Evolution of gene expression during long term coexistence in a bacterial evolution experiment
ABSTRACT: We used microarrays to study the evolution of gene expression in two bacterial ecotypes that coexisted for more than 35000 generations of experimental evolution in an extremely simple environment We sampled four clones from each of two lineages at generations 6,500, 17,000 and 40,000 from the polymorphic Ara-2 population of the E. coli long-term evolution experiment. The four clones from each lineage and generation were mixed equally and used for global expression profiling, growth assays and competition experiments. Global expression profiles and growth assays were also performed on the ancestral strain. RNA extractions were done using cells in mid-exponential growth in the same glucose-limited minimal medium used in the evolution experiment. Global expression profiles were obtained by using Affymetrix arrays, with 5 or 6 biological replicates for each lineage-generation sample.
Project description:We used microarrays to study the evolution of gene expression in two bacterial ecotypes that coexisted for more than 35000 generations of experimental evolution in an extremely simple environment Overall design: We sampled four clones from each of two lineages at generations 6,500, 17,000 and 40,000 from the polymorphic Ara-2 population of the E. coli long-term evolution experiment. The four clones from each lineage and generation were mixed equally and used for global expression profiling, growth assays and competition experiments. Global expression profiles and growth assays were also performed on the ancestral strain. RNA extractions were done using cells in mid-exponential growth in the same glucose-limited minimal medium used in the evolution experiment. Global expression profiles were obtained by using Affymetrix arrays, with 5 or 6 biological replicates for each lineage-generation sample.
Project description:An Eschericia coli strain was constructed in which the PaeR7 restriction-modification gene complex was present under an unstable condition on a partially duplicated chromosomal region so that it may continuously attack the host genome to accelerate its evolution. This strain was grown for more than a hundred of generations through repeated cycles of saturation and dilution. The winners of this competition showed faster growth than the ancestor. Analysis of their genome and transcriptome revealed that they have acquired rearrangements in genes affecting their growth. The global expression pattern changes may have realized growth advantage during the adaptation experiment. Keywords: time course during adaptive evloution Overall design: We used microarrays to know the genetic mechanism underlying adaptive evolution process, and to know the global expression changes that realize advantage in growth during adaptation. Four populations of the parent strain (YA027) and two populations of isogenic r-m+ strain (YA074), for control, were grown and serially propagated in amino acid rich medium (Davis’s MM supplied with 20 amino acids) with Cm (chloramphenicol) and Km (kanamycin), daily with 100-fold dilution. One passage corresponds to 6 - 7 generations, though it might be changed with evolution. Their growth was monitored daily. The growth curve shifted upwards every day in every population, reflecting improvement both in the initial growth rate and the saturation cell density through the evolution. To follow genome changes and global expression changes during adaptive evolution, we selected clones that might represent several stages of evolution with respect to growth from an r+m+ population (population #3) at the 11th, 84th, and 172th passage (corresponding 70, 500, and 1000 generations, respectively): 3-11-2, 3-11-4, 3-11-9, and 3-11-10 from the 11th passage; 3-84-2, 3-84-4, 3-84-6, and 3-84-10 from the 84th passage, and 3-172-1, 3-172-9, and 3-172-10 from the 172nd passage. We subjected them to transcriptome analysis. All gene expression measurements were duplicated or triplicated.
Project description:One of the central goals of evolutionary biology is to explain and predict the molecular basis of adaptive evolution. We studied the evolution of genetic networks in Saccharomyces cerevisiae (budding yeast) populations propagated for more than 200 generations in different nitrogen-limiting conditions. We find that rapid adaptive evolution in nitrogen-poor environments is dominated by the de novo generation and selection of copy number variants (CNVs), a large fraction of which contain genes encoding specific nitrogen transporters including PUT4, DUR3 and DAL4. The large fitness increases associated with these alleles limits the genetic heterogeneity of adapting populations even in environments with multiple nitrogen sources. Complete identification of acquired point mutations, in individual lineages and entire populations, identified heterogeneity at the level of genetic loci but common themes at the level of functional modules, including genes controlling phosphatidylinositol-3-phosphate metabolism and vacuole biogenesis. Adaptive strategies shared with other nutrient-limited environments point to selection of genetic variation in the TORC1 and Ras/PKA signaling pathways as a general mechanism underlying improved growth in nutrient-limited environments. Within a single population we observed the repeated independent selection of a multi-locus genotype, comprised of the functionally related genes GAT1, MEP2 and LST4. By studying the fitness of individual alleles, and their combination, as well as the evolutionary history of the evolving population, we find that the order in which these mutations are acquired is constrained by epistasis. The identification of repeatedly selected variation at functionally related loci that interact epistatically suggests that gene network polymorphisms (GNPs) may be a frequent outcome of adaptive evolution. Our results provide insight into the mechanistic basis by which cells adapt to nutrient-limited environments and suggest that knowledge of the selective environment and the regulatory mechanisms important for growth and survival in that environment greatly increases the predictability of adaptive evolution. mRNA from each evolved clone or from the ancestral strain growing in the specificied nitrogen-limited condition was co-hybridized with mRNA from the ancestral strain grown in ammonium limited media
Project description:Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, deleterious mutations may also have an important role by promoting adaptive genetic changes that are otherwise inaccessible. Here we study the capacity of the baker’s yeast genome to compensate the complete loss of genes during evolution, and explore the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid yeast genotypes, all of which initially displayed slow growth due to the deletion of a single gene. Compensatory adaptation was rapid and pervasive, and it promoted the genomic divergence of parallel evolving populations. The accumulated mutations did not restore wild type genomic expression states and generated diverse growth phenotypes across environments. Taken together, gene loss initiates genomic changes that can influence evolutionary potential upon environmental change. Evolved yeast-lines were generated by growing strains for 400 doublings during 104 days on YPD medium in 96 wells plates, 8 evolved lines were selected for microarray analysis. Two independent colonies of the wild type control, evolved and corresponding ancestor knock-out strains were grown to early midlog and used for transcription profiling by dual channel array against a common reference.
Project description:Although the relationship between phenotypic plasticity and evolutionary dynamics has attracted large interest, very little is known about the contribution of phenotypic plasticity to adaptive evolution. In this study, we analyzed phenotypic and genotypic changes in E. coli cells during adaptive evolution to ethanol stress. To quantify the phenotypic changes, transcriptome analyses were performed. We previously obtained 6 independently evolved ethanol tolerant E. coli strains, strains A through F, by culturing cells under 5% ethanol stress for about 1000 generations and found a significantly larger growth rate than the parent strains (Horinouchi et al, 2010, PMID: 20955615). To elucidate the phenotypic changes that occurred during adaptive evolution, we quantified the time-series of the expression changes by microarray analysis. Starting from frozen stocks obtained at 6 time points (0, 384, 744, 1224, 1824 and 2496 hours) in laboratory evolution, cells were cultured under 5% ethanol stress, and mRNA samples were obtained in the exponential growth phase for microarray analysis.
Project description:A Saccharomyces cerevisiae population was cultured for many generations under conditions to which it is not optimally adapted. These experiments were designed to investigate adaptive evolution under natural selection. This study is described in more detail in Ferea TL, et al. 1999. Proc Natl Acad Sci USA 96:9721-6
Project description:We conducted a set of lab-evolution experiments in yeast and followed the long-term dynamics of aneuploidy under diverse conditions including heat shock and high PH. Each evolution experiment starts with an ancestor strain that was subjected (in several independent repetitions) to certain growth conditions such as high temperature 39°C, permissive temperature 30°C, gradually increasing temperature from 30°C to 39°C, high pH=8.6 and normal PH. In all cases the mRNA extraction was performed on a population sample that was grown for 18 generations under stress-less conditions. This is since we aimed to measure the gene expression changes that are due to stress adaptation and not the physiological response.
Project description:During a 1,000 generations evolution experiment, two types of bacteria (S and L) repeatedly diverged from a common ancestor (A) in a fully sympatric seasonal environment containing glucose and acetate. We compare the transcription profile of the two derived types and the common ancestor in order to investigate the metabolic modifications associated with this adaptive diversification event. Keywords: time point, experimental evolution Overall design: Total RNA was extracted at 4 different time points of the 24H kinetic growth curve of each of the 3 types of bacteria (S, L and A). At each time point, S and L transcription profiles were compared to A. Each comparison was performed in 5 biological replicates.
Project description:microarray experiment to test the gene expression in long term lines of mutator and non-mutator yeast. Here we use an experimental evolution approach to investigate the conditions required for evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ~6700 generations four out of eight experimental mutator lines had evolved a decreased mutation rate. 2 condition experiment, derived experimental evolution strains compared to their ancestor strain. We compared the expression profile of one of the mutator lines (m8) after 6700 generations with its mutator ancestor, and as a control, an evolved non mutator after 6700 generations was compared to to its non-mutator ancestor. In order to prepare cells for expression microarray, glass tubes containing 3 ml of YPD were inoculated from overnight cultures, and grown until the OD600 was approximately 0.3.
Project description:We examined the gene expression changes resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae subjected to two antifungal drugs, fluconazole (FLC) and amphotericin B (AmB). Fluconazole resistance may involve increased efflux or changes in sterol metabolism, while AmB resistance generally involves changes in sterol metabolism; for all of these types of resistance, the gene expression changes are extensive. The goal of these experiments was to test whether failure of gene expression changes all downstream of the original mutation for drug resistance would affect the ability of a mutant cell to evolve and/or to support a drug-resistant phenotype. Overall design: In this study, replicate populations of Saccharomyces cerevisiae were evolved in increasing concentrations of AmB over hundreds of generations to yield resistant types among which there were permanent changes in gene-expression. Evolution experiments were started with 100 µL of an overnight culture that was derived from a single colony inoculated into 10 ml of YPD containing 0.25 µg/ml AmB. Five different populations were evolved in the presence of AmB in two different regimens, long and short. The rational for choosing different evolution regimens was to increase the chance of recruiting different mechanisms of AmB resistance among all of the replicates. In the long regimen, three populations started at 0.25 µg/ml AmB and this concentration was doubled every 100 generations for a total of 1100 generations and a final AmB concentration of 125 µg/ml (sce2468, sce2469, sce2470). In the short regimen, two populations were also started at 0.25 µg/ml AmB, but were given as much time as necessary to grow to high density, usually two to four days. The populations were then transferred a second time into the same AmB concentration. At the next transfer, the AmB concentration was doubled for another two growth cycles, and so on, until the populations had been through two transfers in 128 µg/ml AmB (sce2539 and sce2542).