Search

The Yca1 metacaspase was discovered due to its role in the regulation of apoptosis in Saccharomyces cerevisiae. However, the mechanisms that drive apoptosis in yeast remain poorly understood. Additionally, Yca1 and other metacaspase proteins have recently been recognized for their involvement in other cellular processes, including cellular proteostasis and cell cycle regulation. In this minireview, we outline recent findings on Yca1 that will enable the further study of metacaspase multifunctionality and novel apoptosis pathways in yeast and other nonmetazoans. In addition, we discuss advancements in high-throughput screening technologies that can be applied to answer complex questions surrounding the apoptotic and nonapoptotic functions of metacaspase proteins across a diverse range of species., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

During DNA replication, the newly created sister chromatids are held together until their separation at anaphase. The cohesin complex is in charge of creating and maintaining sister chromatid cohesion (SCC) in all eukaryotes. In Saccharomyces cerevisiae cells, cohesin is composed of two elongated proteins, Smc1 and Smc3, bridged by the kleisin Mcd1/Scc1. The latter also acts as a scaffold for three additional proteins, Scc3/Irr1, Wpl1/Rad61, and Pds5. Although the HEAT-repeat protein Pds5 is essential for cohesion, its precise function is still debated. Deletion of the ELG1 gene, encoding a PCNA unloader, can partially suppress the temperature-sensitive pds5-1 allele, but not a complete deletion of PDS5. We carried out a genetic screen for high-copy-number suppressors and another for spontaneously arising mutants, allowing the survival of a pds5 Δ elg1 Δ strain. Our results show that cells remain viable in the absence of Pds5 provided that there is both an elevation in the level of Mcd1 (which can be due to mutations in the CLN2 gene, encoding a G 1 cyclin), and an increase in the level of SUMO-modified PCNA on chromatin (caused by lack of PCNA unloading in elg1 Δ mutants). The elevated SUMO-PCNA levels increase the recruitment of the Srs2 helicase, which evicts Rad51 molecules from the moving fork, creating single-stranded DNA (ssDNA) regions that serve as sites for increased cohesin loading and SCC establishment. Thus, our results delineate a double role for Pds5 in protecting the cohesin ring and interacting with the DNA replication machinery. IMPORTANCE Sister chromatid cohesion is vital for faithful chromosome segregation, chromosome folding into loops, and gene expression. A multisubunit protein complex known as cohesin holds the sister chromatids from S phase until the anaphase stage. In this study, we explore the function of the essential cohesin subunit Pds5 in the regulation of sister chromatid cohesion. We performed two independent genetic screens to bypass the function of the Pds5 protein. We observe that Pds5 protein is a cohesin stabilizer, and elevating the levels of Mcd1 protein along with SUMO-PCNA accumulation on chromatin can compensate for the loss of the PDS5 gene. In addition, Pds5 plays a role in coordinating the DNA replication and sister chromatid cohesion establishment. This work elucidates the function of cohesin subunit Pds5, the G 1 cyclin Cln2, and replication factors PCNA, Elg1, and Srs2 in the proper regulation of sister chromatid cohesion.

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO 2 )-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO 2 utilising precursors derived from the CBB cycle., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 2 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 CNV adaptation dynamics to estimate the rate at which beneficial CNVs are introduced through de novo mutation and their fitness effects using simulation-based likelihood-free inference approaches. We tested the suitability of 2 evolutionary models: a standard Wright-Fisher model and a chemostat model. We evaluated 2 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 the yeast Saccharomyces cerevisiae to be 10-4.7 to 10-4 CNVs per cell division and a fitness coefficient of 0.04 to 0.1 per generation for GAP1 CNVs in glutamine-limited chemostats. We experimentally validated our inference-based estimates using 2 distinct experimental methods-barcode lineage tracking and pairwise fitness assays-which provide independent confirmation of the accuracy of our approach. Our results are consistent with a beneficial CNV supply rate that is 10-fold greater than the estimated rates of beneficial single-nucleotide mutations, explaining the outsized importance of CNVs in rapid adaptive evolution. More generally, our study demonstrates the utility of novel neural network-based likelihood-free inference methods for inferring the rates and effects of evolutionary processes from empirical data with possible applications ranging from tumor to viral evolution., Competing Interests: The authors have declared that no competing interests exist.

Background: Internationally there are too few suitably skilled registered nurses to meet the demands for dementia care. Research has established low preferences in undergraduate nursing students for working with older people. However, there is limited research on preferences for dementia care. Understanding career preferences is one component of ensuring future workforce capacity., Objective: To assess student nurses' preferences during undergraduate training in relation to working with people with dementia., Methods: Data from a longitudinal survey collected at two UK universities were analysed (n = 488). Measures included career preferences, demographics, participation in a dementia educational intervention, and measures of attitude, knowledge, and empathy to dementia. Open text responses were also included to explore the students' reasons for their preferences., Results: The preference for working with older people and people with dementia was low and decreased during training. A linear regression analysis supports a strong relationship of preferences with attitudes to dementia. Content analysis of students' reasons for their preferences found that perceived difficulty and lack of confidence contributes to the negative evaluation of working with people with dementia., Conclusion: Undergraduate nursing education needs to continue to review its contribution to preparing the dementia workforce and act to support positive attitudes to working with people with dementia across nursing specialties., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Candida Genome Database provides access to biological information about genes and proteins of several medically important Candida species. The website is organized into easily navigable pages that enable data retrieval and analysis. This chapter shows how to explore the CGD Home page and Locus Summary pages, which are the main access points to the database. It also provides a description of how to use the GO analysis tools, GO Term Finder, and GO Slim Mapper and how to browse large-scale datasets using the JBrowse genome browser. Finally, it shows how to search and retrieve data for user-defined sets of genes using the Advanced Search and Batch Download tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Due to limitations on high-resolution strain tracking, selection dynamics during gut microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutations in genes involved in motility and metabolite utilization are reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates revealed linked alleles that demonstrate between-host transmission. A population-genetics model predicts substantial fitness advantages for certain mutants and that migration accounted for ∼10% of the resident microbiota each day. Treatment with ciprofloxacin suggests interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Historical contingency and diminishing returns epistasis have been typically studied for relatively divergent genotypes and/or over long evolutionary timescales. Here, we use Saccharomyces cerevisiae to study the extent of diminishing returns and the changes in the adaptive mutational spectra following a single first adaptive mutational step. We further evolve three clones that arose under identical conditions from a common ancestor. We follow their evolutionary dynamics by lineage tracking and determine adaptive outcomes using fitness assays and whole genome sequencing. We find that diminishing returns manifests as smaller fitness gains during the 2 nd step of adaptation compared to the 1 st step, mainly due to a compressed distribution of fitness effects. We also find that the beneficial mutational spectra for the 2 nd adaptive step are contingent on the 1 st step, as we see both shared and diverging adaptive strategies. Finally, we find that adaptive loss-of-function mutations, such as nonsense and frameshift mutations, are less common in the second step of adaptation than in the first step., (© 2021. The Author(s).)

Background: Microbial evolution experiments can be used to study the tempo and dynamics of evolutionary change in asexual populations, founded from single clones and growing into large populations with multiple clonal lineages. High-throughput sequencing can be used to catalog de novo mutations as potential targets of selection, determine in which lineages they arise, and track the fates of those lineages. Here, we describe a long-term experimental evolution study to identify targets of selection and to determine when, where, and how often those targets are hit., Results: We experimentally evolved replicate Escherichia coli populations that originated from a mutator/nonsense suppressor ancestor under glucose limitation for between 300 and 500 generations. Whole-genome, whole-population sequencing enabled us to catalog 3346 de novo mutations that reached > 1% frequency. We sequenced the genomes of 96 clones from each population when allelic diversity was greatest in order to establish whether mutations were in the same or different lineages and to depict lineage dynamics. Operon-specific mutations that enhance glucose uptake were the first to rise to high frequency, followed by global regulatory mutations. Mutations related to energy conservation, membrane biogenesis, and mitigating the impact of nonsense mutations, both ancestral and derived, arose later. New alleles were confined to relatively few loci, with many instances of identical mutations arising independently in multiple lineages, among and within replicate populations. However, most never exceeded 10% in frequency and were at a lower frequency at the end of the experiment than at their maxima, indicating clonal interference. Many alleles mapped to key structures within the proteins that they mutated, providing insight into their functional consequences., Conclusions: Overall, we find that when mutational input is increased by an ancestral defect in DNA repair, the spectrum of high-frequency beneficial mutations in a simple, constant resource-limited environment is narrow, resulting in extreme parallelism where many adaptive mutations arise but few ever go to fixation.

The environmental conditions of microorganisms' habitats may fluctuate in unpredictable ways, such as changes in temperature, carbon source, pH, and salinity to name a few. Environmental heterogeneity presents a challenge to microorganisms, as they have to adapt not only to be fit under a specific condition, but they must also be robust across many conditions and be able to deal with the switch between conditions itself. While experimental evolution has been used to gain insight into the adaptive process, this has largely been in either unvarying or consistently varying conditions. In cases where changing environments have been investigated, relatively little is known about how such environments influence the dynamics of the adaptive process itself, as well as the genetic and phenotypic outcomes. We designed a systematic series of evolution experiments where we used two growth conditions that have differing timescales of adaptation and varied the rate of switching between them. We used lineage tracking to follow adaptation, and whole genome sequenced adaptive clones from each of the experiments. We find that both the switch rate and the order of the conditions influences adaptation. We also find different adaptive outcomes, at both the genetic and phenotypic levels, even when populations spent the same amount of total time in the two different conditions, but the order and/or switch rate differed. Thus, in a variable environment adaptation depends not only on the nature of the conditions and phenotypes under selection, but also on the complexity of the manner in which those conditions are combined to result in a given dynamic environment., Competing Interests: The authors declare that they have no conflict of interest.

Background: A current issue in workforce planning is ensuring healthcare professionals are both competent and willing to work with older adults with complex needs. This includes dementia care, which is widely recognised as a priority. Yet research suggests that working with older people is unattractive to undergraduate healthcare students., Methods: The aim of this systematic review and narrative synthesis is to explore the factors related to healthcare (medical and nursing) student preferences' for working with older people and people with dementia. Searches were conducted in five databases: MEDLINE, PsycINFO, CINHAL, BNI, ERIC. Screening, data extraction and quality appraisal were conducted by two independent reviewers. A narrative, data-based convergent synthesis was conducted., Results: One thousand twenty-four papers were screened (139 full texts) and 62 papers were included for a narrative synthesis. Factors were grouped into seven categories; student characteristics, experiences of students, course characteristics, career characteristics, patient characteristics, work characteristics and the theory of planned behaviour., Conclusion: Health educators should review their role in cultivating student interest in working with older adults, with consideration of student preparation and the perceived value of this work. There is a lack of evidence about the career preferences of students in relation to dementia, and this warrants further research.

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. 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.

Trade-offs constrain the improvement of performance of multiple traits simultaneously. Such trade-offs define Pareto fronts, which represent a set of optimal individuals that cannot be improved in any one trait without reducing performance in another. Surprisingly, experimental evolution often yields genotypes with improved performance in all measured traits, perhaps indicating an absence of trade-offs at least in the short term. Here we densely sample adaptive mutations in Saccharomyces cerevisiae to ask whether first-step adaptive mutations result in trade-offs during the growth cycle. We isolated thousands of adaptive clones evolved under carefully chosen conditions and quantified their performances in each part of the growth cycle. We too find that some first-step adaptive mutations can improve all traits to a modest extent. However, our dense sampling allowed us to identify trade-offs and establish the existence of Pareto fronts between fermentation and respiration, and between respiration and stationary phases. Moreover, we establish that no single mutation in the ancestral genome can circumvent the detected trade-offs. Finally, we sequenced hundreds of these adaptive clones, revealing new targets of adaptation and defining the genetic basis of the identified trade-offs.

Large-scale genetic interaction (GI) screens in yeast have been invaluable for our understanding of molecular systems biology and for characterizing novel gene function. Owing in part to the high costs and long experiment times required, a preponderance of GI data has been generated in a single environmental condition. However, an unknown fraction of GIs may be specific to other conditions. Here, we developed a pooled-growth CRISPRi-based sequencing assay for GIs, CRISPRiSeq, which increases throughput such that GIs can be easily assayed across multiple growth conditions. We assayed the fitness of approximately 17,000 strains encompassing approximately 7700 pairwise interactions in five conditions and found that the additional conditions increased the number of GIs detected nearly threefold over the number detected in rich media alone. In addition, we found that condition-specific GIs are prevalent and improved the power to functionally classify genes. Finally, we found new links during respiratory growth between members of the Ras nutrient-sensing pathway and both the COG complex and a gene of unknown function. Our results highlight the potential of conditional GI screens to improve our understanding of cellular genetic networks., (© 2019 Jaffe et al.; Published by Cold Spring Harbor Laboratory Press.)

Objectives: Measuring physical activity is complicated particularly in people with dementia, where activity levels are low and subjective measures are susceptible to inaccurate recall. Activity monitors are increasingly being used within research; however, it is unclear how people with dementia view wearing such devices and what aspects of the device effect wear time. The aim of the study was to evaluate the acceptability and feasibility of people with dementia wearing activity monitors., Methods: Twenty-six, community-dwelling, people with mild dementia were asked to wear an activity monitor (GENEactiv Original) over a 1-month period. Perceptions of the device were measured using the Quebec User Evaluation of Satisfaction with Assistive Technology (QUEST) 2.0, alongside qualitative interviews. Device diary and activity monitor data were used to assess compliance., Results: Participants tended to find wearing the activity monitors acceptable, with only three participants (12%) withdrawing prior to the study end date. Participants were generally satisfied with wearing the devices as measured by the QUEST (Mdn = 4.4, IQR = 1.1). Four themes were identified that influenced perceptions of wearing the device: external influences, design, routine, and perceived benefits., Discussion: Asking people with dementia to wear a wrist-worn activity monitor for prolonged periods appears to be both feasible and acceptable. Researchers need to consider the needs and preferences of the sample population prior to selecting activity monitors., (© 2019 John Wiley & Sons, Ltd.)

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 adaptive diversity changes during experimental evolution of Saccharomyces cerevisiae under nitrogen and carbon limitation. We find that, despite differences in beneficial mutational mechanisms and fitness effects, early adaptive genetic diversity increases predictably, driven by the expansion of many single-mutant lineages. However, a crash in adaptive diversity follows, caused by highly fit double-mutant 'jackpot' clones that are fed from exponentially growing single mutants, a process closely related to the classic Luria-Delbrück experiment. The diversity crash is likely to be a general feature of asexual evolution with clonal interference; however, both its timing and magnitude are stochastic and depend on the population size, the distribution of beneficial fitness effects and patterns of epistasis.

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.

Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation.

Few studies have "quantitatively" probed how adaptive mutations result in increased fitness. Even in simple microbial evolution experiments, with full knowledge of the underlying mutations and specific growth conditions, it is challenging to determine where within a growth-saturation cycle those fitness gains occur. A common implicit assumption is that most benefits derive from an increased exponential growth rate. Here, we instead show that, in batch serial transfer experiments, adaptive mutants' fitness gains can be dominated by benefits that are accrued in one growth cycle, but not realized until the next growth cycle. For thousands of evolved clones (most with only a single mutation), we systematically varied the lengths of fermentation, respiration, and stationary phases to assess how their fitness, as measured by barcode sequencing, depends on these phases of the growth-saturation-dilution cycles. These data revealed that, whereas all adaptive lineages gained similar and modest benefits from fermentation, most of the benefits for the highest fitness mutants came instead from the time spent in respiration. From monoculture and high-resolution pairwise fitness competition experiments for a dozen of these clones, we determined that the benefits "accrued" during respiration are only largely "realized" later as a shorter duration of lag phase in the following growth cycle. These results reveal hidden complexities of the adaptive process even under ostensibly simple evolutionary conditions, in which fitness gains can accrue during time spent in a growth phase with little cell division, and reveal that the memory of those gains can be realized in the subsequent growth cycle., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Studying Candida biology requires access to genomic sequence data in conjunction with experimental information that together provide functional context to genes and proteins, and aid in interpreting newly generated experimental data. The Candida Genome Database (CGD) curates the Candida literature, and integrates functional information about Candida genes and their products with a set of analysis tools that facilitate searching for sets of genes and exploring their biological roles. This chapter describes how the various types of information available at CGD can be searched, retrieved, and analyzed. Starting with the guided tour of the CGD Home page and Locus Summary page, this unit shows how to navigate the various assemblies of the C. albicans genome, how to use Gene Ontology tools to make sense of large-scale data, and how to access the microarray data archived at CGD, as well as visualize high-throughput sequencing data through the use of JBrowse.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus., Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli., Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Speciation can occur when a population is split and the resulting subpopulations evolve independently, accumulating mutations over time that make them incompatible with one another. It is thought that such incompatible mutations, known as Bateson-Dobzhansky-Muller (BDM) incompatibilities, may arise when the two populations face different environments, which impose different selective pressures. However, a new study in PLOS Biology by Ono et al. finds that the first-step mutations selected in yeast populations evolving in parallel in the presence of the antifungal drug nystatin are frequently incompatible with one another. This incompatibility is environment dependent, such that the combination of two incompatible alleles can become advantageous under increasing drug concentrations. This suggests that the activity for the affected pathway must have an optimum level, the value of which varies according to the drug concentration. It is likely that many biological processes similarly have an optimum under a given environment and many single-step adaptive ways to reach it; thus, not only should BDM incompatibilities commonly arise during parallel evolution, they might be virtually inevitable, as the combination of two such steps is likely to overshoot the optimum., Competing Interests: The authors have declared that no competing interests exist.

Systematic screens for genetic interactions are a cornerstone of both network and systems biology. However, most screens have been limited to characterizing interaction networks in a single environment. Moving beyond this static view of the cell requires a major technological advance to increase the throughput and ease of replication in these assays. Here, we introduce iSeq-a platform to build large double barcode libraries and rapidly assay genetic interactions across environments. We use iSeq in yeast to measure fitness in three conditions of nearly 400 clonal strains, representing 45 possible single or double gene deletions, including multiple replicate strains per genotype. We show that iSeq fitness and interaction scores are highly reproducible for the same clonal strain across replicate cultures. However, consistent with previous work, we find that replicates with the same putative genotype have highly variable genetic interaction scores. By whole-genome sequencing 102 of our strains, we find that segregating variation and de novo mutations, including aneuploidy, occur frequently during strain construction, and can have large effects on genetic interaction scores. Additionally, we uncover several new environment-dependent genetic interactions, suggesting that barcode-based genetic interaction assays have the potential to significantly expand our knowledge of genetic interaction networks., (Copyright © 2017 Jaffe et al.)

The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available online resource that provides gene, protein and sequence information for multiple Candida species, along with web-based tools for accessing, analyzing and exploring these data. The mission of CGD is to facilitate and accelerate research into Candida pathogenesis and biology, by curating the scientific literature in real time, and connecting literature-derived annotations to the latest version of the genomic sequence and its annotations. Here, we report the incorporation into CGD of Assembly 22, the first chromosome-level, phased diploid assembly of the C. albicans genome, coupled with improvements that we have made to the assembly using additional available sequence data. We also report the creation of systematic identifiers for C. albicans genes and sequence features using a system similar to that adopted by the yeast community over two decades ago. Finally, we describe the incorporation of JBrowse into CGD, which allows online browsing of mapped high throughput sequencing data, and its implementation for several RNA-Seq data sets, as well as the whole genome sequencing data that was used in the construction of Assembly 22., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The characterization of mutational spectra is usually carried out in one of three ways-by direct observation through mutation accumulation (MA) experiments, through parent-offspring sequencing, or by indirect inference from sequence data. Direct observations of spontaneous mutations with MA experiments are limited, given (i) the rarity of spontaneous mutations, (ii) applicability only to laboratory model species with short generation times, and (iii) the possibility that mutational spectra under lab conditions might be different from those observed in nature. Trio sequencing is an elegant solution, but it is not applicable in all organisms. Indirect inference, usually from divergence data, faces no such technical limitations, but rely upon critical assumptions regarding the strength of natural selection that are likely to be violated. Ideally, new mutational events would be directly observed before the biased filter of selection, and without the technical limitations common to lab experiments. One approach is to identify very young mutations from population sequencing data. Here we do so by leveraging two characteristics common to all new mutations-new mutations are necessarily rare in the population, and absent in the genomes of immediate relatives. From 132 clinical yeast strains, we were able to identify 1,425 putatively new mutations and show that they exhibit extremely low signatures of selection, as well as display a mutational spectrum that is similar to that identified by a large scale MA experiment. We verify that population sequencing data are a potential wealth of information for inferring mutational spectra, and should be considered for analysis where MA experiments are infeasible or especially tedious., Competing Interests: The authors have declared that no competing interests exist.

The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species., Importance: Recessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species., (Copyright © 2016 Feri et al.)

The original yeast genome sequencing project was a monumental task, spanning several years, which resulted in the first sequenced eukaryotic genome. The 12 Mbp reference sequence was generated from yeast strain S288c and was of extremely high quality. In the years since it was published, sequencing technology has advanced apace, such that it is within the reach of most labs to sequence yeast strains of interest almost as a matter of standard practice, either via core facilities at their institution or through commercial sequencing services. Because of the availability of the high-quality reference sequence (which itself has received approximately 1500 updates derived from high-throughput sequencing data), reliable identification of differences between a strain of interest and the reference is relatively straightforward, at least for the nonrepetitive regions of the genome. In this introduction, we describe current high-throughput sequencing technology and methods for analysis of the resulting data., (© 2016 Cold Spring Harbor Laboratory Press.)

This protocol provides a detailed description of how to prepare a DNA sequencing library from yeast genomic DNA for use with the Illumina sequencing platform. This method does not require purchase of Illumina kits for library preparation but instead employs specific reagents purchased largely from New England BioLabs, which significantly reduces the cost of library preparation. Although we assume here that users intend to generate libraries with ∼400-bp insert sizes for paired-end sequencing, it is relatively straightforward to modify the shearing and size selection steps for longer or shorter inserts., (© 2016 Cold Spring Harbor Laboratory Press.)

Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Budding yeast has undergone several independent transitions from commercial to clinical lifestyles. The frequency of such transitions suggests that clinical yeast strains are derived from environmentally available yeast populations, including commercial sources. However, despite their important role in adaptive evolution, the prevalence of polyploidy and aneuploidy has not been extensively analyzed in clinical strains. In this study, we have looked for patterns governing the transition to clinical invasion in the largest screen of clinical yeast isolates to date. In particular, we have focused on the hypothesis that ploidy changes have influenced adaptive processes. We sequenced 144 yeast strains, 132 of which are clinical isolates. We found pervasive large-scale genomic variation in both overall ploidy (34% of strains identified as 3n/4n) and individual chromosomal copy numbers (36% of strains identified as aneuploid). We also found evidence for the highly dynamic nature of yeast genomes, with 35 strains showing partial chromosomal copy number changes and eight strains showing multiple independent chromosomal events. Intriguingly, a lineage identified to be baker's/commercial derived with a unique damaging mutation in NDC80 was particularly prone to polyploidy, with 83% of its members being triploid or tetraploid. Polyploidy was in turn associated with a >2× increase in aneuploidy rates as compared to other lineages. This dataset provides a rich source of information on the genomics of clinical yeast strains and highlights the potential importance of large-scale genomic copy variation in yeast adaptation., (Copyright © 2016 Zhu et al.)

Adaptation in diploids is predicted to proceed via mutations that are at least partially dominant in fitness. Recently, we argued that many adaptive mutations might also be commonly overdominant in fitness. Natural (directional) selection acting on overdominant mutations should drive them into the population but then, instead of bringing them to fixation, should maintain them as balanced polymorphisms via heterozygote advantage. If true, this would make adaptive evolution in sexual diploids differ drastically from that of haploids. The validity of this prediction has not yet been tested experimentally. Here, we performed four replicate evolutionary experiments with diploid yeast populations (Saccharomyces cerevisiae) growing in glucose-limited continuous cultures. We sequenced 24 evolved clones and identified initial adaptive mutations in all four chemostats. The first adaptive mutations in all four chemostats were three copy number variations, all of which proved to be overdominant in fitness. The fact that fitness overdominant mutations were always the first step in independent adaptive walks supports the prediction that heterozygote advantage can arise as a common outcome of directional selection in diploids and demonstrates that overdominance of de novo adaptive mutations in diploids is not rare., (Copyright © 2016 by the Genetics Society of America.)

Studying Candida biology requires access to genomic sequence data in conjunction with experimental information that provides functional context to genes and proteins. The Candida Genome Database (CGD) integrates functional information about Candida genes and their products with a set of analysis tools that facilitate searching for sets of genes and exploring their biological roles. This chapter describes how the various types of information available at CGD can be searched, retrieved, and analyzed. Starting with the guided tour of the CGD Home page and Locus Summary page, this unit shows how to navigate the various assemblies of the C. albicans genome, how to use Gene Ontology tools to make sense of large-scale data, and how to access the microarray data archived at CGD.

Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding., (© 2015 Schep et al.; Published by Cold Spring Harbor Laboratory Press.)

Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages.

Evolution of large asexual cell populations underlies ∼30% of deaths worldwide, including those caused by bacteria, fungi, parasites, and cancer. However, the dynamics underlying these evolutionary processes remain poorly understood because they involve many competing beneficial lineages, most of which never rise above extremely low frequencies in the population. To observe these normally hidden evolutionary dynamics, we constructed a sequencing-based ultra high-resolution lineage tracking system in Saccharomyces cerevisiae that allowed us to monitor the relative frequencies of ∼500,000 lineages simultaneously. In contrast to some expectations, we found that the spectrum of fitness effects of beneficial mutations is neither exponential nor monotonic. Early adaptation is a predictable consequence of this spectrum and is strikingly reproducible, but the initial small-effect mutations are soon outcompeted by rarer large-effect mutations that result in variability between replicates. These results suggest that early evolutionary dynamics may be deterministic for a period of time before stochastic effects become important.

The fitness landscape is a powerful metaphor for describing the relationship between genotype and phenotype for a population under selection. However, empirical data as to the topography of fitness landscapes are limited, owing to difficulties in measuring fitness for large numbers of genotypes under any condition. We previously reported a case of reciprocal sign epistasis (RSE), where two mutations individually increased yeast fitness in a glucose-limited environment, but reduced fitness when combined, suggesting the existence of two peaks on the fitness landscape. We sought to determine whether a ridge connected these peaks so that populations founded by one mutant could reach the peak created by the other, avoiding the low-fitness "Valley-of-Death" between them. Sequencing clones after 250 generations of further evolution provided no evidence for such a ridge, but did reveal many presumptive beneficial mutations, adding to a growing body of evidence that clonal interference pervades evolving microbial populations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cryptococcus, a major cause of disseminated infections in immunocompromised patients, kills over 600,000 people per year worldwide. Genes involved in the virulence of the meningitis-causing fungus are being characterized at an increasing rate, and to date, at least 648 Cryptococcus gene names have been published. However, these data are scattered throughout the literature and are challenging to find. Furthermore, conflicts in locus identification exist, so that named genes have been subsequently published under new names or names associated with one locus have been used for another locus. To avoid these conflicts and to provide a central source of Cryptococcus gene information, we have collected all published Cryptococcus gene names from the scientific literature and associated them with standard Cryptococcus locus identifiers and have incorporated them into FungiDB (www.fungidb.org). FungiDB is a panfungal genome database that collects gene information and functional data and provides search tools for 61 species of fungi and oomycetes. We applied these published names to a manually curated ortholog set of all Cryptococcus species currently in FungiDB, including Cryptococcus neoformans var. neoformans strains JEC21 and B-3501A, C. neoformans var. grubii strain H99, and Cryptococcus gattii strains R265 and WM276, and have written brief descriptions of their functions. We also compiled a protocol for gene naming that summarizes guidelines proposed by members of the Cryptococcus research community. The centralization of genomic and literature-based information for Cryptococcus at FungiDB will help researchers communicate about genes of interest, such as those related to virulence, and will further facilitate research on the pathogen., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community's underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an "engine" generating diversity by creating new metabolic niches, but not the occupants themselves.

Though sequence differences between alleles are often limited to a few polymorphisms, these differences can cause large and widespread allelic variation at the expression level. Such allele-specific expression (ASE) has been extensively explored at the level of transcription but not translation. Here we measured ASE in the diploid yeast Candida albicans at both the transcriptional and translational levels using RNA-seq and ribosome profiling, respectively. Since C. albicans is an obligate diploid, our analysis isolates ASE arising from cis elements in a natural, nonhybrid organism, where allelic effects reflect evolutionary forces. Importantly, we find that ASE arising from translation is of a similar magnitude as transcriptional ASE, both in terms of the number of genes affected and the magnitude of the bias. We further observe coordination between ASE at the levels of transcription and translation for single genes. Specifically, reinforcing relationships--where transcription and translation favor the same allele--are more frequent than expected by chance, consistent with selective pressure tuning ASE at multiple regulatory steps. Finally, we parameterize alleles based on a range of properties and find that SNP location and predicted mRNA-structure stability are associated with translational ASE in cis. Since this analysis probes more than 4000 allelic pairs spanning a broad range of variations, our data provide a genome-wide view into the relative impact of cis elements that regulate translation., (© 2014 Muzzey et al.; Published by Cold Spring Harbor Laboratory Press.)

Different organisms have independently and recurrently evolved similar phenotypic traits at different points throughout history. This phenotypic convergence may be caused by genotypic convergence and in addition, constrained by historical contingency. To investigate how convergence may be driven by selection in a particular environment and constrained by history, we analyzed nine life-history traits and four metabolic traits during an experimental evolution of six yeast strains in four different environments. In each of the environments, the population converged toward a different multivariate phenotype. However, the evolution of most traits, including fitness components, was constrained by history. Phenotypic convergence was partly associated with the selection of mutations in genes involved in the same pathway. By further investigating the convergence in one gene, BMH1, mutated in 20% of the evolved populations, we show that both the history and the environment influenced the types of mutations (missense/nonsense), their location within the gene itself, as well as their effects on multiple traits. However, these effects could not be easily predicted from ancestors' phylogeny or past selection. Combined, our data highlight the role of pleiotropy and epistasis in shaping a rugged fitness landscape., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

The Aspergillus Genome Database (AspGD; http://www.aspgd.org) is a freely available web-based resource that was designed for Aspergillus researchers and is also a valuable source of information for the entire fungal research community. In addition to being a repository and central point of access to genome, transcriptome and polymorphism data, AspGD hosts a comprehensive comparative genomics toolbox that facilitates the exploration of precomputed orthologs among the 20 currently available Aspergillus genomes. AspGD curators perform gene product annotation based on review of the literature for four key Aspergillus species: Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus and Aspergillus niger. We have iteratively improved the structural annotation of Aspergillus genomes through the analysis of publicly available transcription data, mostly expressed sequenced tags, as described in a previous NAR Database article (Arnaud et al. 2012). In this update, we report substantive structural annotation improvements for A. nidulans, A. oryzae and A. fumigatus genomes based on recently available RNA-Seq data. Over 26 000 loci were updated across these species; although those primarily comprise the addition and extension of untranslated regions (UTRs), the new analysis also enabled over 1000 modifications affecting the coding sequence of genes in each target genome.

The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available online resource that provides gene, protein and sequence information for multiple Candida species, along with web-based tools for accessing, analyzing and exploring these data. The goal of CGD is to facilitate and accelerate research into Candida pathogenesis and biology. The CGD Web site is organized around Locus pages, which display information collected about individual genes. Locus pages have multiple tabs for accessing different types of information; the default Summary tab provides an overview of the gene name, aliases, phenotype and Gene Ontology curation, whereas other tabs display more in-depth information, including protein product details for coding genes, notes on changes to the sequence or structure of the gene and a comprehensive reference list. Here, in this update to previous NAR Database articles featuring CGD, we describe a new tab that we have added to the Locus page, entitled the Homology Information tab, which displays phylogeny and gene similarity information for each locus.

We identify the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle-regulated genes. FOXM1 is bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle transcription factors (E2F1, E2F4, E2F6, and GABPA) from the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle-regulated genes in a second cancer-derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.

Molecular signaling networks are ubiquitous across life and likely evolved to allow organisms to sense and respond to environmental change in dynamic environments. Few examples exist regarding the dispensability of signaling networks, and it remains unclear whether they are an essential feature of a highly adapted biological system. Here, we show that signaling network function carries a fitness cost in yeast evolving in a constant environment. We performed whole-genome, whole-population Illumina sequencing on replicate evolution experiments and find the major theme of adaptive evolution in a constant environment is the disruption of signaling networks responsible for regulating the response to environmental perturbations. Over half of all identified mutations occurred in three major signaling networks that regulate growth control: glucose signaling, Ras/cAMP/PKA and HOG. This results in a loss of environmental sensitivity that is reproducible across experiments. However, adaptive clones show reduced viability under starvation conditions, demonstrating an evolutionary tradeoff. These mutations are beneficial in an environment with a constant and predictable nutrient supply, likely because they result in constitutive growth, but reduce fitness in an environment where nutrient supply is not constant. Our results are a clear example of the myopic nature of evolution: a loss of environmental sensitivity in a constant environment is adaptive in the short term, but maladaptive should the environment change., Competing Interests: The authors have declared that no competing interests exist.

Candida albicans is an opportunistic fungal pathogen that can cause disseminated infection in patients with indwelling catheters or other implanted medical devices. A common resident of the human microbiome, C. albicans responds to environmental signals, such as cell contact with catheter materials and exposure to serum or CO2, by triggering the expression of a variety of traits, some of which are known to contribute to its pathogenic lifestyle. Such traits include adhesion, biofilm formation, filamentation, white-to-opaque (W-O) switching, and two recently described phenotypes, finger and tentacle formation. Under distinct sets of environmental conditions and in specific cell types (mating type-like a [MTLa]/alpha cells, MTL homozygotes, or daughter cells), C. albicans utilizes (or reutilizes) a single signal transduction pathway-the Ras pathway-to affect these phenotypes. Ras1, Cyr1, Tpk2, and Pde2, the proteins of the Ras signaling pathway, are the only nontranscriptional regulatory proteins that are known to be essential for regulating all of these processes. How does C. albicans utilize this one pathway to regulate all of these phenotypes? The regulation of distinct and yet related processes by a single, evolutionarily conserved pathway is accomplished through the use of downstream transcription factors that are active under specific environmental conditions and in different cell types. In this minireview, we discuss the role of Ras signaling pathway components and Ras pathway-regulated transcription factors as well as the transcriptional regulatory networks that fine-tune gene expression in diverse biological contexts to generate specific phenotypes that impact the virulence of C. albicans.

Wine has been made for thousands of years. In modern times, as the importance of yeast as an ingredient in winemaking became better appreciated, companies worldwide have collected and marketed specific yeast strains for enhancing positive and minimizing negative attributes in wine. It is generally believed that each yeast strain contributes uniquely to fermentation performance and wine style because of its genetic background; however, the impact of metabolic diversity among wine yeasts on aroma compound production has not been extensively studied. We characterized the metabolic footprints of 69 different commercial wine yeast strains in triplicate fermentations of identical Chardonnay juice, by measuring 29 primary and secondary metabolites; we additionally measured seven attributes of fermentation performance of these strains. We identified up to 1000-fold differences between strains for some of the metabolites and observed large differences in fermentation performance, suggesting significant metabolic diversity. These differences represent potential selective markers for the strains that may be important to the wine industry. Analysis of these metabolic traits further builds on the known genomic diversity of these strains and provides new insights into their genetic and metabolic relatedness., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)