Experimental evolution of resistance in Paramecium caudatum against the bacterial parasite Holospora undulata

Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by frequency-dependent selection. This requires that different parasite genotypes perform differently on different host genotypes. Such genotype-by-genotype interactions arise if adaptation to one host (or parasite) genotype reduces performance on others. These direct costs of adaptation can maintain genetic polymorphism and generate geographic patterns of local host or parasite adaptation. Fixation of all-resistant (or all-infective) genotypes is further prevented if adaptation trades off with other host (or parasite) life-history traits. For the host, such indirect costs of resistance refer to reduced fitness of resistant genotypes in the absence of parasites. We studied (co)evolution in experimental microcosms of several clones of the freshwater protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. After two and a half years of culture, inoculation of evolved and naive (never exposed to the parasite) hosts with evolved and founder parasites revealed an increase in host resistance, but not in parasite infectivity. A cross-infection experiment showed significant host clone-by-parasite isolate interactions, and evolved hosts tended to be more resistant to their own (local) parasites than to parasites from other hosts. Compared to naive clones, evolved host clones had lower division rates in the absence of the parasite. Thus, our study indicates de novo evolution of host resistance, associated with both direct and indirect costs. This illustrates how interactions with parasites can lead to the genetic divergence of initially identical populations.     

Reproductive Flexibility: Genetic Variation,Genetic Costs and Long-Term Evolution in a Collembola

In a variable yet predictable world, organisms may use environmental cues to make adaptive adjustments to their phenotype. Such phenotypic flexibility is expected commonly to evolve in life history traits, which are closely tied to Darwinian fitness. Yet adaptive life history flexibility remains poorly documented. Here we introduce the collembolan Folsomia candida, a soil-dweller, parthenogenetic (all-female) microarthropod, as a model organism to study the phenotypic expression, genetic variation, fitness consequences and long-term evolution of life history flexibility. We demonstrate that collembola have a remarkable adaptive ability for adjusting their reproductive phenotype: when transferred from harsh to good conditions (in terms of food ration and crowding), a mother can fine-tune the number and the size of her eggs from one clutch to the next. The comparative analysis of eleven clonal populations of worldwide origins reveals (i) genetic variation in mean egg size under both good and bad conditions; (ii) no genetic variation in egg size flexibility, consistent with convergent evolution to a common physiological limit; (iii) genetic variation of both mean reproductive investment and reproductive investment flexibility, associated with a reversal of the genetic correlation between egg size and clutch size between environmental conditions ; (iv) a negative genetic correlation between reproductive investment flexibility and adult lifespan. Phylogenetic reconstruction shows that two life history strategies, called HIFLEX and LOFLEX, evolved early in evolutionary history. HIFLEX includes six of our 11 clones, and is characterized by large mean egg size and reproductive investment, high reproductive investment flexibility, and low adult survival. LOFLEX (the other five clones) has small mean egg size and low reproductive investment, low reproductive investment flexibility, and high adult survival. The divergence of HIFLEX and LOFLEX could represent different adaptations to environments differing in mean quality and variability, or indicate that a genetic polymorphism of reproductive investment reaction norms has evolved under a physiological tradeoff between reproductive investment flexibility and adult lifespan.     

The repeatability of adaptive radiation during long-term experimental evolution of Escherichia coli in a multiple nutrient environment

Adaptive radiations occur when a species diversifies into different ecological specialists due to competition for resources and trade-offs associated with the specialization. The evolutionary outcome of an instance of adaptive radiation cannot generally be predicted because chance (stochastic events) and necessity (deterministic events) contribute to the evolution of diversity. With increasing contributions of chance, the degree of parallelism among different instances of adaptive radiations and the predictability of an outcome will decrease. To assess the relative contributions of chance and necessity during adaptive radiation, we performed a selection experiment by evolving twelve independent microcosms of Escherichia coli for 1000 generations in an environment that contained two distinct resources. Specialization to either of these resources involves strong trade-offs in the ability to use the other resource. After selection, we measured three phenotypic traits: 1) fitness, 2) mean colony size, and 3) colony size diversity. We used fitness relative to the ancestor as a measure of adaptation to the selective environment; changes in colony size as a measure of the evolution of new resource specialists because colony size has been shown to correlate with resource specialization; and colony size diversity as a measure of the evolved ecological diversity. Resource competition led to the rapid evolution of phenotypic diversity within microcosms. Measurements of fitness, colony size, and colony size diversity within and among microcosms showed that the repeatability of adaptive radiation was high, despite the evolution of genetic variation within microcosms. Consistent with the observation of parallel evolution, we show that the relative contributions of chance are far smaller and less important than effects due to adaptation for the traits investigated. The two-resource environment imposed similar selection pressures in independent populations and promoted parallel phenotypic adaptive radiations in all independently evolved microcosms.     

Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12

Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.     

Myriocin-induced adaptive laboratory evolution of an industrial strain of Saccharomyces cerevisiae reveals its potential to remodel lipid composition and heat tolerance

The modification of lipid composition allows cells to adjust membrane biophysical properties in response to changes in environmental temperature. Here, we use adaptive laboratory evolution (ALE) in the presence of myriocin, a sphingolipid (SLs) biosynthesis inhibitor, to remodel the lipid profile of an industrial yeast strain (LH) of Saccharomyces cerevisiae. The approach enabled to obtain a heterogeneous population (LHev) of myriocin-tolerant evolved clones characterized by its growth capacity at high temperature. Myriocin exposure also caused tolerance to soraphen A, an inhibitor of the acetyl-CoA carboxylase Acc1, the rate-limiting enzyme in fatty acid de novo production, supporting a change in lipid metabolism during ALE. In line with this, characterization of two randomly selected clones, LH03 and LH09, showed the presence of lipids with increased saturation degree and reduced acyl length. In addition, the clone LH03, which displays the greater improvement in fitness at 40°C, exhibited higher SL content as compared with the parental strain. Analysis of the LH03 and LH09 genomes revealed a loss of chromosomes affecting genes that have a role in fatty acid synthesis and elongation. The link between ploidy level and growth at high temperature was further supported by the analysis of a fully isogenic set of yeast strains with ploidy between 1N and 4N which showed that the loss of genome content provides heat tolerance. Consistent with this, a thermotolerant evolved population (LH40°) generated from the parental LH strain by heat-driven ALE exhibited a reduction in the chromosome copy number. Thus, our results identify myriocin-driven evolution as a powerful approach to investigate the mechanisms of acquired thermotolerance and to generate improved strains.     

Patchy distribution of flexible genetic elements in bacterial populations mediates robustness to environmental uncertainty

The generation and maintenance of genetic variation seems to be a general ecological strategy of bacterial populations. Thereby they gain robustness to irregular environmental change, which is primarily the result of the dynamic evolution of biotic interactions. A benefit of maintaining population heterogeneity is that only a fraction of the population has to bear the cost of not (yet) beneficial deviation. On evolutionary time frames, an added value of the underlying mechanisms is evolvability, i.e. the heritable ability of an evolutionary lineage to generate and maintain genetic variants that are potentially adaptive in the course of evolution. Horizontal gene transfer is an important mechanism that can lead to differences between individuals within bacterial populations. Broad host-range plasmids foster this heterogeneity because they are typically present in only a fraction of the population and provide individual cells with genetic modules newly acquired from other populations or species. We postulate that the benefit of robustness on population level could balance the cost of transfer and replication functions that plasmids impose on their hosts. Consequently, mechanisms that make a subpopulation conducive to specific conjugative plasmids may have evolved, which could explain the persistence of even cryptic plasmids that do not encode any traits.     

P1 bacteriophage and tellurite sensitivity in Klebsiella pneumoniae and Escherichia coli

Klebsiella pneumoniae and Escherichia coli respond inversely toward P1 bacteriophage or TeO3(-2). Klebsiella pneumoniae is resistant to both antagonists and E. coli is sensitive. However, P1 cmts lysogens (P1 cmts resistant) of K. pneumoniae became sensitive to tellurite and when cured from P1 cmts regained resistance. Escherichia coli spontaneous mutants selected for resistance to either P1 or TeO3(-2) were collaterally resistant to the other. As well, TeO3(-3) enhanced the adsorption of P1 vir to both E. coli and K. pneumoniae. Several outer membrane proteins were enhanced in the K. pneumoniae lysogens and were reduced in E. coli lysogens; one of which was the same molecular weight (77 000) in both bacteria. When partially purified it enhanced the plaque efficiency of P1 vir. Lipopolysaccharide (LPS) from E. coli C600 inactivated P1 vir, but neither the P1 lysogens nor LPS derived from the lysogens inactivated P1 vir. Escherichia coli P1 lysogens produced only heptose-deficient LPS. It is suggested that both LPS and outer membrane protein(s) comprise the P1 receptor. TeO3(-2) may interact with one or both components.     

Adaptive evolution of baker's yeast in a dough‐like environment enhances freeze and salinity tolerance

We used adaptive evolution to improve freeze tolerance of industrial baker's yeast. Our hypothesis was that adaptation to low temperature is accompanied by enhanced resistance of yeast to freezing. Based on this hypothesis, yeast was propagated in a flour-free liquid dough model system, which contained sorbitol and NaCl, by successive batch refreshments maintained constantly at 12°C over at least 200 generations. Relative to the parental population, the maximal growth rate (µ_max) under the restrictive conditions, increased gradually over the time course of the experiment. This increase was accompanied by enhanced freeze tolerance. However, these changes were not the consequence of genetic adaptation to low temperature, a fact that was confirmed by prolonged selection of yeast cells in YPD at 12°C. Instead, the experimental populations showed a progressive increase in NaCl tolerance. This phenotype was likely achieved at the expense of others traits, since evolved cells showed a ploidy reduction, a defect in the glucose derepression mechanism and a loss in their ability to utilize gluconeogenic carbon sources. We discuss the genetic flexibility of S. cerevisiae in terms of adaptation to the multiple constraints of the experimental design applied to drive adaptive evolution and the technologically advantageous phenotype of the evolved population.     

Rates of DNA sequence evolution in experimental populations of Escherichia coli during 20,000 generations

We examined rates of DNA sequence evolution in 12 populations of Escherichia coli propagated in a glucose minimal medium for 20,000 generations. Previous work saw mutations mediated by mobile elements in these populations, but the extent of other genomic changes was not investigated. Four of the populations evolved defects in DNA repair and became mutators. Some 500 bp was sequenced in each of 36 genes for 50 clones, including 2 ancestral variants, 2 clones from each population at generation 10,000, and 2 from each at generation 20,000. Ten mutations were found in total, all point mutations including mostly synonymous substitutions and nonsynonymous polymorphisms; all 10 were found in mutator populations. We compared the observed sequence evolution to predictions based on different scenarios. The number of synonymous substitutions is lower than predicted from measured mutation rates in E. coli, but the number is higher than rates based on comparing E. coli and Salmonella genomes. Extrapolating to the entire genome, these data predict about 250 synonymous substitutions on average per mutator population, but only about 3 synonymous substitutions per nonmutator population, during 20,000 generations. These data illustrate the challenge of finding sequence variation among bacterial isolates that share such a recent ancestor. However, this limited variation also provides a useful baseline for research aimed at finding the beneficial substitutions in these populations.     

Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain

Fermentation of glucose to D-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a D-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.     

Identification of adaptive changes in an evolving population of Escherichia coli: the role of changes with regulatory and highly pleiotropic effects

A population of Escherichia coli initiated with a single clone developed extensive morphological and physiological polymorphism after being maintained for 773 generations in glucose-limited continuous culture. To understand the mechanisms of adaptation to this environment, total protein patterns of four adaptive clones and of the parent strains were examined by two-dimensional gel electrophoresis. Approximately 20% of the proteins (approximately 160 in absolute numbers) showed significantly different levels of expression in pairwise comparisons of parent and adapted clones. The extent of these changes points to the importance of mutations with regulatory and/or highly pleiotropic effects in the adaptive process. The four evolved clones all expressed fewer proteins than did the parent strain, supporting the hypothesis of energy conservation during evolutionary change. Forty-two proteins that could be assigned to known cellular functions were identified. The changes in some of them indicated that the evolved clones developed different adaptive mechanisms to glucose-limited environment. Changes were observed in the expression levels of proteins associated with translation, membrane composition, shock response, and active transport. A fraction of the changes could not be either explained or predicted from a consideration of the nature of the environment in which the clones evolved.     

Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.     

Testing optimality with experimental evolution: lysis time in a bacteriophage

Optimality models collapse the vagaries of genetics into simple trade-offs to calculate phenotypes expected to evolve by natural selection. Optimality approaches are commonly criticized for this neglect of genetic details, but resolution of this disagreement has been difficult. The importance of genetic details may be tested by experimental evolution of a trait for which an optimality model exists and in which genetic details can be studied. Here we evolved lysis time in bacteriophage T7, a virus of Escherichia coli. Lysis time is equivalent to the age of reproduction in an organism that reproduces once and then dies. Delaying lysis increases the number of offspring but slows generation time, and this trade-off renders the optimum sensitive to environmental conditions: earlier lysis is favored when bacterial hosts are dense, later lysis is favored when hosts are sparse. In experimental adaptations, T7 evolved close to the optimum in conditions favoring early lysis but not in conditions favoring late lysis. One of the late lysis adaptations exhibited no detectable phenotypic evolution despite genetic evolution; the other evolved only partly toward the expected optimum. Overall, the lysis time of the adapted phages remained closer to their starting values than predicted by the model. From the perspective of the optimality model, the experimental conditions were expected to select changes only along the postulated trade-off, but a trait outside the trade-off evolved as well. Evidence suggests that the model's failure ultimately stems from a violation of the trade-off, rather than a paucity of mutations.     

The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.     

Adaptation to fluctuations in temperature by nine species of bacteria

Rapid environmental fluctuations are ubiquitous in the wild, yet majority of experimental studies mostly consider effects of slow fluctuations on organism. To test the evolutionary consequences of fast fluctuations, we conducted nine independent experimental evolution experiments with bacteria. Experimental conditions were same for all species, and we allowed them to evolve either in fluctuating temperature alternating rapidly between 20°C and 40°C or at constant 30°C temperature. After experimental evolution, we tested the performance of the clones in both rapid fluctuation and in constant environments (20°C, 30°C and 40°C). Results from experiments on these nine species were combined meta‐analytically. We found that overall the clones evolved in the fluctuating environment had evolved better efficiency in tolerating fluctuations (i.e., they had higher yield in fluctuating conditions) than the clones evolved in the constant environment. However, we did not find any evidence that fluctuation‐adapted clones would have evolved better tolerance to any measured constant environments (20°C, 30°C, and 40°C). Our results back up recent empirical findings reporting that it is hard to predict adaptations to fast fluctuations using tolerance curves.