High-throughput analysis of adaptation using barcoded strains of Saccharomyces cerevisiae

Background: Experimental evolution of microbes can be used to empirically address a wide range of questions about evolution and is increasingly employed to study complex phenomena ranging from genetic evolution to evolutionary rescue. Regardless of experimental aims, fitness assays are a central component of this type of research, and low-throughput often limits the scope and complexity of experimental evolution studies. We created an experimental evolution system in Results: We first confirm that barcode insertions do not alter fitness and that barcode sequencing can be used to efficiently detect fitness differences via pooled competition-based fitness assays. Next, we examine the effects of ploidy, chemical stress, and population bottleneck size on the evolutionary dynamics and fitness gains (adaptation) in a total of 76 experimentally evolving, asexual populations by conducting 1,216 fitness assays and analyzing 532 longitudinal-evolutionary samples collected from the evolving populations. In our analysis of these data we describe the strengths of this experimental evolution system and explore sources of error in our measurements of fitness and evolutionary dynamics. Conclusions: Our experimental treatments generated distinct fitness effects and evolutionary dynamics, respectively quantified via multiplexed fitness assays and barcode lineage tracking. These findings demonstrate the utility of this new resource for designing and improving high-throughput studies of experimental evolution. The approach described here provides a framework for future studies employing experimental designs that require high-throughput multiplexed fitness measurements

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance

Hsp90 interaction networks in fungi – tools and techniques

Heat-shock protein 90 (Hsp90) is a central regulator of cellular proteostasis. It stabilizes numerous proteins that are involved in fundamental processes of life, including cell growth, cell-cycle progression and the environmental response. In addition to stabilizing proteins, Hsp90 governs gene expression and controls the release of cryptic genetic variation. Given its central role in evolution and development, it is important to identify proteins and genes that interact with Hsp90. This requires sophisticated genetic and biochemical tools, including extensive mutant collections, suitable epitope tags, proteomics approaches and Hsp90-specific pharmacological inhibitors for chemogenomic screens. These usually only exist in model organisms, such as the yeast Saccharomyces cerevisiae. Yet, the importance of other fungal species, such as Candida albicans and Cryptococcus neoformans, as serious human pathogens accelerated the development of genetic tools to study their virulence and stress response pathways. These tools can also be exploited to map Hsp90 interaction networks. Here, we review tools and techniques for Hsp90 network mapping available in different fungi and provide a summary of existing mapping efforts. Mapping Hsp90 networks in fungal species spanning >500 million years of evolution provides a unique vantage point, allowing tracking of the evolutionary history of eukaryotic Hsp90 networks

Genetic Basis of Thermal Divergence in Saccharomyces species

The genetic architecture of phenotypic divergence is a central question in evolutionary biology. Genetic architecture is impacted by whether evolution occurs through accumulation of many small-effect or a few large-effect changes, the relative contribution of coding and cis-regulatory changes, and the prevalence of epistatic effects. Our empirical understanding of the genetic basis of evolutionary change remains incomplete, largely because reproductive barriers limit genetic analysis to those phenotypes that distinguish closely related species. In this dissertation, I use hybrid genetic analysis to examine the basis of thermal divergence between two post-zygotically isolated species, Saccharomyces cerevisiae and S. uvarum. S. cerevisiae is relatively heat tolerant, whereas S. uvarum is heat sensitive but outperforms S. cerevisiae at 4 degree C. Gene expression analysis with an S. cerevisiae and S. uvarum hybrid revealed a small set of 136 genes with temperature-dependent cis-acting differences, suggesting that the temperature divergence has not caused widespread cis-regulatory divergence. Using a genome-wide non-complementation screen, I found a single nuclear-encoded gene with a modest contribution to heat tolerance, and a large effect of the species\u27 mitochondrial DNA (mitotype). Recombinant mitotypes and allele replacements indicate multiple mitochondria-encoded genes contribute to thermal divergence, with the coding sequence of COX1 showing a moderate effect on both heat and cold tolerance. The non-complementation approach also identified allele differences of CUP2, a copper-binding transcription factor, in copper resistance of S. cerevisiae and S. uvarum. Chimeric alleles showed that multiple changes underlie the resistance of S. cerevisiae CUP2, with cis-regulatory changes having a larger effect than coding changes. Taken together, my findings suggest that evolution of interspecific phenotypic differences often involves accumulation of small-to-medium effect changes, such as those in mitochondrial DNA and CUP2, and can occur through both coding and cis-regulatory changes

Using Rb-TnSeq to Analyze Natural Variation in Saccharomyces cerevisiae

One of the main challenges in biology today is the characterization of millions of genes of unknown function being continuously identified in sequencing studies. Transposon mutagenesis is a technique that has been widely used for annotating gene function and has now been combined with next-generation sequencing (Tn-Seq) to assess mutant fitness on a genome wide basis. However, Tn-Seq approaches are often constrained by laborious library preparation protocols which limit the number of organisms or conditions that can be assessed. Random bar code transposon-site sequencing (RB-TnSeq), is a transposon sequencing technique that streamlines library preparation and increases the throughput of mutant fitness profiling by incorporating random DNA barcodes into Tn5 Transposons. Rb-TnSeq has been successfully used for high throughput mutant fitness assays in diverse bacterial species. However, this technique is yet to be applied to a eukaryotic model organism. The goal of this study is to develop tools that allow the construction of barcoded mutant libraries in saccharomyces cerevisiae and describes methods for producing barcoded mutant libraries using a plasmid based or transposome based approach. These library construction protocols can prove to be powerful tools for studying gene function in S. cerevisiae on a genome wide basis

Next-generation sequencing and its potential impact on food microbial genomics

Recent efforts of researchers to elucidate the molecular mechanisms of biological systems have been revolutionized greatly with the use of high throughput and cost-effective techniques such as next generation sequencing (NGS). Application of NGS to microbial genomics is not just limited to predict the prevalence of microorganisms in food samples but also to elucidate the molecular basis of how microorganisms respond to different food-associated conditions, which in turn offers tremendous opportunities to predict and control the growth and survival of desirable or undesirable microorganisms in food. Concurrently, NGS has facilitated the development of new genome-assisted approaches for correlating genotype and phenotype. The aim of this review is to provide a snapshot of the various possibilities that these new technologies are opening up in area of food microbiology, focusing the discussion mainly on lactic acid bacteria and yeasts associated with fermented food. The contribution of NGS to a system level understanding of food microorganisms is also discussed

Candida albicans repetitive elements display epigenetic diversity and plasticity

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30?°C, while robust heterochromatin is assembled over these regions at 39?°C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships