Saccharomyces Cerevisiae S288c Has a Mutation in Flo8, a Gene Required for Filamentous Growth

Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae. This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen. Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen. Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene. This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation. Since feral strains of S. cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.     

Deciphering the hybridisation history leading to the Lager lineage based on the mosaic genomes of Saccharomyces bayanus strains NBRC1948 and CBS380

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380(T) and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380(T) harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380(T) and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380(T) or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Isolation and characterization of the HO gene from the yeast Saccharomyces paradoxus

A DNA fragment homologous to the homothallism (HO) gene of Saccharomyces cerevisiae was isolated from Saccharomyces paradoxus and was found to contain an open reading frame that was 90.9% identical to the coding sequence of the S. cerevisiae HO gene. The putative HO gene was shown to induce diploidization in a heterothallic haploid strain from S. cerevisiae. Phylogenetic analysis revealed that the coding and 5'-upstream regulatory regions from five Saccharomyces sensu stricto HO genes have coevolved, and that S. paradoxus is phylogenetically closer to S. cerevisiae than to S. bayanus. Finally, heterothallic haploid strains were isolated from the original homothallic type strain of S. paradoxus by disrupting the S. paradoxus HO gene with the S. cerevisiae URA3 gene.     

Chromosomal rearrangements during vegetative growth of a wild strain of Saccharomyces cerevisiae.

Electrophoretic karyotypes of two strains of Saccharomyces cerevisiae, a haploid laboratory strain and a wild strain known to be at least diploid, have been checked during vegetative growth. The karyotype of the haploid strain was very stable; however, the diploid strain underwent frequent modifications. In most cases the number of bands was reduced, but occasionally we observed one band splitting into two. In one case, chromosomal rearrangements took place between differently sized copies of chromosomes I and VI. We concluded that the chromosome length polymorphism observed among wild strains of S. cerevisiae could be explained partly by chromosomal structure reorganization occurring during mitosis.     

Comparative proteomics of industrial lager yeast reveals differential expression of the cerevisiae and non-cerevisiae parts of their genomes

The proteomes of three industrial lager beer strains, CMBS33, OG2252 and A15, were analysed under standardised laboratory growth conditions. Protein spots in the 2-DE pattern of the lager strains were subjected to MS/MS to identify protein variants. We found the protein composition of the three lager strains to be qualitatively rather similar, while being substantially different from the Saccharomyces cerevisiae strain BY4742. Database searches using several fully sequenced genomes from the Saccharomyces genera indicated that the non-cerevisiae proteins in the 2-D pattern of lager strains were most closely related to S. bayanus. For many proteins the regulation of the bayanus-like protein and its cerevisiae counterpart varied in a strain-dependent manner, e.g. the bayanus-like form of Tdh3p was roughly eight-fold more abundant than the cerevisiae form in the OG2252 strain. We also found differential regulation of cerevisiae- and bayanus-like proteins during various stress conditions like low temperature growth, and adaptation to high temperatures or high salinity, e.g. for Arg1p, Sti1p and Pdc1p. Our data on the differential regulation of the two genomes in these hybrid strains may have important industrial implications for strain improvement and strain protection.     

Genetic structure and genealogy in the Sphagnum subsecundum complex (Sphagnaceae: Bryophyta)

Allopolyploidy is probably the most extensively studied mode of plant speciation and allopolyploid species appear to be common in the mosses (Bryophyta). The Sphagnum subsecundum complex includes species known to be gametophytically haploid or diploid, and it has been proposed that the diploids (i.e., with tetraploid sporophytes) are allopolyploids. Nucleotide sequence and microsatellite variation among haploids and diploids from Newfoundland and Scandinavia indicate that (1) the diploids exhibit fixed or nearly fixed heterozygosity at the majority of loci sampled, and are clearly allopolyploids, (2) diploids originated independently in North America and Europe, (3) the European diploids appear to have the haploid species, S. subsecundum, as the maternal parent based on shared chloroplast DNA haplotypes, (4) the North American diploids do not have the chloroplast DNA of any sampled haploid, (5) both North American and European diploids share nucleotide and microsatellite similarities with S. subsecundum, (6) the diploids harbor more nucleotide and microsatellite diversity than the haploids, and (7) diploids exhibit higher levels of linkage disequilibrium among microsatellite loci. An experiment demonstrates significant artifactual recombination between interspecific DNAs coamplified by PCR, which may be a complicating factor in the interpretation of sequence-based analyses of allopolyploids.     

Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae)

The yeast, Saccharomyces cerevisiae, was used as a model to investigate theories of ploidy evolution. Mutagenesis experiments using the alkylating agent EMS (ethane methyl sulphonate) were conducted to assess the relative importance that masking of deleterious mutations has on response to and recovery from DNA damage. In particular, we tested whether cells with higher ploidy levels have relatively higher fitnesses after mutagenesis, whether the advantages of masking are more pronounced in tetraploids than in diploids, and whether purging of mutations allows more rapid recovery of haploid cells than cells with higher ploidy levels. Separate experiments were performed on asexually propagating stationary phase cells using (1) prototrophic haploid (MAT alpha) and diploid (MATa/alpha) strains and (2) isogenic haploid, diploid and tetraploid strains lacking a functional mating type locus. In both sets of experiments, haploids showed a more pronounced decrease in apparent growth rate than diploids, but both haploids and diploids appeared to recover very rapidly. Tetraploids did not show increased benefits of masking compared with diploids but volume measurements and FACScan analyses on the auxotrophic strains indicated that all treated tetraploid strains decreased in ploidy level and that some of the treated haploid lines increased in ploidy level. Results from these experiments confirm that while masking deleterious mutations provides an immediate advantage to higher ploidy levels in the presence of mutagens, selection is extremely efficient at removing induced mutations, leading growth rates to increase rapidly over time at all ploidy levels. Furthermore, ploidy level is itself a mutable trait in the presence of EMS, with both haploids and tetraploids often evolving towards diploidy (the ancestral state of S. cerevisiae) during the course of the experiment.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

Generation of a large set of genetically tractable haploid and diploid Saccharomyces strains

Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics studies. Despite several hundreds of isolates already available, the scientific community relies on the use of only a handful of unrelated strains. The lack of sequence information, haploid derivatives and genetic markers has prevented novel strains from being used. Here, we release a set of 55 S. cerevisiae and Saccharomyces paradoxus genetically tractable strains, previously sequenced in the Saccharomyces Genome Resequencing Project. These strains are stable haploid derivatives and ura3 auxotrophs tagged with a 6-bp barcode, recognized by a restriction enzyme to allow easy identification. We show that the specific barcode can be used to accurately measure the prevalence of different strains during competition experiments. These strains are now amenable to a wide variety of genetic experiments and can be easily crossed with each other to create hybrids and segregants, providing a valuable resource for breeding programmes and quantitative genetic studies. Three versions of each strain (haploid Mat a and Mat α and diploid Mat a /α all as ura3 ∷ KanMX-Barcode ) are available through the National Culture Yeast Collection.     

The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S.?cerevisiae and non-S.?cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S.?cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S.?cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S.?cerevisiae and S.?kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation.     

The inheritance of mtDNA in lager brewing strains

In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus.     

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.     

Mutants of Saccharomyces cerevisiae and Candida utilis with increased susceptibility to digestive enzymes.

Mutants of Candida utilis and a haploid strain of Saccharomyces cerevisiae were isolated, after ultraviolet light mutagenesis, which had increased sensitivities to snail gut enzymes (ses). Three of the five S. cerevisiae mutants tested had increased sensitivities to porcine pepsin, all were more susceptible to a sequential treatment with pepsin, lipase, peptidase, and trypsin, four were sensitive to osmotic shock, and two had increased glucan/mannan ratios in their cell walls. All combinations of mutants showed positive complementation in heterozygous diploids, although complementation between one pair, which had the same phenotype, was incomplete, indicating that four to five different cistrons were involved. All mutations were found to be recessive. Haploid strains bearing pairs of ses mutations were not markedly more sensitive to mammalian digestive enzymes than strains with single mutations. Rat-feeding experiments with three mutants and the parental strains indicated that the protein was efficiently utilized in all cases. Net protein ratios for the two mutants of S. cerevisiae tested were slightly higher than that for their parent, but the differences were of marginal significance.     

Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species

Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.     

Schistosoma mansoni: the IMP4 gene is involved in DNA repair/tolerance after treatment with alkylating agent methyl methane sulfonate

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.     

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

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.