Biology subjects

Stuecker, T. N.

Publications and source records attributed to Stuecker, T. N..

7 recordsLinked to original sources

bioRxiv2025

Surprising regulatory plasticity for the conserved HOG pathway in diverse Saccharomyces cerevisiae strains

Mitogen-activated protein kinases (MAPKs) display remarkable regulatory plasticity across evolution, ranging from highly specialized pathways to broadly responsive global signaling hubs. In the budding yeast Saccharomyces cerevisiae, the high-osmolarity glycerol (HOG) network has served as a paradigm for largely stress-specific MAPK signaling, where the Hog1 MAPK coordinates osmoadaptation. This stands in sharp contrast to Hog1 orthologs in other fungi and humans, which respond not only to osmotic stress, but also diverse stresses including UV, heat shock, oxidative stress, and pathogen signals. Whether the relative osmospecificity of S. cerevisiae Hog1 represents an ancestral feature or lineage-specific evolution remains unclear. The majority of foundational work on S. cerevisiae HOG signaling has been performed in laboratory strains that are known to be genetic and phenotypic outliers, and we have been leveraging wild S. cerevisiae strains to understand aspects of stress signaling that may have been lost in laboratory strains. Here, we examined the phenotypic effects of hog1{Delta} mutations in a commonly-used laboratory S. cerevisiae strain and diverse wild strains on stress cross protection and gene expression. Our findings demonstrate an expanded role in cross-stress protection for Hog1 in wild yeast strains compared to the laboratory strain. More strikingly, we identified a large number of Hog1-dependent genes for non-osmotic stresses in the wild strains that were completely absent in the lab strain. Notably, the Hog1 regulon in wild strains responding to non-osmotic stresses is largely distinct from the canonical osmotic stress response, which we show likely occurs through non-canonical cytoplasmic functions. These findings reveal surprising within-species plasticity for the highly conserved HOG network, suggesting that evolutionary transitions between specialist to generalist stress signaling may occur with relative ease.

genetics↗

bioRxiv2025

MIC*: A Framework for Interpretable Analysis of Ordinal Viability Data

Microbial survival assays frequently use ordered categorical (ordinal) scores, such as semi-quantitative viability scores across drug concentrations. While these ordinal data provide rich information about dose-response dynamics, they require appropriate statistical approaches for proper analysis. Proportional-odds (PO) ordinal regression is specifically designed for such data, modeling the ordered nature of scores while accommodating continuous variables such as concentration. However, despite its advantages, PO regression remains underutilized in microbiology because its cumulative log-odds outputs are abstract and biologically unintuitive. Consequently, researchers often resort to flawed alternatives: collapsing scores into binary outcomes (growth/no growth), treating scores as continuous values for t-tests or ANOVA, or applying nonparametric tests that ignore dose-response structure. Each approach sacrifices power or validity, risking unreliable conclusions. To allow researchers to better leverage the power of ordinal datasets, we introduce MIC*, a summary measure that translates PO regression results into biologically interpretable units. MIC* is defined as the treatment concentration where the predicted probability of "no viability" equals 0.5, and is thus conceptually related to the minimum inhibitory dose (MIC) widely used in antimicrobial research. MIC* retains the rigor of ordinal regression while providing more biologically intuitive effect size measures. The MIC* framework enables formal comparisons as absolute differences ({Delta}MIC*) or relative fold-changes ({Delta}log2MIC*), allowing for robust statistical comparisons between samples. Monte Carlo simulations demonstrate that MIC* yields robust estimates with superior power compared to conventional statistical methods, while case studies demonstrate practical utility. To ensure wide adoption, we provide a suite of open-source tools: a BLInded Scoring System (BLISS) for generating ordinal viability scores, and web-based (MICalculator) and R package (ordinalMIC) alternatives for performing complete MIC* analyses, thus reducing barriers to appropriate analysis of ordinal phenotypes in microbiology. ImportanceMicrobiologists routinely use visual scoring systems to assess cell viability for antimicrobial and stress resistance studies. However, current analysis approaches tend to be problematic, with researchers seeking simple approaches that either discard valuable information or violate important statistical assumptions. While rigorous and appropriate statistical approaches exist, they are rarely used in microbiology because they produce outputs that are non-intuitive and require specialized expertise to implement. Our MIC* framework overcomes these longstanding barriers by translating abstract outputs into an intuitive, concentration-based metric akin to the minimum inhibitory concentration (MIC) well Known to microbiologists. MIC* allows for biologically interpretable comparisons on both absolute ({Delta}MIC*) and relative ({Delta}log2MIC*) scales, allowing for facile between-sample comparisons. MIC* outperforms conventional statistical approaches, and we offer user-friendly software tools to enable broad adoption by the community.

microbiology↗

bioRxiv2025

Dynamic global acetylation remodeling during the yeast heat shock response

All organisms experience stress and must rapidly respond to changing conditions. Thus, cells have evolved sophisticated rapid-response mechanisms such as post-translational protein modification to rapidly and reversibly modulate protein activity. One such post-translational modification is reversible lysine acetylation, where proteomic studies have identified thousands of acetylated proteins across diverse organisms. While the sheer size of the acetylome is striking, the function of acetylation for the vast majority of proteins remains largely obscure. Here, we show that global acetylation plays a previously unappreciated role in the heat shock response of Saccharomyces cerevisiae. We find that dysregulated acetylation renders cells heat sensitive, and moreover, that the acetylome is globally remodeled during heat shock over time. Using quantitative acetyl-proteomics, we identified [~]400 high-confidence acetyl marks across [~]200 proteins that significantly change in acetylation when cells are shifted to elevated temperature. Proteins with significant changes in lysine acetylation during heat shock strongly overlap with genes induced or repressed by stress. Thus, we hypothesize that protein acetylation augments the heat shock response by activating induced proteins and inactivating repressed proteins. Intriguingly, we find nearly 40 proteins with at least two acetyl marks that significantly change in the opposite directions. These proteins are strongly enriched for chaperones and ribosomal proteins, suggesting that these two key processes are coordinately regulated by protein acetylation during heat shock. Moreover, we hypothesize that the same type of activating and inactivating marks that exist on histones may be a general feature of proteins regulated by acetylation. Overall, this work has identified a new layer of post-translational regulation that likely augments the classic heat shock response.

genomics↗

bioRxiv2024

Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas cutting combined with homologous recombination of a repair template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a {beta}-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.

genetics↗

bioRxiv2023

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance

BackgroundOrganisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild primary doses of one stress can enhance tolerance to severe doses of a different secondary stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. ResultsDuring the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. ConclusionsUltimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms. Author SummaryOrganisms in nature frequently experience environmental stress in predictable patterns. For example, during the summer months in temperate climates, the warmth of the morning sun gradually gives way to high afternoon temperatures. Organisms that can anticipate these predictable patterns to mobilize stress defenses would have an advantage in nature. One way organisms anticipate future stress is through cross protection, where cells exposed to a mild dose of one stress gain the ability to survive an otherwise lethal dose of different stress. To better understand the molecular mechanisms that are responsible cross protection, we have been taking advantage of wild yeast strains that are either more resilient or more sensitive to stresses. During the course of this study, we found that strains with superficially similar levels of cross protection differ in the precise molecular mechanisms that underlie the trait. Our study suggests that different molecular strategies may be important for yielding similar stress resistances under different environmental constraints, and highlights the power of harnessing natural genetic diversity to understand the molecular mechanisms underlying differences in environmental responses.

genomics↗

bioRxiv2018

Independent mechanisms for acquired salt tolerance versus growth resumption induced by mild ethanol pretreatment in Saccharomyces cerevisiae

All living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells pre-exposed to a mild sub-lethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains of Saccharomyces cerevisiae to investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross-protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of the ENA genes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation in ENA expression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that the ENA genes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, the ENA genes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack of ENA induction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant.\n\nIMPORTANCEMicrobes in nature frequently experience \"boom or bust\" cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability survive an otherwise lethal dose of a subsequent stress. In the budding yeast Saccharomyces cerevisiae, certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquisition of higher stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms.

microbiology↗

bioRxiv2017

Linkage mapping of yeast cross protection connects gene expression variation to a higher-order organismal trait

Gene expression variation is extensive in nature, and is hypothesized to play a major role in shaping phenotypic diversity. However, connecting differences in gene expression across individuals to higher-order organismal traits is not trivial. In many cases, gene expression variation may be evolutionarily neutral, and in other cases expression variation may only affect phenotype under specific conditions. To understand connections between gene expression variation and stress defense phenotypes, we have been leveraging extensive natural variation in the gene expression response to acute ethanol in laboratory and wild Saccharomyces cerevisiae strains. Previous work found that the genetic architecture underlying these expression differences included dozens of \"hotspot\" loci that affected many transcripts in trans. In the present study, we provide new evidence that one of these expression QTL hotspot loci is responsible for natural variation in one particular stress defense phenotype--ethanol-induced cross protection against severe doses of H2O2. The causative polymorphism is in the heme-activated transcription factor Hap1p, which we show directly impacts cross protection, but not the basal H2O2 resistance of unstressed cells. This provides further support that distinct cellular mechanisms underlie basal and acquired stress resistance. We also show that the Hap1p-dependent cross protection relies on novel regulation of cytosolic catalase T (Ctt1p) during ethanol stress in wild strains. Because ethanol accumulation precedes aerobic respiration and accompanying reactive oxygen species formation, wild strains with the ability to anticipate impending oxidative stress would likely be at an advantage. This study highlights how strategically chosen traits that better correlate with gene expression changes can improve our power to identify novel connections between gene expression variation and higher-order organismal phenotypes.

genetics↗