Biology subjects

Sherlock, G. J.

Publications and source records attributed to Sherlock, G. J..

11 recordsLinked to original sources

bioRxiv2026

Experimental Evolution of Yeast Reveals Trade-offs Between Early and Late Stationary Phase

Stationary phase in yeast and other microorganisms begins when a limiting nutrient in the environment is exhausted and cell division ceases. Most cells subsequently enter quiescence and lose viability. In spent media, without metabolic byproducts being diluted, cellular processes can modify the environment and cause the relative growth rates of different genotypes to vary over the course of stationary phase. In this work we experimentally evolve S. cerevisiae in batch culture, varying the time spent in stationary phase between growth cycles. We measure the relative fitness of the resulting adaptive clones across a range of environments: with different amounts of time in stationary phase and in two different carbon sources. By comparing the inferred performance (relative growth rate during a period of the growth cycle) of a mutant to that of its ancestor, we can estimate the effects of each observed mutation on performance during various phases of growth. We show that when an adaptive mutation emerges in growth cycles that include a stationary phase, its effect on stationary phase performance is largely independent of the type of carbon source provided. However, for the same group of mutants, mutational effects on performance in early stationary phase are negatively correlated with those effects in late stationary phase, suggesting a trade-off. We also show that increased intervals of stationary phase result in larger fitness effects of adaptive mutations and distinct routes of adaptation. Together, these results demonstrate that stationary phase consists of more than one distinct fitness-related phenotype, and that the phenotypes that allow for high performance in the first few days of stationary phase trade off with those that allow for high performance in later stationary phase.

evolutionary biology↗

bioRxiv2025

Centromeres evolve progressively through selection at the kinetochore interface

During mitosis, stable but dynamic interactions between the centromere DNA and kinetochore complex enable accurate and efficient chromosome segregation. Even though many proteins of the kinetochore are highly conserved, centromeres are among the fastest evolving regions within a genome, showing extensive variation even on short evolutionary timescales. Here, we sought to understand how new types of centromeres emerge and reach fixation by mapping centromere evolution across 138 budding yeast species and over 2,500 natural strain isolates. We show that new centromeres spread progressively via drift and subsequent selection, and that the kinetochore interface, which is evolving slowly in relative terms, determines which new centromere variants are tolerated. Together, our findings provide insight into the evolutionary constraints and trajectories shaping centromere evolution.

evolutionary biology↗

bioRxiv2024

High-throughput DNA engineering by mating bacteria

To reduce the operational friction and scale DNA engineering, we report here an in vivo DNA assembly technology platform called SCRIVENER (Sequential Conjugation and Recombination for In Vivo Elongation of Nucleotides with low ERrors). SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and in vivo homologous recombination to seamlessly stitch blocks of DNA together by mating E. coli in large arrays or pools. This workflow is simpler, cheaper, and higher throughput than current DNA assembly approaches that require DNA to be moved in and out of cells at different procedural steps. We perform over 5,000 assemblies with two to 13 DNA blocks that range from 240 bp to 8 kb and show that SCRIVENER is capable of assembling constructs as long as 23 kb at relatively high throughput and fidelity. Most SCRIVENER errors are deletions between long interspersed repeats. However, SCRIVENER can overcome these errors by enabling assembly and sequence verification at high replication at a nominal additional cost per replicate. We show that SCRIVENER can be used to build combinatorial libraries in arrays or pools, and that DNA blocks onboarded into the platform can be repurposed and reused with any other DNA block in high throughput without a PCR step. Because of these features, DNA engineering with SCRIVENER has the potential to accelerate design-build-test-learn cycles of DNA products.

synthetic biology↗

bioRxiv2024

A shift from pleiotropic to modular adaptation revealed by a high-resolution two-step adaptive walk

Evolution by natural selection is expected to be a slow and gradual process. In particular, the mutations that drive evolution are predicted to be small and modular, incrementally improving a small number of traits. However, adaptive mutations identified early in microbial evolution experiments, cancer, and other systems often provide substantial fitness gains and pleiotropically improve multiple traits at once. We asked whether such pleiotropically adaptive mutations are common throughout adaptation or are instead a rare feature of early steps in evolution that tend to target key signaling pathways. To do so, we conducted barcoded second-step evolution experiments initiated from five first-step mutations identified from a prior yeast evolution experiment. We then isolated hundreds of second-step mutations from these evolution experiments, measured their fitness and performance in several growth phases, and conducted whole-genome sequencing of the second-step clones. Here, we found that while the vast majority of mutants isolated from the first-step of evolution in this condition show patterns of pleiotropic adaptation - improving both performance in fermentation and respiration growth phases - second-step mutations show a shift towards modular adaptation, mostly improving respiration performance and only rarely improving fermentation performance. We also identified a shift in the molecular basis of adaptation from genes in cellular signaling pathways towards genes involved in respiration and mitochondrial function. Our results suggest that the genes in cellular signaling pathways are particularly capable of providing large, adaptively pleiotropic benefits to the organism due to their ability to coherently affect many phenotypes at once. As such, these genes may serve as the source of pleiotropic adaptation in the early stages of evolution, and once these become exhausted, organisms then adapt more gradually, acquiring smaller, more modular mutations.

evolutionary biology↗

bioRxiv2024

Analysis and culturing of the prototypic crAssphage reveals a phage-plasmid lifestyle

The prototypic crAssphage (Carjivirus communis) is one of the most abundant, prevalent, and persistent gut bacteriophages, yet it remains uncultured and its lifestyle uncharacterized. For the last decade, crAssphage has escaped plaque-dependent culturing efforts, leading us to investigate alternative lifestyles that might explain its widespread success. Through genomic analyses and culturing, we find that crAssphage uses a phage-plasmid lifestyle to persist extrachromosomally. Plasmid-related genes are more highly expressed than those implicated in phage maintenance. Leveraging this finding, we use a plaque-free culturing approach to measure crAssphage replication in culture with Phocaeicola vulgatus, Phocaeicola dorei, and Bacteroides stercoris, revealing a broad host range. We demonstrate that crAssphage persists with its hosts in culture without causing major cell lysis events or integrating into host chromosomes. The ability to switch between phage and plasmid lifestyles within a wide range of hosts contributes to the prolific nature of crAssphage in the human gut microbiome.

microbiology↗

bioRxiv2023

Spindle architecture constrains karyotype in budding yeast

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cells chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

cell biology↗

bioRxiv2023

Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

Adaptation is driven by the selection for beneficial mutations that provide a fitness advantage in the specific environment in which a population is evolving. However, environments are rarely constant or predictable. When an organism well adapted to one environment finds itself in another, pleiotropic effects of mutations that made it well adapted to its former environment will affect its success. To better understand such pleiotropic effects, we evolved both haploid and diploid barcoded budding yeast populations in multiple environments, isolated adaptive clones, and then determined the fitness effects of adaptive mutations in "non-home" environments in which they were not selected. We find that pleiotropy is common, with most adaptive evolved lineages showing fitness effects in non-home environments. Consistent with other studies, we find that these pleiotropic effects are unpredictable: they are beneficial in some environments and deleterious in others. However, we do find that lineages with adaptive mutations in the same genes tend to show similar pleiotropic effects. We also find that ploidy influences the observed adaptive mutational spectra in a condition-specific fashion. In some conditions, haploids and diploids are selected with adaptive mutations in identical genes, while in others they accumulate mutations in almost completely disjoint sets of genes.

evolutionary biology↗

bioRxiv2022

No evidence that synonymous mutations in yeast genes are mostly deleterious

In a recent paper1, Shen et al. reported that most mutations in the coding regions of 21 yeast genes were strongly deleterious, and that the distributions of fitness effects were similar for synonymous and nonsynonymous mutations. Taken at face value, these results would conflict with well-established findings from a broad range of fields and approaches. Here, we argue that these results arose from a lack of appropriate controls for the impacts of background genetic effects in edited strains. A re-examination of the data in Shen et al. strongly suggests that it is entirely consistent with the expectation that most nonsynonymous and nearly all synonymous mutations have no detectable effects on fitness. We present analyses which show that the data is inconsistent with the proposed explanation that pervasive fitness effects of synonymous mutations arise from their effects on mRNA levels, that the sequence-based fitness assay overestimates fitness effects compared to the growth-based fitness assay, and that the observed wide fitness distributions for nonsense mutations are consistent with off-target effects or other uncontrolled sources of biological variation contributing to measured fitness. We conclude by discussing the essential controls and other experimental design considerations that are required to produce interpretable results regarding the fitness effects of mutations in large-scale screens.

genetics↗

bioRxiv2021

Simulation-based inference of evolutionary parameters from adaptation dynamics using neural networks

The rate of adaptive evolution depends on the rate at which beneficial mutations are introduced into a population and the fitness effects of those mutations. The rate of beneficial mutations and their expected fitness effects is often difficult to empirically quantify. As these two parameters determine the pace of evolutionary change in a population, the dynamics of adaptive evolution may enable inference of their values. Copy number variants (CNVs) are a pervasive source of heritable variation that can facilitate rapid adaptive evolution. Previously, we developed a locus-specific fluorescent CNV reporter to quantify CNV dynamics in evolving populations maintained in nutrient-limiting conditions using chemostats. Here, we use the observed CNV adaptation dynamics to estimate the rate at which beneficial CNVs are introduced through de novo mutation and their fitness effects using simulation-based Bayesian likelihood-free inference approaches. We tested the suitability of two evolutionary models: a standard Wright-Fisher model and a chemostat growth model. We evaluated two likelihood-free inference algorithms: the well-established Approximate Bayesian Computation with Sequential Monte Carlo (ABC-SMC) algorithm, and the recently developed Neural Posterior Estimation (NPE) algorithm, which applies an artificial neural network to directly estimate the posterior distribution. By systematically evaluating the suitability of different inference methods and models we show that NPE has several advantages over ABC-SMC and that a Wright-Fisher evolutionary model suffices in most cases. Using our validated inference framework, we estimate the CNV formation rate at the GAP1 locus in yeast as 10-4.7 -10-4 per cell division, and a selection coefficient of 0.04 - 0.1 per generation for GAP1 CNVs in glutamine-limited chemostats. We experimentally validated our estimates using barcode lineage tracking and pairwise fitness assays. Our results are consistent with a high beneficial CNV supply rate that is 10-fold greater than the estimated rates of beneficial single-nucleotide mutations, explaining their outsized importance in rapid adaptive evolution. More generally, our study demonstrates the utility of novel simulation-based likelihood-free inference methods for inferring the rates and effects of evolutionary processes from empirical data.

evolutionary biology↗

bioRxiv2018

Transmission and persistence of crAssphage, a ubiquitous human-associated bacteriophage

CrAss-like phages are double-stranded DNA viruses that are prevalent in human gut microbiomes. Here, we analyze gut metagenomic data from mother-infant pairs and patients undergoing fecal microbiota transplantation to evaluate the patterns of acquisition, transmission and strain diversity of crAss-like phages. We find that crAss-like phages are rarely detected at birth but are increasingly prevalent in the infant microbiome after one month of life. We observe nearly identical genomes in 50% of cases where the same crAss-like clade is detected in both the mother and the infant, suggesting vertical transmission. In cases of putative transmission of prototypical crAssphage (p-crAssphage), we find that a subset of strains present in the mother are detected in the infant, and that strain diversity in infants increases with time. Strain diversity in the p-crAssphage population in mothers is generally low, with a median of 50 variable sites in the 97kb genome. Putative tail fiber proteins are enriched for nonsynonymous strain variation compared to other genes, suggesting a potential evolutionary benefit to maintaining strain diversity in specific genes. Finally, we show that p-crAssphage can be acquired through fecal microbiota transplantation. These results enhance our understanding of the acquisition, individual-level strain diversity, and transmission of crAss-like phages, which have emerged as some of the most successful human-associated microbes.

microbiology↗

bioRxiv2017

The dynamics of adaptive genetic diversity during the early stages of clonal evolution

The dynamics of genetic diversity in large clonally-evolving cell populations are poorly understood, despite having implications for the treatment of cancer and microbial infections. Here, we combine barcode lineage tracking, sequencing of adaptive clones, and mathematical modelling of mutational dynamics to understand diversity changes during experimental evolution. We find that, despite differences in beneficial mutational mechanisms and fitness effects between two environments, early adaptive genetic diversity increases predictably, driven by the expansion of many single-mutant lineages. However, a crash in diversity follows, caused by highly-fit double-mutants fed from exponentially growing single-mutants, a process closely related to the classic Luria-Delbruck experiment. The diversity crash is likely to be a general feature of clonal evolution, however its timing and magnitude is stochastic and depends on the population size, the distribution of beneficial fitness effects, and patterns of epistasis.

evolutionary biology↗