Project description:In some of the earliest uses of genome-wide gene-expression microarrays and array-based Comparative Genomic Hybridization (aCGH), a set of diploid yeasts that had undergone experimental evolution under aerobic glucose limitation was used to explore how gene expression and genome structure had responded to this selection pressure. To more deeply understand how adaptation to one environment might constrain or enhance performance in another we have now identified the adaptive mutations in this set of clones using whole-genome sequencing, and have assessed whether the evolved clones had become generalists or specialists by assaying their fitness under three contrasting growth environments: aerobic and anaerobic glucose limitation and aerobic acetate limitation. Additionally, evolved clones and their common ancestor were assayed for gene expression, biomass estimates and residual substrate levels under the alternative growth conditions. Relative fitnesses were evaluated by competing each clone against a common reference strain in each environment. Unexpectedly, we found that the evolved clones also outperformed their ancestor under strictly fermentative and strictly oxidative growth conditions. We conclude that yeasts evolving under aerobic glucose limitation become generalists for carbon limitation, as the mutations selected for in one environment are advantageous in others. High-throughput sequencing of the evolved clones uncovered mutations in genes involved in glucose sensing, signaling, and transport that in part explain these physiological phenotypes, with different sets of mutations found in independently-evolved clones. Earlier gene expression data from aerobic glucose-limited cultures had revealed a shift from fermentation towards respiration in all evolved clones explaining increased fitness in that condition. However, because the evolved clones also show higher fitness under strictly anaerobic conditions and under conditions requiring strictly respirative growth, this switch cannot be the sole source of adaptive benefit. Furthermore, because independently evolved clones are genetically distinct we conclude that there are multiple mutational paths leading to the generalist phenotype. Strain Name: Parental strain (CP1AB) or evolved clones (E1 - E5) Media: aerobic / anaerobic 36 hybridizations
Project description:Exploring molecular details of carbon utilization trade-offs in galactose-evolved yeast Adaptively evolved yeast mutants on galactose for around 400 generations showed diminished growth and carbon uptake rates on glucose. Genome-scale approaches were applied to characterize the molecular genetic basis of these trade-offs in carbon source utilization. Engineered mutants showing trade-offs in a specific carbon uptake rate between both carbons were used as controls. The transcriptional responses of the evolved mutants were almost identical during growth on both carbon sources. These carbon-independent conserved patterns were clearly observed in specific pathways and genes. Up-regulation of PGM2, a confirmed beneficial genetic change for improving galactose utilization was preserved on both carbons. In addition, HXK1, GLK1 and genes involved in reserve carbohydrate metabolism were up-regulated, while HXK2 was down-regulated. Genes that have a transcription factor binding site for Gis1p, Rph1p, Msn2/4p and Nrg1p were up-regulated. These results indicated changes in the metabolic pathways involved in metabolism of both carbons and in nutrient signaling pathway. The concentration profile of trehalose and glycogen supported these findings. Mutations in RAS2 and ERG5 genes were selected because of their beneficial and neutral effect on galactose utilization, respectively in our previous study. Site-directed mutants containing galactose-beneficial mutations in RAS2 only resulted in a significant decrease in glucose utilization. Integration of all these analyses clearly suggest an antagonistic pleiotropic trade-off in carbon source utilization caused by changes in regulatory region, and we hereby demonstrate how systems biology can be used to gain insight into evolutionary processes at the molecular level. Yeast galactose evolved mutants having improved galactose availability were grown on aerobic batch with glucose as carbon source
Project description:Aneuploidy is a hallmark of tumor cells and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild-type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains each containing a different telomeric amplicon (Tamp) ranging in size from 0.4 to 1,000kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations. Each of these arrays is a Comparative Genomic Hybridization experiment to detect copy number differences between a reference strain and a strain of interest.
Project description:The hypothesis that increased fitness within a selective environment must be accompanied by a loss of fitness in other non-selective environments leads to the notion of evolutionary tradeoffs. Experimental evolution provides an approach to test the existence of evolutionary tradeoffs, characterize their general quality, and reveal their genetic origins. To examine the underlying mechanism for a fitness trade-off, we constructed the evolutionary trajectories of Escherichia coli K-12 at increasing temperatures up to 45.3°C, and found diverging mutational histories that led to adaptive phenotypes with and without fitness trade-offs at low temperatures. We identified genetic changes in cellular respiration, iron metabolism and methionine biosynthesis that regulated gene expression to achieve thermal adaptation and determined the presence and absence of a fitness trade-off. Our results suggested that evolutionary trade-off could be generated by a regulatory protein mutation that was beneficial in the selective conditions but forced suboptimal proteome allocation under non-selective environments.
Project description:In some of the earliest uses of genome-wide gene-expression microarrays and array-based Comparative Genomic Hybridization (aCGH), a set of diploid yeasts that had undergone experimental evolution under aerobic glucose limitation was used to explore how gene expression and genome structure had responded to this selection pressure. To more deeply understand how adaptation to one environment might constrain or enhance performance in another we have now identified the adaptive mutations in this set of clones using whole-genome sequencing, and have assessed whether the evolved clones had become generalists or specialists by assaying their fitness under three contrasting growth environments: aerobic and anaerobic glucose limitation and aerobic acetate limitation. Additionally, evolved clones and their common ancestor were assayed for gene expression, biomass estimates and residual substrate levels under the alternative growth conditions. Relative fitnesses were evaluated by competing each clone against a common reference strain in each environment. Unexpectedly, we found that the evolved clones also outperformed their ancestor under strictly fermentative and strictly oxidative growth conditions. We conclude that yeasts evolving under aerobic glucose limitation become generalists for carbon limitation, as the mutations selected for in one environment are advantageous in others. High-throughput sequencing of the evolved clones uncovered mutations in genes involved in glucose sensing, signaling, and transport that in part explain these physiological phenotypes, with different sets of mutations found in independently-evolved clones. Earlier gene expression data from aerobic glucose-limited cultures had revealed a shift from fermentation towards respiration in all evolved clones explaining increased fitness in that condition. However, because the evolved clones also show higher fitness under strictly anaerobic conditions and under conditions requiring strictly respirative growth, this switch cannot be the sole source of adaptive benefit. Furthermore, because independently evolved clones are genetically distinct we conclude that there are multiple mutational paths leading to the generalist phenotype. Strain Name: Parental strain (CP1AB) or evolved clones (E1 - E5) Media: aerobic / anaerobic
Project description:Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.
Project description:Divergent adaptation can be associated with reproductive isolation in the process of speciation. We recently demonstrated the link between divergent adaptation and the onset of reproductive isolation in experimental populations of the yeast Saccharomyces cerevisiae evolved from a single progenitor in either a high-salt or a low-glucose environment. However, the genetic basis for adaptation and reproductive isolation remained unknown in this system. Here, we use whole-genome re-sequencing of representatives of three populations to identify 15 candidate mutations, six of which explained the adaptive increases in mitotic fitness in the two environments. In two populations evolved in high salt, two different mutations occurred in the proton efflux pump gene PMA1 and the global transcriptional repressor gene CYC8; the ENA genes encoding sodium efflux pumps were over-expressed once through expansion of this gene cluster and once as a result of the mutation in the regulator CYC8. In the population from low glucose, one mutation occurred in MDS3, which modulates growth at high pH, and one in MKT1, a global regulator of mRNAs encoding mitochondrial proteins, the latter recapitulating a naturally-occurring variant. A Dobzhansky-Muller (DM) incompatibility between the evolved alleles of PMA1 and MKT1 strongly depressed fitness in the low-glucose environment. This DM interaction is the first reported between experimentally evolved alleles of known genes and shows how reproductive isolation can arise rapidly when divergent selection is strong. Evolved and progenitor strains were grown in stressed and unstressed conditions to identify role of mutations in gene expression.
Project description:We studied adaptation of the metabolically versatile bacterium Pseudomonas aeruginosa to standard laboratory conditions by propagating mismatch repair-deficient P. aeruginosa in exponential phase for 24 days in rich medium. In the selective environment of this large-bottleneck mutation accumulation experiment, the bacteria developed shorter lag phases, higher growth rates and higher maximum cell densities. Transcriptional profiling and phenotyping for growth in different media revealed that higher fitness under laboratory conditions evolved via different pathways. Although common adaptive mutations or mutations that define trade-offs were not identified, there was a convergent evolution of transcriptional profiles associated with a shift from biofilm-associated to planktonic lifestyles. Our results indicate that under constant planktonic conditions P. aeruginosa uses several genetic pathways in order to fine-tune adaptation towards faster growth. The selected mutations in the different genetic pathways show a great variety of biofilm, virulence and motility phenotypic trade-offs, thus implying that on the population level, the adaptation of P. aeruginosa to constant conditions does not compromise its versatility. Methods: mRNA profiles were generated for Pseudomonas aeruginosa samples derived from LB-cultures grown to an OD600 =0.4-0.6. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina). The samples were sequenced in single end mode on an Illumina HiSeq 2500 device and mRNA reads were trimmed and mapped to the NC_008463.1 (PA14) reference genome from NCBI using Stampy pipeline with default settings.