Genetic modification of Candida maltosa, a non-pathogenic CTG species, reveals EFG1 function.

Candida maltosa is closely related to important pathogenic Candida species, especially C. tropicalis and C. albicans, but it has been rarely isolated from humans. For this reason, through comparative studies, it could be a powerful model to understand the genetic underpinnings of the pathogenicity of Candida species. Here, we generated a cohesive assembly of the C. maltosa genome and developed genetic engineering tools that will facilitate studying this species at a molecular level. We used a combination of short and long-read sequencing to build a polished genomic draft composed of 14 Mbp, 45 contigs and close to 5700 genes. This assembly represents a substantial improvement from the currently available sequences that are composed of thousands of contigs. Genomic comparison with C. albicans and C. tropicalis revealed a substantial reduction in the total number of genes in C. maltosa. However, gene loss seems not to be associated to the avirulence of this species given that most genes that have been previously associated with pathogenicity were also present in C. maltosa. To be able to edit the genome of C. maltosa we generated a set of triple auxotrophic strains so that gene deletions can be performed similarly to what has been routinely done in pathogenic Candida species. As a proof of concept, we generated gene knockouts of EFG1, a gene that encodes a transcription factor that is essential for filamentation and biofilm formation in C. albicans and C. tropicalis. Characterization of these mutants showed that Efg1 also plays a role in biofilm formation and filamentous growth in C. maltosa, but it seems to be a repressor of filamentation in this species. The genome assembly and auxotrophic mutants developed here are a key step forward to start using C. maltosa for comparative and evolutionary studies at a molecular level.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Draft Genome Sequence of a Kazachstania humilis Strain Isolated from Agave Fermentation.

The ascomycetous yeast Kazachstania humilis is an active species in backslopped sourdough and in the spontaneous fermentation of several traditional foods and beverages. Here, we report the draft genome sequence of a K. humilis strain isolated from agave must from a traditional distillery in Mexico.

Evolution of the complex transcription network controlling biofilm formation in Candida species.

We examine how a complex transcription network composed of seven 'master' regulators and hundreds of target genes evolved over a span of approximately 70 million years. The network controls biofilm formation in several Candida species, a group of fungi that are present in humans both as constituents of the microbiota and as opportunistic pathogens. Using a variety of approaches, we observed two major types of changes that have occurred in the biofilm network since the four extant species we examined last shared a common ancestor. Master regulator 'substitutions' occurred over relatively long evolutionary times, resulting in different species having overlapping but different sets of master regulators of biofilm formation. Second, massive changes in the connections between the master regulators and their target genes occurred over much shorter timescales. We believe this analysis is the first detailed, empirical description of how a complex transcription network has evolved.

Corrigendum: Genetic Modification of Closely Related Candida Species.

[This corrects the article DOI: 10.3389/fmicb.2019.00357.].

Genetic Modification of Closely Related Candida Species.

Species from the genus Candida are among the most important human fungal pathogens. Several of them are frequent commensals of the human microbiota but are also able to cause a variety of opportunistic infections, especially when the human host becomes immunocompromised. By far, most of the research to understand the molecular underpinnings of the pathogenesis of these species has focused on Candida albicans, the most virulent member of the genus. However, epidemiological data indicates that related Candida species are also clinically important. Here, we describe the generation of a set of strains and plasmids to genetically modify C. dubliniensis and C. tropicalis, the two pathogenic species most closely related to C. albicans. C. dubliniensis is an ideal model to understand C. albicans pathogenesis since it is the closest species to C. albicans but considerably less virulent. On the other hand, C. tropicalis is ranked among the four most common causes of infections by Candida species. Given that C. dubliniensis and C. tropicalis are obligate diploids with no known conventional sexual cycle, we generated strains that are auxotrophic for at least two amino acids which allows the tandem deletion of both alleles of a gene by complementing the two auxotrophies. The strains were generated in two different genetic backgrounds for each species - one for which the genomic sequence is available and a second clinically important one. In addition, we have adapted plasmids developed to delete genes and epitope/fluorophore tag proteins in C. albicans so that they can be employed in C. tropicalis. The tools generated here allow for efficient genetic modification of C. dubliniensis and C. tropicalis, and thus facilitate the study of the molecular basis of pathogenesis in these medically relevant fungi.

Gene regulatory network plasticity predates a switch in function of a conserved transcription regulator.

The rewiring of gene regulatory networks can generate phenotypic novelty. It remains an open question, however, how the large number of connections needed to form a novel network arise over evolutionary time. Here, we address this question using the network controlled by the fungal transcription regulator Ndt80. This conserved protein has undergone a dramatic switch in function-from an ancestral role regulating sporulation to a derived role regulating biofilm formation. This switch in function corresponded to a large-scale rewiring of the genes regulated by Ndt80. However, we demonstrate that the Ndt80-target gene connections were undergoing extensive rewiring prior to the switch in Ndt80's regulatory function. We propose that extensive drift in the Ndt80 regulon allowed for the exploration of alternative network structures without a loss of ancestral function, thereby facilitating the formation of a network with a new function.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues.

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming.

Systematic Genetic Screen for Transcriptional Regulators of the Candida albicans White-Opaque Switch.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

Finding a Missing Gene: EFG1 Regulates Morphogenesis in Candida tropicalis.

Fungi from the genus Candida are common members of the human microbiota; however, they are also important opportunistic pathogens in immunocompromised hosts. Several morphological transitions have been linked to the ability of these fungi to occupy the different ecological niches in the human body. The transcription factor Efg1 from the APSES family plays a central role in the transcription circuits underlying several of these morphological changes. In Candida albicans, for example, Efg1 is a central regulator of filamentation, biofilm formation, and white-opaque switching, processes associated with survival in the human host. Orthologs of Efg1 are present throughout the Candida clade but, surprisingly, the genome sequence of Candida tropicalis failed to uncover a gene coding for Efg1. One possibility was that the paralog of Efg1, Efh1, had assumed the function of Efg1 in C. tropicalis. However, we show that this gene has only a minor role in the morphological transitions mentioned above. Instead, we report here that C. tropicalis does have an ortholog of the EFG1 gene found in other Candida species. The gene is located in a different genomic position than EFG1 in C. albicans, in a region that contains a gap in the current genome assembly of C. tropicalis. We show that the newly identified C. tropicalis EFG1 gene regulates filamentation, biofilm formation, and white-opaque switching. Our results highlight the conserved role of Efg1 in controlling morphogenesis in Candida species and remind us that published genome sequences are drafts that require continuous curation and careful scrutiny.

Multiple genomic changes associated with reorganization of gene regulation and adaptation in yeast.

Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.

Genome-wide survey of post-meiotic segregation during yeast recombination.

BACKGROUND: When mismatches in heteroduplex DNA formed during meiotic recombination are left unrepaired, post-meiotic segregation of the two mismatched alleles occurs during the ensuing round of mitosis. This gives rise to somatic mosaicism in multicellular organisms and leads to unexpected allelic combinations among progeny. Despite its implications for inheritance, post-meiotic segregation has been studied at only a few loci. RESULTS: By genotyping tens of thousands of genetic markers in yeast segregants and their clonal progeny, we analyzed post-meiotic segregation at a genome-wide scale. We show that post-meiotic segregation occurs in close to 10% of recombination events. Although the overall number of markers affected in a single meiosis is small, the rate of post-meiotic segregation is more than five orders of magnitude larger than the base substitution mutation rate. Post-meiotic segregation took place with equal relative frequency in crossovers and non-crossovers, and usually at the edges of gene conversion tracts. Furthermore, post-meiotic segregation tended to occur in markers that are isolated from other heterozygosities and preferentially at polymorphism types that are relatively uncommon in the yeast species. CONCLUSIONS: Overall, our survey reveals the genome-wide characteristics of post-meiotic segregation. The results show that post-meiotic segregation is widespread in meiotic recombination and could be a significant determinant of allelic inheritance and allele frequencies at the population level.

The baker's yeast diploid genome is remarkably stable in vegetative growth and meiosis.

Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1-2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.

Antagonistic changes in sensitivity to antifungal drugs by mutations of an important ABC transporter gene in a fungal pathogen.

Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding cassette) efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may represent an interesting case of adaptive loss of gene function with significant clinical implications.

Genetic analysis of variation in transcription factor binding in yeast.

Variation in transcriptional regulation is thought to be a major cause of phenotypic diversity. Although widespread differences in gene expression among individuals of a species have been observed, studies to examine the variability of transcription factor binding on a global scale have not been performed, and thus the extent and underlying genetic basis of transcription factor binding diversity is unknown. By mapping differences in transcription factor binding among individuals, here we present the genetic basis of such variation on a genome-wide scale. Whole-genome Ste12-binding profiles were determined using chromatin immunoprecipitation coupled with DNA sequencing in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains and their parental lines. We identified extensive Ste12-binding variation among individuals, and mapped underlying cis- and trans-acting loci responsible for such variation. We showed that most transcription factor binding variation is cis-linked, and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several proposed Ste12 cofactors. We also identified two trans-factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than ten genes under alpha-factor treatment. Neither of these two genes was previously known to regulate Ste12, and we suggest that they may be mediators of gene activity and phenotypic diversity. Ste12 binding strongly correlates with gene expression for more than 200 genes, indicating that binding variation is functional. Many of the variable-bound genes are involved in cell wall organization and biogenesis. Overall, these studies identified genetic regulators of molecular diversity among individuals and provide new insights into mechanisms of gene regulation.

Array-based genotyping in S.cerevisiae using semi-supervised clustering.

MOTIVATION: Microarrays provide an accurate and cost-effective method for genotyping large numbers of individuals at high resolution. The resulting data permit the identification of loci at which genetic variation is associated with quantitative traits, or fine mapping of meiotic recombination, which is a key determinant of genetic diversity among individuals. Several issues inherent to short oligonucleotide arrays -- cross-hybridization, or variability in probe response to target -- have the potential to produce genotyping errors. There is a need for improved statistical methods for array-based genotyping. RESULTS: We developed ssGenotyping (ssG), a multivariate, semi-supervised approach for using microarrays to genotype haploid individuals at thousands of polymorphic sites. Using a meiotic recombination dataset, we show that ssG is more accurate than existing supervised classification methods, and that it produces denser marker coverage. The ssG algorithm is able to fit probe-specific affinity differences and to detect and filter spurious signal, permitting high-confidence genotyping at nucleotide resolution. We also demonstrate that oligonucleotide probe response depends significantly on genomic background, even when the probe's specific target sequence is unchanged. As a result, supervised classifiers trained on reference strains may not generalize well to diverged strains; ssG's semi-supervised approach, on the other hand, adapts automatically.

High-resolution mapping of meiotic crossovers and non-crossovers in yeast.

Meiotic recombination has a central role in the evolution of sexually reproducing organisms. The two recombination outcomes, crossover and non-crossover, increase genetic diversity, but have the potential to homogenize alleles by gene conversion. Whereas crossover rates vary considerably across the genome, non-crossovers and gene conversions have only been identified in a handful of loci. To examine recombination genome wide and at high spatial resolution, we generated maps of crossovers, crossover-associated gene conversion and non-crossover gene conversion using dense genetic marker data collected from all four products of fifty-six yeast (Saccharomyces cerevisiae) meioses. Our maps reveal differences in the distributions of crossovers and non-crossovers, showing more regions where either crossovers or non-crossovers are favoured than expected by chance. Furthermore, we detect evidence for interference between crossovers and non-crossovers, a phenomenon previously only known to occur between crossovers. Up to 1% of the genome of each meiotic product is subject to gene conversion in a single meiosis, with detectable bias towards GC nucleotides. To our knowledge the maps represent the first high-resolution, genome-wide characterization of the multiple outcomes of recombination in any organism. In addition, because non-crossover hotspots create holes of reduced linkage within haplotype blocks, our results stress the need to incorporate non-crossovers into genetic linkage analysis.

Systematic screens for human disease genes, from yeast to human and back.

Systematic screens for human disease genes have emerged in recent years, due to the wealth of information provided by genome sequences and large scale datasets. Here we review how integration of genomic data in yeast and human is helping to elucidate the genetic basis of mitochondrial diseases. The identification of nearly all yeast mitochondrial proteins and many of their functional interactions provides insight into the role of mitochondria in cellular processes. This information enables prioritization of the candidate genes underlying mitochondrial disorders. In an iterative fashion, the link between predicted human candidate genes and their disease phenotypes can be experimentally tested back in yeast.

Generation of variability by in vivo recombination of halves of a (beta/alpha)8 barrel protein.

Similar to what has been achieved with nucleic acids, directed evolution of proteins would be greatly facilitated by the availability of large libraries and efficient selection methods. So far, host cell transformation efficiency has been a bottleneck, practically limiting libraries to sizes less than 10(9). One way to circumvent this problem has been implemented with antibody systems, where contribution to the binding site is provided by two different polypeptides (light and heavy chains). The central concept is the construction of binary systems in which the gene from the two chains are separated by a cre-lox recombinase recognition site, packaged in a phage, and subsequently introduced, by multiple infection, into a recombinase expressing cell [Sblattero D, Bradbury A. Nat Biotechnol 2000;18(1):75-80]. Here, we describe the development of a system which applies the same concept to a single-domain enzyme, the cytoplasmic (beta/alpha)8 barrel protein phosphoribosyl anthranilate isomerase (PRAI) from E. coli. For that purpose, we identified the site at which a loop containing the recognition sequence for cre-lox recombinase could be inserted yielding a functional enzyme. We evaluated the effect of this insertion on the capability of the engineered gene to complement a trp F-E. coli strain and the efficiency of the system to recover the original sequence from an abundance of non-functional mutant genes.