Biology subjects

Buskirk, S. W.

Publications and source records attributed to Buskirk, S. W..

9 recordsLinked to original sources

bioRxiv2022

G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts Syndrome (RBS) and Cornelia de Lange Syndrome (CdLS), respectively. In yeast, deletion of ECO1 results in cell inviability. Co-deletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2 and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

cell biology↗

bioRxiv2022

Long-term adaptation to galactose as a sole carbon source selects for mutations in nutrient signaling pathways

Galactose is a secondary fermentable sugar that requires specific regulatory and structural genes for its assimilation, which are under catabolite repression by glucose. When glucose is absent, the catabolic repression is attenuated, and the structural GAL genes are fully activated. In Saccharomyces cerevisiae, the GAL pathway is under selection in environments where galactose is present. However, it is unclear the adaptive strategies in response to long-term propagation in galactose as a sole carbon source in laboratory evolution experiments. Here, we performed a 4,000-generation evolution experiment using 48 diploid Saccharomyces cerevisiae populations to study adaptation in galactose. We show that fitness gains were greater in the galactose-evolved population than in identically evolved populations with glucose as a sole carbon source. Whole-genome sequencing of 96 evolved clones revealed recurrent de novo single nucleotide mutations in candidate targets of selection, copy number variations, and ploidy changes. We find that most mutations that improve fitness in galactose lie outside of the canonical GAL pathway and are involved in nutrient signaling. Reconstruction of specific evolved alleles in candidate target of selection, SEC23 and IRA1, showed a significant increase in fitness in galactose compared to glucose. In addition, most of our evolved populations (28/46; 61%) fixed aneuploidies on Chromosome VIII, suggesting a parallel adaptive amplification. Finally, we show greater loss of extrachromosomal elements in our glucose-evolved lineages compared with previous glucose evolution. Broadly, these data further our understanding of the evolutionary pressures that drive adaptation to less-preferred carbon sources.

evolutionary biology↗

bioRxiv2021

Overdominant and partially dominant mutations drive short-term adaptation in diploid yeast

Identification of adaptive targets in experimental evolution typically relies on extensive replication and allele reconstructions. An alternative approach is to directly assay all mutations in an evolved clone by generating pools of segregants that contain random combinations of the evolved mutations. Here, we apply this method to 6 clones isolated from 4 diploid populations that were clonally evolved for 2,000 generations in rich glucose medium. Each clone contains [~]20-25 mutations relative to the ancestor. We derived intermediate genotypes between the founder and the evolved clones by bulk mating sporulated cultures of each evolved clone to a barcoded haploid version of the founder. We competed the barcoded segregants en masse and quantified the fitness of each barcode. We estimated average fitness effects of evolved mutations using barcode fitness and whole genome sequencing for a subset of segregants or time-course whole population whole genome sequencing. In contrast to our previous work in haploid populations, we find that diploids carry fewer evolved mutations with a detectable fitness effect (6%), contributing a modest fitness advantage (up to 5.4%). In agreement with theoretical expectations, reconstruction experiments show that all adaptive mutations manifest some degree of dominance over the ancestral allele, and most are overdominant. Competition assays under conditions that deviated from the evolutionary environment show that adaptive mutations are often pleiotropic.

evolutionary biology↗

bioRxiv2021

Exploring a local genetic interaction network using evolutionary replay experiments

Understanding how genes interact is a central challenge in biology. Experimental evolution provides a useful, but underutilized, tool for identifying genetic interactions, particularly those that involve non-loss-of-function mutations or mutations in essential genes. We previously identified a strong positive genetic interaction between specific mutations in KEL1 (P344T) and HSL7 (A695fs) that arose in an experimentally-evolved Saccharomyces cerevisiae population. Because this genetic interaction is not phenocopied by gene deletion, it was previously unknown. Using "evolutionary replay" experiments we identified additional mutations that have positive genetic interactions with the kel1-P344T mutation. We replayed the evolution of this population 672 times from six timepoints. We identified 30 populations where the kel1-P344T mutation reached high frequency. We performed whole-genome sequencing on these populations to identify genes in which mutations arose specifically in the kel1-P344T background. We reconstructed mutations in the ancestral and kel1-P344T backgrounds to validate positive genetic interactions. We identify several genetic interactors with KEL1, we validate these interactions by reconstruction experiments, and we show these interactions are not recapitulated by loss-of-function mutations. Our results demonstrate the power of experimental evolution to identify genetic interactions that are positive, allele specific, and not readily detected by other methods, and sheds light on a previously under-explored region of the yeast genetic interaction network.

evolutionary biology↗

bioRxiv2020

One gene, multiple ecological strategies: a biofilm regulator is a capacitor for sustainable diversity

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR. RpfR uniquely integrates two major signaling systems -- quorum sensing and the motile-sessile switch mediated by cyclic-d-GMP -- by two domains that sense, respond to, and control synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions. Significance statementMany organisms including bacteria live in fluctuating environments requiring attachment and dispersal. These lifestyle decisions require multiple external signals to be processed by several genetic pathways, but how they are integrated is largely unknown. We conducted multiple evolution experiments totaling >20,000 generations with Burkholderia cenocepacia populations grown in a model of the biofilm life cycle and identified parallel mutations in one gene, rpfR, that is a conserved central regulator. Because RpfR has multiple sensor and catalytic domains, different mutations can produce different ecological strategies that can coexist and even increase net growth. This study demonstrates that a single gene may coordinate complex life histories in biofilm-dwelling bacteria and that selection in defined environments can reshape niche breadth by single mutations.

microbiology↗

bioRxiv2019

Negative frequency-dependent selection maintains coexisting genotypes during fluctuating selection

Natural environments are rarely static; rather selection can fluctuate on time scales 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.

evolutionary biology↗

bioRxiv2019

Adaptive evolution of a rock-paper-scissors sequence along a direct line of descent

Nontransitivity - commonly illustrated by the rock-paper-scissors game - is well documented among extant species as a contributor to biodiversity. However, it is unclear if nontransitive interactions also arise by way of genealogical succession, and if so, through what mechanisms. Here we identify a nontransitive evolutionary sequence in the context of yeast experimental evolution in which a 1,000-generation evolved clone outcompetes a recent ancestor but loses in direct competition with a distant ancestor. We show that nontransitivity arises due to the combined forces of adaptation in the yeast nuclear genome and the stepwise deterioration of an intracellular virus. We show that, given the initial conditions of the experiment, this outcome likely to arise: nearly half of all populations experience multilevel selection, fixing adaptive mutations in both the nuclear and viral genomes. In contrast to conventional views of virus-host coevolution, we find no evidence that viral mutations (including loss of the virus) increase the fitness of the host. Instead, the evolutionary success of evolved viral variants results from their selective advantage over viral competitors within the context of individual cells. Our results provide the first mechanistic case-study of the adaptive evolution of nontransitivity, in which a series of adaptive replacements produce organisms that are less fit when compared to a distant genealogical ancestor.

evolutionary biology↗

bioRxiv2018

Cause and consequences of genome duplication in haploid yeast populations

Whole genome duplications (WGD) represent important evolutionary events that shape future adaptation. WGDs are known to have occurred in the lineages leading to plants, fungi, and vertebrates. Changes to ploidy level impact the rate and spectrum of beneficial mutations and thus the rate of adaptation. Laboratory evolution experiments initiated with haploid Saccharomyces cerevisiae cultures repeatedly experience WGD. We report recurrent genome duplication in 46 haploid yeast populations evolved for 4,000 generations. We find that WGD confers a fitness advantage, and this immediate fitness gain is accompanied by a shift in genomic and phenotypic evolution. The presence of ploidy-enriched targets of selection and structural variants reveals that autodiploids utilize adaptive paths inaccessible to haploids. We find that autodiploids accumulate recessive deleterious mutations, indicating an increased capacity for neutral evolution. Finally, we report that WGD results in a reduced adaptation rate, indicating a trade-off between immediate fitness gains and long term adaptability.

evolutionary biology↗

bioRxiv2017

Hitchhiking and epistasis give rise to cohort dynamics in adapting populations

Beneficial mutations are the driving force of adaptive evolution. In asexual populations, the identification of beneficial alleles is confounded by the presence of genetically-linked hitchhiker mutations. Parallel evolution experiments enable the recognition of common targets of selection, yet these targets are inherently enriched for genes of large target size and mutations of large effect. A comprehensive study of individual mutations is necessary to create a realistic picture of the evolutionarily significant spectrum of beneficial mutations. Here we utilize a bulk-segregant approach to identify the beneficial mutations across 11 lineages of experimentally-evolved yeast populations. We report that most genome sequence evolution is non-adaptive: nearly 80% of detected mutations have no discernable effects on fitness and less than 1% are deleterious. We determine the distribution of driver and hitchhiker mutations in 31 mutational cohorts, groups of up to ten mutations that arise synchronously from low frequency and track tightly with one another. Surprisingly, we find that one-third of cohorts lack identifiable driver mutations. In addition, we identify intra-cohort synergistic epistasis between mutations in hsl7 and kel1, which arose together in a low frequency lineage.

evolutionary biology↗