RNA viruses, M satellites, chromosomal killer genes, and killer/nonkiller phenotypes in the 100-genomes S. cerevisiae strains.

We characterized previously identified RNA viruses (L-A, L-BC, 20S, and 23S), L-A-dependent M satellites (M1, M2, M28, and Mlus), and M satellite-dependent killer phenotypes in the Saccharomyces cerevisiae 100-genomes genetic resource population. L-BC was present in all strains, albeit in 2 distinct levels, L-BChi and L-BClo; the L-BC level is associated with the L-BC genotype. L-BChi, L-A, 20S, 23S, M1, M2, and Mlus (M28 was absent) were in fewer strains than the similarly inherited 2µ plasmid. Novel L-A-dependent phenotypes were identified. Ten M+ strains exhibited M satellite-dependent killing (K+) of at least 1 of the naturally M0 and cured M0 derivatives of the 100-genomes strains; in these M0 strains, sensitivities to K1+, K2+, and K28+ strains varied. Finally, to complement our M satellite-encoded killer toxin analysis, we assembled the chromosomal KHS1 and KHR1 killer genes and used naturally M0 and cured M0 derivatives of the 100-genomes strains to assess and characterize the chromosomal killer phenotypes.

A novel narnavirus is widespread in Saccharomyces cerevisiae and impacts multiple host phenotypes.

RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions, and are not known to affect host fitness. In this study, we describe a new S. cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in 1 strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Furthermore, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.

Mitochondrial Genome Variation Affects Multiple Respiration and Nonrespiration Phenotypes in Saccharomyces cerevisiae.

Mitochondrial genome variation and its effects on phenotypes have been widely analyzed in higher eukaryotes but less so in the model eukaryote Saccharomyces cerevisiae Here, we describe mitochondrial genome variation in 96 diverse S. cerevisiae strains and assess associations between mitochondrial genotype and phenotypes as well as nuclear-mitochondrial epistasis. We associate sensitivity to the ATP synthase inhibitor oligomycin with SNPs in the mitochondrially encoded ATP6 gene. We describe the use of iso-nuclear F1 pairs, the mitochondrial genome equivalent of reciprocal hemizygosity analysis, to identify and analyze mitochondrial genotype-dependent phenotypes. Using iso-nuclear F1 pairs, we analyze the oligomycin phenotype-ATP6 association and find extensive nuclear-mitochondrial epistasis. Similarly, in iso-nuclear F1 pairs, we identify many additional mitochondrial genotype-dependent respiration phenotypes, for which there was no association in the 96 strains, and again find extensive nuclear-mitochondrial epistasis that likely contributes to the lack of association in the 96 strains. Finally, in iso-nuclear F1 pairs, we identify novel mitochondrial genotype-dependent nonrespiration phenotypes: resistance to cycloheximide, ketoconazole, and copper. We discuss potential mechanisms and the implications of mitochondrial genotype and of nuclear-mitochondrial epistasis effects on respiratory and nonrespiratory quantitative traits.

Introducing MX Cassettes into Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome can be readily and precisely modified with the use of knock out (KO) marker cassettes to delete genes. The most frequently used family of KO cassettes is the MX cassettes. This protocol describes how to use the different types of MX cassettes by selecting for prototrophy, utilization of cytosine or acetamide as a sole nitrogen source, or resistance to one of six different drugs.

Popping Out MX Cassettes from Saccharomyces cerevisiae.

MX cassettes are frequently used to generate knockout (KO) mutations in Saccharomyces cerevisiae. The recycling or "popping out" of an MX cassette flanked by direct repeats allows the same cassette to be reused in a strain to generate additional KO mutations. Popping out MX cassettes also eliminates MX homology in a strain, which facilitates the subsequent generation of additional KO mutations with other MX cassettes. MX cassettes can be recycled or "popped out" of the genome by spontaneous recombination between large, cassette-borne MX3 or PR direct repeats and by Cre-mediated, site-specific recombination between small, cassette-borne loxP direct repeats. Both of these techniques leave a mutation with a cassette-encoded "scar." For the URA3MX, LYS5MX, FCA1/FCY1MX, and amdSYM cassettes, there are counterselections. Counterselections are extremely useful as they allow for positive selection for plasmid shuffling, transplacement of mutant alleles into the genome, and recycling or popping out cassettes flanked by cassette-encoded direct repeats to yield mutations with a cassette-encoded scar. Finally, after amplifying with the appropriately designed primers, integrated counterselectable MX cassettes can be popped out to generate seamless or "scar-free" deletion mutations, as well as indel and point mutations.

MX Cassettes for Knocking Out Genes in Yeast.

Precise modifications of the Saccharomyces cerevisiae genome use marker cassettes, most often in the form of "knockout" (KO) marker cassettes, to delete genes. Many different KO marker cassettes exist, some of which require strains with specific genotypes, such as auxotrophic mutations, and others that have no strain genotype requirements, such as selections for drug resistance and one of two selections for nitrogen source utilization. This introduction focuses on the most frequently used family of KO cassettes-the MX cassettes. In particular, we focus on and describe the different types of MX cassettes and selections; specifically, selections for prototrophy; selections for utilization of cytosine or acetamide as sole nitrogen sources; and selections for resistance to six different drugs. The use of cassettes to place genes under regulated control is briefly discussed. Also discussed are strain genotype requirements (where applicable); media requirements; how to "recycle" or "pop out" cassettes; and counterselections against specific KO cassettes.

2µ plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

We determined that extrachromosomal 2µ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2µ, we identified three distinct classes of 2µ. We identified 2µ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2µ. Similar to S. cerevisiae, we found no integrated 2µ sequences in any S. paradoxus strains. However, we identified part of 2µ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2µ. We identified extrachromosomal 2µ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2µ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2µ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2µ, consistent with interspecific transfer.

The 100-genomes strains, an S. cerevisiae resource that illuminates its natural phenotypic and genotypic variation and emergence as an opportunistic pathogen.

Saccharomyces cerevisiae, a well-established model for species as diverse as humans and pathogenic fungi, is more recently a model for population and quantitative genetics. S. cerevisiae is found in multiple environments-one of which is the human body-as an opportunistic pathogen. To aid in the understanding of the S. cerevisiae population and quantitative genetics, as well as its emergence as an opportunistic pathogen, we sequenced, de novo assembled, and extensively manually edited and annotated the genomes of 93 S. cerevisiae strains from multiple geographic and environmental origins, including many clinical origin strains. These 93 S. cerevisiae strains, the genomes of which are near-reference quality, together with seven previously sequenced strains, constitute a novel genetic resource, the "100-genomes" strains. Our sequencing coverage, high-quality assemblies, and annotation provide unprecedented opportunities for detailed interrogation of complex genomic loci, examples of which we demonstrate. We found most phenotypic variation to be quantitative and identified population, genotype, and phenotype associations. Importantly, we identified clinical origin associations. For example, we found that an introgressed PDR5 was present exclusively in clinical origin mosaic group strains; that the mosaic group was significantly enriched for clinical origin strains; and that clinical origin strains were much more copper resistant, suggesting that copper resistance contributes to fitness in the human host. The 100-genomes strains are a novel, multipurpose resource to advance the study of S. cerevisiae population genetics, quantitative genetics, and the emergence of an opportunistic pathogen.

Structures of naturally evolved CUP1 tandem arrays in yeast indicate that these arrays are generated by unequal nonhomologous recombination.

An important issue in genome evolution is the mechanism by which tandem duplications are generated from single-copy genes. In the yeast Saccharomyces cerevisiae, most strains contain tandemly duplicated copies of CUP1, a gene that encodes a copper-binding metallothionein. By screening 101 natural isolates of S. cerevisiae, we identified five different types of CUP1-containing repeats, as well as strains that only had one copy of CUP1. A comparison of the DNA sequences of these strains indicates that the CUP1 tandem arrays were generated by unequal nonhomologous recombination events from strains that had one CUP1 gene.

Nature and distribution of large sequence polymorphisms in Saccharomyces cerevisiae.

To obtain a better understanding of the genome-wide distribution and the nature of large sequence polymorphisms (LSPs) in Saccharomyces cerevisiae, we hybridized genomic DNA of 88 haploid or homozygous diploid S. cerevisiae strains of diverse geographic origins and source substrates onto high-density tiling arrays. On the basis of loss of hybridization, we identified 384 LSPs larger than 500 bp that were located in 188 non-overlapping regions of the genome. Validation by polymerase chain reaction-amplification and/or DNA sequencing revealed that 39 LSPs were due to deletions, whereas 74 LSPs involved sequences diverged far enough from the S288c reference genome sequence as to prevent hybridization to the microarray features. The LSP locations were biased toward the subtelomeric regions of chromosomes, where high genetic variation in genes involved in transport or fermentation is thought to facilitate rapid adaptation of S. cerevisiae to new environments. The diverged LSP sequences appear to have different allelic ancestries and were in many cases identified as Saccharomyces paradoxus introgressions.

Genomic structure of and genome-wide recombination in the Saccharomyces cerevisiae S288C progenitor isolate EM93.

The diploid isolate EM93 is the main ancestor to the widely used Saccharomyces cerevisiae haploid laboratory strain, S288C. In this study, we generate a high-resolution overview of the genetic differences between EM93 and S288C. We show that EM93 is heterozygous for >45,000 polymorphisms, including large sequence polymorphisms, such as deletions and a Saccharomyces paradoxus introgression. We also find that many large sequence polymorphisms (LSPs) are associated with Ty-elements and sub-telomeric regions. We identified 2,965 genetic markers, which we then used to genotype 120 EM93 tetrads. In addition to deducing the structures of all EM93 chromosomes, we estimate that the average EM93 meiosis produces 144 detectable recombination events, consisting of 87 crossover and 31 non-crossover gene conversion events. Of the 50 polymorphisms showing the highest levels of non-crossover gene conversions, only three deviated from parity, all of which were near heterozygous LSPs. We find that non-telomeric heterozygous LSPs significantly reduce meiotic recombination in adjacent intervals, while sub-telomeric LSPs have no discernable effect on recombination. We identified 203 recombination hotspots, relatively few of which are hot for both non-crossover gene conversions and crossovers. Strikingly, we find that recombination hotspots show limited conservation. Some novel hotspots are found adjacent to heterozygous LSPs that eliminate other hotspots, suggesting that hotspots may appear and disappear relatively rapidly.

Homoserine toxicity in Saccharomyces cerevisiae and Candida albicans homoserine kinase (thr1Delta) mutants.

In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding homoserine kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, homoserine is also toxic for Candida albicans thr1Delta mutants. Since increasing levels of threonine, but not other amino acids, overcome the homoserine toxicity of thr1Delta mutants, homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Delta mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Delta mutant homoserine toxicity. Since the doa4Delta and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to homoserine toxicity.

Fungal homoserine kinase (thr1Delta) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation.

The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae homoserine kinase (thr1Delta) and threonine synthase (thr4Delta) mutants. In striking contrast to aspartate kinase (hom3Delta) mutants, S. cerevisiae thr1Delta and thr4Delta mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Delta mutants, but not hom3Delta mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Delta and thr4Delta mutants as well as C. albicans thr1Delta mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Delta mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.

Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production.

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Microsatellite analysis of genetic diversity among clinical and nonclinical Saccharomyces cerevisiae isolates suggests heterozygote advantage in clinical environments.

The genetic structure of a global sample of 170 clinical and nonclinical Saccharomyces cerevisiae isolates was analysed using 12 microsatellite markers. High levels of genetic diversity were revealed both among the clinical and among the nonclinical S. cerevisiae isolates without significant differentiation between these two groups of isolates, rendering a single origin of pathogenic isolates unlikely. This suggests that S. cerevisiae is a true opportunistic pathogen, with a diversity of unrelated genetic backgrounds able to cause infections in humans, and that the ability of S. cerevisiae isolates to cause infections is likely due to a combination of their phenotypic plasticity and the immune system status of the exposed individuals. As was previously reported for bread, beer and wine strains and for environmental S. cerevisiae isolates, the microsatellite genotypes indicated ploidy level variation, from possibly haploid up to tetraploid, among clinical S. cerevisiae isolates. However, rather than haploid, sporulation proficiency and spore viability data indicated that most S. cerevisiae isolates that were mono-allelic at all examined microsatellite loci were likely homothallic and self-diploidized. Interestingly, the proportion of heterozygous clinical isolates was found to be significantly higher than the proportion of heterozygous nonclinical isolates, suggesting a selective advantage of heterozygous S. cerevisiae yeasts in clinical environments.

A multispecies-based taxonomic microarray reveals interspecies hybridization and introgression in Saccharomyces cerevisiae.

A multispecies-based taxonomic microarray targeting coding sequences of diverged orthologous genes in Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces kudriavzevii, Naumovia castellii, Lachancea kluyveri and Candida glabrata was designed to allow identification of isolates of these species and their interspecies hybrids. Analysis of isolates of several Saccharomyces species and interspecies hybrids demonstrated the ability of the microarray to differentiate these yeasts on the basis of their specific hybridization patterns. Subsequent analysis of 183 supposed S. cerevisiae isolates of various ecological and geographical backgrounds revealed one misclassified S. bayanus or Saccharomyces uvarum isolate and four aneuploid interspecies hybrids, one between S. cerevisiae and S. bayanus and three between S. cerevisiae and S. kudriavzevii. Furthermore, this microarray design allowed the detection of multiple introgressed S. paradoxus DNA fragments in the genomes of three different S. cerevisiae isolates. These results show the power of multispecies-based microarrays as taxonomic tools for the identification of species and interspecies hybrids, and their ability to provide a more detailed characterization of interspecies hybrids and recombinants.

Sequential elimination of major-effect contributors identifies additional quantitative trait loci conditioning high-temperature growth in yeast.

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

Threonine biosynthetic genes are essential in Cryptococcus neoformans.

We identified and attempted to disrupt the Cryptococcus neoformans homoserine and/or threonine biosynthetic genes encoding aspartate kinase (HOM3), homoserine kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.

Genome sequencing and comparative analysis of Saccharomyces cerevisiae strain YJM789.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the approximately 12-Mb genome of YJM789 contains approximately 60,000 SNPs and approximately 6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.