constant environments has been well characterized, the effects of environmental complexity remain seldom studied. One simple but understudied factor is the rate of environmental change. Here we used experimental evolution with RNA viruses to investigate whether evolutionary dynamics varied based on the rate of environmental turnover. We used whole‐genome next‐generation sequencing to characterize evolutionary dynamics in virus populations adapting to a sudden versus gradual shift onto a novel host cell type. In support of theoretical models, we found that when populations evolved in response to a sudden environmental change, mutations of large beneficial effect tended to fix early, followed by mutations of smaller beneficial effect; as predicted, this pattern broke down in response to a gradual environmental change. Early mutational steps were highly parallel across replicate populations in both treatments. The fixation of single mutations was less common than sweeps of associated “cohorts” of mutations, and this pattern intensified when the environment changed gradually. Additionally, clonal interference appeared stronger in response to a gradual change. Our results suggest that the rate of environmental change is an important determinant of evolutionary dynamics in asexual populations.     

Pervasive,yet idiosyncratic,epistatic pleiotropy during adaptation in a behaviourally complex microbe

Understanding how multiple mutations interact to jointly impact multiple ecologically important traits is critical for creating a robust picture of organismal fitness and the process of adaptation. However, this is complicated by both environmental heterogeneity and the complexity of genotype‐to‐phenotype relationships generated by pleiotropy and epistasis. Moreover, little is known about how pleiotropic and epistatic relationships themselves change over evolutionary time. The soil bacterium Myxococcus xanthus employs several distinct social traits across a range of environments. Here, we use an experimental lineage of M. xanthus that evolved a novel form of social motility to address how interactions between epistasis and pleiotropy evolve. Specifically, we test how mutations accumulated during selection on soft agar pleiotropically affect several other social traits (hard agar motility, predation and spore production). Relationships between changes in swarming rate in the selective environment and the four other traits varied greatly over time in both direction and magnitude, both across timescales of the entire evolutionary lineage and individual evolutionary time steps. We also tested how a previously defined epistatic interaction is pleiotropically expressed across these traits. We found that phenotypic effects of this epistatic interaction were highly correlated between soft and hard agar motility, but were uncorrelated between soft agar motility and predation, and inversely correlated between soft agar motility and spore production. Our results show that ‘epistatic pleiotropy’ varied greatly in magnitude, and often even in sign, across traits and over time, highlighting the necessity of simultaneously considering the interacting complexities of pleiotropy and epistasis when studying the process of adaptation.     

Metabolic constraints on the evolution of antibiotic resistance

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint‐based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition‐dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome‐based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin‐resistant strains. Overall, resolving metabolic adaptation throughout antibiotic‐driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.     

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.     

Laboratory evolution and multi-platform genome re-sequencing of the cellulolytic actinobacterium Thermobifida fusca

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.     

Parallelism in adaptive radiations of experimental Escherichia coli populations

Adaptive radiations are major contributors to species diversity. Although the underlying mechanisms of adaptive radiations, specialization and trade‐offs, are relatively well understood, the tempo and repeatability of adaptive radiations remain elusive. Ecological specialization can occur through the expansion into novel niches or through partitioning of an existing niche. To test how the mode of resource specialization affects the tempo and repeatability of adaptive radiations, we selected replicate bacterial populations in environments that promoted the evolution of diversity either through niche expansion or through niche partitioning, and in a third low‐quality single‐resource environment, in which diversity was not expected to evolve. Colony size diversity evolved equally fast in environments that provided ecological opportunities regardless of the mode of resource specialization. In the low‐quality environments, diversity did not consistently evolve. We observed the largest fitness improvement in the low‐quality environment and the smallest the glucose‐limited environment. We did not observe a change in the rate of evolutionary change in either trait or environment, suggesting that the pool of beneficial mutations was not exhausted. Overall, the mode of resource specialization did not affect the tempo or repeatability of adaptive radiations. These results demonstrate the limitations of eco‐evolutionary feedbacks to affect evolutionary outcomes.     

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products.