A Structural and Phylogenetic Study of the HO Gene from Saccharomyces bayanus var. uvarum

A novel HO gene (Uv-HO) was cloned from the Saccharomyces bayanus var. uvarum (abbreviated as S. uvarum in this study) type strain. The coding region of Uv-HO showed relatively high homology (95%) to that of the Sb-HO gene (S. bayanus var. bayanus HO), but not to the HO genes of other Saccharomyces sensu stricto species. However, the 5′ and 3′ non-coding region of Uv-HO showed less similarity (79% and 76% respectively) even to those of the most homologous gene Sb-HO. Motifs of the mating-type control and the cell-cycle control were conserved in the 5′ non-coding region of Uv-HO, but numbers and positions of motifs were different from those of Sb-HO. CHEF-Southern analysis showed that all tested strains of S. bayanus species, including S. uvarum, carried the HO gene on the 1,100-kb chromosome. By HO-typing PCR using mixed primers, which provided a rapid and convenient tool for yeast identification, either the Uv-HO gene or the Sb-HO gene was detected in strains of S. bayanus species, but two strains were found to have both types of HO gene in each genome. These results suggest that S. uvarum has a unique sequence, but might share the same chromosome constitution within S. bayanus species, and that S. bayanus is a heterogeneous species, of which some strains might be natural hybrid.     

Analysis of temperature‐mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D‐DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC–MS was performed. The proteomic and “aromatomic” results are discussed in relation to the oenological properties of S. bayanus var. uvarum.     

Mitochondrial DNA polymorphism of the yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RPLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S. cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

On the Complexity of the Saccharomyces bayanus Taxon: Hybridization and Potential Hybrid Speciation

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99–100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus. Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Molecular Analysis of the α-Galactosidase MEL Genes in Yeast Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the -galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1–99.3%), comparable with interspecific homology (94.8–100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae(MELj) was 76.2–81.7%, suggesting certain species specificity. On this evidence, the -galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum

Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain SCPP (CLIB 2025) of S. bayanus var. uvarum is known as a producer of three pectinolytic enzymes. Received: 28 April 2000 / Received revision: 20 July 2000 / Accepted: 25 July 2000     

Some remarks on “a taxonomic key for the genus Saccharomyces” (Vaughan Martini and Martini 1993)

The practicability of Vaughan Martini and Martini's Taxonomic Key for the Genus Saccharomyces for the separation of S. bayanus from other Saccharomyces sensu stricto species was studied. It was concluded that the ability to grow in vitamin free medium was not a suitable character for this purpose. A new wild S. bayanus strain, isolated from exudate of Carpinus betulus was also included in this study. This appears to be the third documented strain of that species isolated outside of an artificial fermentation environment.     

Three newly delimited species of Saccharomyces sensu stricto

Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species. S. cerevisiae and S. bayanus strains had only 22% of their genomes in common. S. pastorianus, with intermediate hybridization values between S. cerevisiae and S. bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species. S. pastorianus replaces S. carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904. These data do not agree with the results of traditional physiological tests.     

Genetic identification of naturalSaccharomyces sensu stricto yeasts from Finland,Holland and Slovakia

Genetic and karyotypic studies of naturalSaccharomyces sensu stricto yeasts from Finland, Holland and Slovakia revealed three wild sibling-species:Saccharomyces cerevisiae, Saccaromyces bayanus andSaccharomyces paradoxus.     

Application of a substrate inhibition model to estimate the effect of fructose concentration on the growth of diverse <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis> strains

In this study, we performed an analysis of the ability of four Saccharomyces cerevisiae and one S. bayanus var. uvarum strains, isolated from different industrial processes, to ferment increasing amounts of fructose (from 0 to 70%, w/v). Overall yeast growth was estimated by integration of the area under optical density vs. time curves. Subsequently, this parameter was modeled by means of a substrate inhibition model. All strains showed a similar behavior against fructose concentration in spite of their different origins, but with slight differences. The optimum fructose concentrations to stimulate yeast growth were obtained between 4.33 and 6.05%, while the maximum concentrations above which growth was completely inhibited were attained between 59.56 and 63.85%. Statistically, model parameters calculated for wine yeast strains were significantly different than those obtained for yeasts from Agave and table olive fermentations, except for the maximum inhibitory concentration. The methodology used in this work could be useful for the industry in general as a first procedure to select yeast strains with higher fructose preferences or tolerances, and especially for winemakers, where the risk of spoilage increases by the presence of a marked residual fructose concentration in the finished wine.     

Evolution of C6, C8 and C10 acids and their ethyl esters in cells and musts during fermentation with threeSaccharomyces cerevisiae races

Summary The evolution of the cell and must contents of three short-chain fatty acids (C₆, C₈ and C₁₀) and their ethyl esters during fermentations withSaccharomyces cerevisiae racescerevisiae, bayanus andcapensis were studied. The former is a fermentative yeast and the last two are flor film yeasts. The acid concentrations in the musts increased throughout the alcoholic fermentations, and maximum cell concentrations of the fatty acids were reached after 48 h of fermentation. Maximum ester concentrations in the cells were attained after 48–72 h of fermentation. In the musts, ethyl octanoate and ethyl decanoate reached a peak also at this point, and ethyl hexanoate after 10 days. After 134 days,S. cerevisiae racecapensis formed a thick flor film whileS. cerevisiae racebayanus developed a thin film andS. cerevisiae racecerevisiae formed no film. At this point, acid contents remained constant in the wines produced byS. cerevisiae racescerevisiae andbayanus, and decreased in those obtained with racecapensis. The ethyl ester contents tended to decrease with the exception of ethyl decanoate in the fermentations carried out byS. cerevisiae racescerevisiae andbayanus.     

Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA ofSaccharomyces

Polymorphism in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains ofSaccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding toS. bayanus andS. cerevisiae. The type culture ofS. carlsbergensis (ATCC 76529), which could not be separated from theS. cerevisiae group by small subunit (SSU) rDNA patterns, showed aScrfI profile that was distinct from all the other strains. The type culture ofs. intermedius var.turicensis (ATCC 76519) is assigned toS. bayanus on the basis of the combined results of SSU and ITS restriction analyses.S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.     

Genetic relationship and biological status of the industrially important yeast <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.     

Enzyme and colour variation in the hoplonemertean Tetrastemma nigrifrons from the Sea of Japan

Five sympatric colour varieties of the hoplonemertean Tetrastemma nigrifrons (var. pallidum, var. bicolor, var. purpureum, var. punctata, var. albino) were found in Vostok Bay (the Sea of Japan). The taxonomic status of the varieties, some of which have been known for almost a century, was uncertain. Starch gel electrophoresis was used to compare allele frequencies of these varieties at 19 isozyme loci. Neither fixed allelic differences nor statistically significant differences in allele frequencies between different colour varieties was revealed. It was concluded therefore that all the colour varieties studied are conspecific. As estimated from the electrophoretic screening of 24 isozyme loci, the species is highly variable at the molecular level. It has high mean values of observed and expected heterozygosity estimates, H _o =0.313 ± 0.049 and H _e =0.323 ± 0.050, and is one of the most variable species among invertebrates.     

A genetically isolated population of Saccharomyces cerevisiae in Malaysia

A divergent population of Saccharomyces cerevisiae has been identified in Malaysia by molecular and genetic analysis. It has also demonstrated that the yeast S. bayanus may be found in South America. The origin of S. cerevisiae is discussed.     

Comparison of ecophysiological responses to heavy snow in two varieties ofAucuba japonica with different areas of distribution

Aucuba japonica varieties are common evergreen understory shrubs in Japan.Aucuba japonica var.borealis is distributed on the Sea of Japan side of Honshu and Hokkaido where heavy snow cover lasts for more than 3 months in winter.Aucuba japonica var.japonica is distributed in areas with shallow or no snow on the Pacific Ocean side of Honshu and Shikoku. The ecophysiological characteristics of var.borealis were compared with those of var.japonica to examine the effects of heavy and long-term snow cover on the life cycle of var.borealis. Shoots of both varieties were shaded in crushed ice for 110 days, but their photosynthetic activities, chlorophyll contents and the chlorophylla/b ratio was not affected. The leaves of var.borealis were no less frost tolerant than those of var.japonica. In spite of the difference in environmental factors, both varieties had similar characteristics in seasonal changes of photosynthesis, respiration and chlorophylla/b ratio. These results suggest that var.japonica could survive in areas with heavy snow where it does not normally occur. Leaf net production (LNP) was estimated based on the microclimatic data and seasonal photosynthetic and respiration rates. The difference in the annual LNP between the two varieties was equivalent to the difference in the LNP during the snow season. One of the major effects of snow cover is to interrupt and reduce the production period of var.borealis.     

Sulfite formation by wine yeasts

Four strains of wine yeasts of two different species (Saccharomyces cerevisiae var. ellipsoideus and S. bayanus) were investigated with respect to regulation of NADPH- and benzyl viologen-dependent sulfite reductases by various sulfur sources. The enzyme activity was followed over a growth period of 96 h. The low sulfite-producing strains showed an increased biosynthesis of NADPH-dependent sulfite reductase during the exponential growth phase in the presence of sulfate, sulfite and djencolic-acid. This increase was not observed in the high sulfite-producing strains. Methionine and cysteine prevented this derepression. At the end of the exponential growth phase, enzyme biosynthesis was repressed again, presumably by sulfur-containing amino acids which were produced during growth. The regulatory influence of the various sulfur sources on benzyl viologen dependent sulfitereductase activity is obviously much weaker.Abbreviation BV benzyl viologen     

Cloning and Nucleotide Sequence of the Alcohol Acetyltransferase II Gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery lager yeast have one ATF2 gene, while S. bayanus has no similar gene.     

Taxonomy,ecology, and genetics of the yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis>: A new object for science and practice

The review considers various aspects of the biology of the yeast Saccharomyces bayanus, which is distantly related to the cultured yeast S. cerevisiae. The cryotolerant S. bayanus strains found in wine-making became the second most important yeast for basic and applied studies. Introduction of natural and experimental hybrids of S. cerevisiae × S. bayanus in a range of fermentation processes indicates the high breeding importance of S. bayanus. The biological species S. bayanus acts as a new gene pool for the scientific and breeding projects.