Temperature adaptation of enzymes: lessons from laboratory evolution.
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Evolutionary engineering and molecular characterization of an antimycin A-resistant Saccharomyces cerevisiae strain: the key role of pleiotropic drug resistance (PDR1).
Antimycin A, an antifungal agent that inhibits mitochondrial respiration, provides a useful model for studying resistance mechanisms. Antifungal resistance is an escalating clinical concern with limited treatment options available. To understand the molecular mechanisms of antimycin A resistance, a genetically stable, antimycin A-resistant Saccharomyces cerevisiae strain was successfully developed for the first time through an evolutionary engineering strategy, based on long-term systematic application of gradually increasing antimycin A stress in repetitive batch cultures without prior chemical mutagenesis. Comparative whole genome resequencing analysis of the evolved strain ant905-9 revealed two missense mutations in PDR1 and PRP8 genes involved in pleiotropic drug resistance and RNA splicing, respectively. Using CRISPR/Cas9 genome editing tools, the identified mutations were introduced individually and together into the reference strain, and it was confirmed that the Pdr1p.M732R mutation alone confers antimycin A-resistance in S. cerevisiae. Comparative transcriptomic analysis of the reverse-engineered Pdr1p.M732R strain showed alterations in PDR (pleiotropic drug resistance), transmembrane transport, vesicular trafficking, and autophagy pathways. Our results highlight the potential key role of PDR1 in antifungal drug resistance. This study provides new insights into mitochondrial drug resistance and the adaptive potential of yeast under respiratory stress.
Saccharomyces cerevisiae↗
Directed evolution study of temperature adaptation in a psychrophilic enzyme.
We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins.
Adaptation, Physiological↗
Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution.
Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.
Adaptation, Physiological↗
Parallel adaptive evolution cultures of Escherichia coli lead to convergent growth phenotypes with different gene expression states.
Laboratory evolution can be used to address fundamental questions about adaptation to selection pressures and, ultimately, the process of evolution. In this study, we investigated the reproducibility of growth phenotypes and global gene expression states during adaptive evolution. The results from parallel, replicate adaptive evolution experiments of Escherichia coli K-12 MG1655 grown on either lactate or glycerol minimal media showed that (1) growth phenotypes at the endpoint of evolution are convergent and reproducible; (2) endpoints of evolution have different underlying gene expression states; and (3) the evolutionary gene expression response involves a large number of compensatory expression changes and a smaller number of adaptively beneficial expression changes common across evolution strains. Gene expression changes initially showed a large number of differentially expressed genes in response to an environmental change followed by a return of most genes to a baseline expression level, leaving a relatively small set of differentially expressed genes at the endpoint that varied between evolved populations.
Biological Evolution↗
Adaptation of a South American malaria vector to laboratory colonization suggests faster-male evolution for mating ability.
BACKGROUND: Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) is one of the very few South American mosquito vectors of malaria successfully colonized in the laboratory. These vectors are very hard to breed because they rarely mate in artificial conditions. A few years ago a free-mating laboratory colony of An. albitarsis sensu stricto was established after about 30 generations of artificial-mating. To begin to understand the process of adaptation of these malaria vectors to the laboratory we have compared the insemination rates of colony mosquitoes to those from the original population in both artificial and free-mating crosses. We also carried out crossing experiments between the two types of mosquitoes for a preliminary analysis of the genetic basis of such adaptation. RESULTS: We show that, compared to the original population, colony males but not females have increased their insemination rates in the laboratory in both types of mating, suggesting that faster-male evolution of mating ability might have occurred during the colonization process. CONCLUSIONS: The results are consistent with the faster-male theory, which predicts that sexual selection will cause faster rates of evolution of genes expressed in males. The data also suggests that attempts to colonize other South American malaria mosquitoes will be more successful if special attention is given to the male ability to mate in a confined space.
Acclimatization↗
Natural selection on body size in Tribolium: possible genetic constraints on adaptive evolution.
To determine whether genetic constraints on adaptive evolution were operating in a laboratory population of a flour beetle, Tribolium castaneum, we first estimated the direct selection acting on each of several body size traits. Strong selection in males for an increase in pupal weight and a decrease in the ratio of adult to pupal weight occurred. In addition, a non-significant trend for a decrease in adult width was found. No significant selection on females was detected, although there were trends toward an increase in pupal weight and a decrease in adult width. These estimates were then combined with estimates of the genetic variances and covariances of the traits to predict the multivariate response to selection, that is, the evolutionary change in the traits across one generation. These projections showed only a small predicted change in male pupal weight in spite of the strong selection on pupal weight, and a relatively large predicted increase in width in spite of the possible negative direct selection on this trait. Both of these results were due in part to the positive genetic covariance between pupal weight and width, and they therefore suggest the possibility of genetic constraints on adaptive evolution of these traits.
Animals↗
The genetic basis of thermal reaction norm evolution in lab and natural phage populations.
Two major goals of laboratory evolution experiments are to integrate from genotype to phenotype to fitness, and to understand the genetic basis of adaptation in natural populations. Here we demonstrate that both goals are possible by re-examining the outcome of a previous laboratory evolution experiment in which the bacteriophage G4 was adapted to high temperatures. We quantified the evolutionary changes in the thermal reaction norms--the curves that describe the effect of temperature on the growth rate of the phages--and decomposed the changes into modes of biological interest. Our analysis indicated that changes in optimal temperature accounted for almost half of the evolutionary changes in thermal reaction norm shape, and made the largest contribution toward adaptation at high temperatures. Genome sequencing allowed us to associate reaction norm shape changes with particular nucleotide mutations, and several of the identified mutations were found to be polymorphic in natural populations. Growth rate measures of natural phage that differed at a site that contributed substantially to adaptation in the lab indicated that this mutation also underlies thermal reaction norm shape variation in nature. In combination, our results suggest that laboratory evolution experiments may successfully predict the genetic bases of evolutionary responses to temperature in nature. The implications of this work for viral evolution arise from the fact that shifts in the thermal optimum are characterized by tradeoffs in performance between high and low temperatures. Optimum shifts, if characteristic of viral adaptation to novel temperatures, would ensure the success of vaccine development strategies that adapt viruses to low temperatures in an attempt to reduce virulence at higher (body) temperatures.
Base Sequence↗
Improved dissection efficiency in the human gross anatomy laboratory by the integration of computers and modern technology.
The need to increase the efficiency of dissection in the gross anatomy laboratory has been the driving force behind the technologic changes we have recently implemented. With the introduction of an integrated systems-based medical curriculum and a reduction in laboratory teaching hours, anatomy faculty at the University of North Texas Health Science Center (UNTHSC) developed a computer-based dissection manual to adjust to these curricular changes and time constraints. At each cadaver workstation, Apple iMac computers were added and a new dissection manual, running in a browser-based format, was installed. Within the text of the manual, anatomical structures required for dissection were linked to digital images from prosected materials; in addition, for each body system, the dissection manual included images from cross sections, radiographs, CT scans, and histology. Although we have placed a high priority on computerization of the anatomy laboratory, we remain strong advocates of the importance of cadaver dissection. It is our belief that the utilization of computers for dissection is a natural evolution of technology and fosters creative teaching strategies adapted for anatomy laboratories in the 21st century. Our strategy has significantly enhanced the independence and proficiency of our students, the efficiency of their dissection time, and the quality of laboratory instruction by the faculty.
Anatomy↗
Evolution to predominance of swine influenza virus hemagglutinin mutants of predictable phenotype during single infections of the natural host.
L and H2 mutants of the A/NJ/11/76 H1N1 strain of swine influenza virus differ by having either a lysine or a glutamic acid at position 153 of the hemagglutinin glycoprotein of the virus. In two separate experiments, experimental infection of swine with various doses of the H2 mutant resulted in the emergence in 11 of 20 animals of virus with the L phenotype. All evidence indicates that the H2----L mutation, selection, and evolution to predominance occurred within the 7-day span of individual infections. L and H2 mutations appear to act as alleles in the adaptation of virus, respectively, to natural and laboratory hosts. Although the gradual evolution of mutants during sequential infections is commonplace, the present recognition of rapid and predictable evolution of mutants of increased replication efficiency and specific phenotype in the natural host, to our knowledge, is unprecedented.
Animals↗
Constraints on adaptive evolution: the functional trade-off between reproduction and fast-start swimming performance in the Trinidadian guppy (Poecilia reticulata).
The empirical study of natural selection reveals that adaptations often involve trade-offs between competing functions. Because natural selection acts on whole organisms rather than isolated traits, adaptive evolution may be constrained by the interaction between traits that are functionally integrated. Yet, few attempts have been made to characterize how and when such constraints are manifested or whether they limit the adaptive divergence of populations. Here we examine the consequences of adaptive life-history evolution on locomotor performance in the live-bearing guppy. In response to increased predation from piscivorous fish, Trinidadian guppies evolve an increased allocation of resources toward reproduction. These populations are also under strong selection for rapid fast-start swimming performance to evade predators. Because embryo development increases a female's wet mass as she approaches parturition, an increased investment in reproductive allocation should impede fast-start performance. We find evidence for adaptive but constrained evolution of fast-start swimming performance in laboratory trials conducted on second-generation lab-reared fish. Female guppies from high-predation localities attain a faster acceleration and velocity and travel a greater distance during fast-start swimming trials. However, velocity and distance traveled decline more rapidly over the course of pregnancy in these same females, thus reducing the magnitude of divergence in swimming performance between high- and low-predation populations. This functional trade-off between reproduction and swimming performance reveals how different aspects of the phenotype are integrated and highlights the complexity of adaptation at the whole-organism level.
Adaptation, Physiological↗
Three-butterfly system provides a field test of müllerian mimicry.
In 1879, Müller proposed that two brightly coloured distasteful butterfly species (co-models) that share a single warning-colour pattern would benefit by spreading the selective burden of educating predators. The mutual benefit of sharing warning signals among distasteful species, so-called müllerian mimicry, is supported by comparative evidence, theoretical studies and laboratory simulations; however, to date, this key exemplar of adaptive evolution has not been experimentally tested in the field. To measure natural selection generated by müllerian mimicry, I exploited the unusual polymorphism of Heliconius cydno (Lepidoptera: Nymphalidae). Here I show increased survival of H. cydno morphs that match locally abundant monomorphic co-model species. This study demonstrates müllerian mimicry in the field. It also shows that müllerian mimicry with several co-models generates geographically divergent selection, which explains the existence of polymorphism in distasteful species with warning coloration.
Animals↗
Natural selection in a bottle.
The study of natural selection in laboratory systems undergoing experimental evolution can provide important insights into the relationship between natural selection and adaptation. We studied selection on the norm of reaction of age at first reproduction in a laboratory population of Drosophila melanogaster. This population had been selected on a discrete generation schedule in the laboratory for more than 600 generations. Using genetically marked strains, we studied development time, size, female fecundity, and viability of flies that began development at different times relative to the initiation of each bottle. Only flies that began development within 30 h of the initiation of the bottle were reliably able to eclose before the next transfer. Theory predicts that flies initiating development around this critical time should decrease size at maturity to ensure eclosion by the 14-d deadline, but late flies are not smaller. This result suggests an unknown constraint on response to selection on age at maturity in this population. Ultimately, laboratory systems provide the best opportunity for the study of natural selection, genetic variation, and evolutionary response in the same population.
Aging↗
Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains.
Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.
Acetate-CoA Ligase↗
Evolution of fitness. V. Rate of evolution of irradiated populations of drosophila.
Measurable evolution occurs in laboratory populations of Drosophila under strong natural selection. The rate of adaptation to the experimental environment is significantly greater in populations with genetic variability increased by radiation than in the control, nonirradiated populations.
Adaptation, Biological↗
Experimental Evolution of Poxviruses.
Experimental evolution is the process of exposing virus populations to defined selective pressures in a laboratory setting to identify adaptive changes. Coupled with deep sequencing, this experimental approach allows for nucleotide-level resolution of poxvirus adaptive strategies over time. Here, we present a general method of poxvirus experimental evolution, Illumina-based deep sequencing, and bioinformatic analyses to identify structural changes (e.g., gene duplication) as well as local adaptive changes (e.g., small indels and single nucleotide polymorphisms).
Poxviridae↗
Long-term experimental evolution in Escherichia coli. X. Quantifying the fundamental and realized niche.
BACKGROUND: Twelve populations of the bacterium, Escherichia coli, adapted to a simple, glucose-limited, laboratory environment over 10,000 generations. As a consequence, these populations tended to lose functionality on alternative resources. I examined whether these populations in turn became inferior competitors in four alternative environments. These experiments are among the first to quantify and compare dimensions of the fundamental and realized niches. RESULTS: Three clones were isolated from each of the twelve populations after 10,000 generations of evolution. Direct competition between these clones and the ancestor in the selective environment revealed average fitness improvements of approximately 50%. When grown in the wells of Biolog plates, however, evolved clones grew 25% worse on average than the ancestor on a variety of different carbon sources. Next, I competed each evolved population versus the ancestor in four foreign environments (10-fold higher and lower glucose concentration, added bile salts, and dilute LB media). Surprisingly, nearly all populations were more fit than the ancestor in each foreign environment, though the margin of improvement was least in the most different environment. Most populations also evolved increased sensitivity to novobiocin. CONCLUSIONS: Reduced functionality on numerous carbon sources suggested that the fundamental niche of twelve E. coli populations had narrowed after adapting to a specific laboratory environment. However, in spite of these results, the same populations were competitively superior in four novel environments. These findings suggest that adaptation to certain dimensions of the environment may compensate for other functional losses and apparently enhance the realized niche.
Adaptation, Physiological↗
Budding yeast as a model organism for population genetics.
Population genetics is a highly theoretical field in which many models and theories of broad significance have received little experimental testing. Microbes are well-suited for empirical population genetics since populations of almost any size may be studied genetically, and because many have easily controlled life cycles. Saccharomyces cerevisiae is almost ideal for such studies as the growing body of knowledge and techniques that have made it the best characterized eukaryote genome also allow the experimental manipulation and analysis of its population genetics. In experiments to date, the evolution of laboratory yeast populations has been observed for up to 1000 generations. In several cases, adaptation has occurred by gene duplications. The interaction between mutation, selection and genetic drift at varying population sizes is a major area of theoretical study in which yeast experiments can provide particularly valuable data. Conflicts between gene-level and among-cell selection, and co-evolution between genes within a genome, are additional topics in which a population genetics perspective may be particularly helpful. The growing field of genomics is increasingly complementary with that of population genetics. The characterization of the yeast genome presents unprecedented opportunities for the detailed study of evolutionary and population genetics. Conversely, the redundancy of the yeast genome means that, for many open reading frames, deletion has only a quantitative effect that is most readily observed in competitions with a wild-type strain.
Adaptation, Biological↗