Search

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

In the present article we describe the fermentation characteristics of some novel, natural yeast hybrids (S. cerevisiaexS. kudriavzevii), isolated from Austrian vineyards, and their significance for the aroma spectra of wines they produced. S. cerevisiae, S. bayanus var. uvarum and S. kudriavzevii were used for comparison. Fermentation took place at four different temperatures (14 degrees C, 18 degrees C, 22 degrees C and 26 degrees C) in two grape must varieties, Blauburger and Muskat Ottonell. The fermentation performed by the hybrids occurred more harmoniously than that carried out by the reference yeasts. At any temperature the fermentation rate was in the upper range, especially that of fructose fermentation. Furthermore, the production of ethanol was remarkable. The aroma compositions of wines created by hybrids resemble those of the parental species, but certain aroma constituents (depending on the must) are significantly more concentrated in the hybrid-produced wines. These novel criteria may be advantageous for wine making.

beta-d-Fructofuranosidase (FFase), an important enzyme of the confectionery and fructose syrup industry, is produced by several microorganisms. However, yeasts are the most used source because of their high sucrose fermentation capacity. In this work, production of FFase was carried out in submerged fermentation using a high enzyme-producing yeast strain. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effects of 10 nitrogen sources were studied in a 16-run experimental design. Beef extract, yeast extract, N-Z-amine, tryptone, meat extract, and ammonium acetate were found to have significant effects on enzyme production. Among these, yeast extract, N-Z-amine, and ammonium acetate were the most significant. A maximum FFase activity of 299.4 U/mL was obtained after a 24 h fermentation period.

The Saccharomyces sensu stricto genus contains many species that are industrially important for fermentation of wines, beers and ales. The molecular characterization of the genomes of yeasts involved in these processes reveals that the majority arose from interspecific hybridization between two and sometimes three yeast species. The hybridization events generated allopolyploid genomes, and subsequent recombination events between the parental genomes resulted in the formation of mosaic chromosomes. The polyploid and hybrid nature of the genomes confers robust characteristics such as tolerance to environmental stress to these industrial yeasts and provides a means for adaptive evolution.

Since the completion of the genome sequence of Saccharomyces cerevisiae in 1996 (refs 1, 2), there has been a large increase in complete genome sequences, accompanied by great advances in our understanding of genome evolution. Although little is known about the natural and life histories of yeasts in the wild, there are an increasing number of studies looking at ecological and geographic distributions, population structure and sexual versus asexual reproduction. Less well understood at the whole genome level are the evolutionary processes acting within populations and species that lead to adaptation to different environments, phenotypic differences and reproductive isolation. Here we present one- to fourfold or more coverage of the genome sequences of over seventy isolates of the baker's yeast S. cerevisiae and its closest relative, Saccharomyces paradoxus. We examine variation in gene content, single nucleotide polymorphisms, nucleotide insertions and deletions, copy numbers and transposable elements. We find that phenotypic variation broadly correlates with global genome-wide phylogenetic relationships. S. paradoxus populations are well delineated along geographic boundaries, whereas the variation among worldwide S. cerevisiae isolates shows less differentiation and is comparable to a single S. paradoxus population. Rather than one or two domestication events leading to the extant baker's yeasts, the population structure of S. cerevisiae consists of a few well-defined, geographically isolated lineages and many different mosaics of these lineages, supporting the idea that human influence provided the opportunity for cross-breeding and production of new combinations of pre-existing variations.

As hamster scrapie cannot infect mice, due to sequence differences in their PrP proteins, we find "species barriers" to transmission of the [URE3] prion in Saccharomyces cerevisiae among Ure2 proteins of S. cerevisiae, paradoxus, bayanus, cariocanus, and mikatae on the basis of differences among their Ure2p prion domain sequences. The rapid variation of the N-terminal Ure2p prion domains results in protection against the detrimental effects of infection by a prion, just as the PrP residue 129 Met/Val polymorphism may have arisen to protect humans from the effects of cannibalism. Just as spread of bovine spongiform encephalopathy prion variant is less impaired by species barriers than is sheep scrapie, we find that some [URE3] prion variants are infectious to another yeast species while other variants (with the identical amino acid sequence) are not. The species barrier is thus prion variant dependent as in mammals. [URE3] prion variant characteristics are maintained even on passage through the Ure2p of another species. Ure2p of Saccharomyces castelli has an N-terminal Q/N-rich "prion domain" but does not form prions (in S. cerevisiae) and is not infected with [URE3] from Ure2p of other Saccharomyces. This implies that conservation of its prion domain is not for the purpose of forming prions. Indeed the Ure2p prion domain has been shown to be important, though not essential, for the nitrogen catabolism regulatory role of the protein.

The bottom fermenting yeast Saccharomyces pastorianus is reported to have arisen as a natural hybrid of two yeast strains, S. cerevisiae and S. bayanus. The S. pastorianus genome includes S. cerevisiae-type (Sc-type) genes and orthologous lager-fermenting-yeast specific-type (Lg-type) genes derived from S. cerevisiae and S. bayanus, respectively. To gain insights into the physiological properties of S. pastorianus, we developed an in situ synthesized 60-mer oligonucleotide microarray for gene expression monitoring of these orthologous genes, consisting of approximately 6600 Sc-type genes and 3200 Lg-type genes. A comparison of the transcriptional profile of orthologous genes (e.g. Sc-type and Lg-type genes) in S. cerevisiae or S. bayanus demonstrated the feasibility of performing gene expression studies with this microarray. Genome-wide analysis of S. pastorianus with this microarray could clearly distinguish more than 67% of the expressed orthologous genes. Furthermore, it was shown that the gene expression of particular Lg-type genes differed from that of the orthologous Sc-type genes, suggesting that some Lg-type and Sc-type genes may have different functional roles. We conclude that the oligonucleotide microarray that we constructed is a powerful tool for the monitoring of gene expression of the orthologous genes of S. pastorianus.

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and beta-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the beta-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that beta-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the beta-tubulin gene.

Yeasts used in the production of lagers belong to the genus Saccharomyces pastorianus. Species within this genus arose from a natural hybridization event between two yeast species that appear to be closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. The resultant hybrids contain complex allopolyploid genomes and retain genetic characteristics of both parental species. Recent genome analysis using both whole genome sequencing and competitive genomic hybridization techniques has revealed the underlying composition of lager yeasts genomes. There appear to be at least 36 unique chromosomes, many of which are lager specific, resulting from recombination events between the homeologous parental chromosomes. The recombination events are limited to a defined set of genetic loci, which are highly conserved within strains of lager yeasts. In addition to the hybrid chromosomes, several non-reciprocal chromosomal translocations and inversions are also observed. Remarkably, in response to exposure to environmental stresses such as high temperatures and high osmotic pressure, the genomes appear to be highly dynamic and undergo recombination events at defined loci and alterations in the telomeric regions. The ability of environmental stress to alter the structure and composition of the genomes of lager yeasts may point to mechanisms of adaptive evolution in these species.

In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism (RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.

The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.

The essential protein Cdc13p binds the single-stranded telomeric 3' overhangs in Saccharomyces cerevisiae and takes part in the regulation of telomere length. The DNA-binding domain (DBD) of Cdc13p is structurally established by an oligonucleotide/oligosaccharide-binding (OB)-fold domain. The sequence homolog in Saccharomyces castellii (scasCDC13) was characterized previously, and the full-length protein was found to bind telomeric DNA specifically. Here, the DBD of scasCdc13p was defined to the central part (402-658) of the protein. The region necessary for forming the scasCdc13p-DBD is larger than the minimal DBD of S. cerevisiae Cdc13p. Deletion of this extended DBD region from the full-length protein completely abolished the DNA binding, indicating the importance of the extended region for the correct formation of a binding-competent DBD. The scasCdc13p-DBD bound the same 8-mer minimal binding site as the full-length protein, but an extension of the target site in the 3' end increased the stability of the DNA-protein complex. Significantly, scasCdc13p-DBD showed a retained high sequence specific binding, where the four nucleotides of most importance for the sequence specificity are highly conserved in eukaryotic telomeric repeats. Thus, the unique single-stranded DNA-binding properties of the full-length protein are entirely retained within the isolated scasCdc13p-DBD.

The aim of the present study was to evaluate the autochthonous yeast population during spontaneous fermentations of grape musts in Austrian wine-producing areas. Investigation of genomic and genetic variations among wine yeasts was a first step towards a long-term goal of selecting strains with valuable enological properties typical for this geographical region. An approach, combining sequences of the D1/D2 domain of the 26S rRNA gene and random amplified polymorphic DNA fingerprinting, was used to characterize yeasts at the species level, whereas the differentiation of Saccharomyces strains was accomplished by amplified fragment length polymorphism fingerprinting. At the beginning of fermentation, representatives of nine genera were identified, with Hanseniaspora and Metschnikowia species characterized most frequently. Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum strains, which were identified throughout the entire fermentation process, showed a high level of genetic diversity. A number of S. cerevisiae strains were common at multiple wineries, but a wide range of strains with characteristic profiles were characterized at individual locations. This biodiversity survey represents a contribution to the investigation and preservation of genetic diversity of biotechnologically relevant yeasts in Austrian wine-making areas.

The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto complex are a group of closely related species that are isolated by a postzygotic barrier. They can easily hybridize; and their allodiploid hybrids propagate by mitotic divisions as efficiently as the parental strains, but can barely divide by meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The postzygotic isolation is not effective in allotetraploids that are able to carry out meiosis and produce viable spores (fertile interspecies hybrids). By application of molecular identification methods, double (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces kudriavzevii) and triple (S. cerevisiae x S. uvarum x S. kudriavzevii) hybrids were recently identified in yeast populations of fermenting grape must and cider in geographically distinct regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed great variability of hybrid genome structures and demonstrated that the alloploid genome of the zygote can undergo drastic changes during mitotic and meiotic divisions of the hybrid cells. This genome-stabilization process involves loss of chromosomes and genes and recombination between the partner genomes. This article briefly reviews the results of the analysis of interspecies hybrids, proposes a model for the mechanism of genome stabilization and highlights the potential of interspecies hybridization in winemaking.

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.

Inter-specific hybridization leading to abrupt speciation is a well-known, common mechanism in angiosperm evolution; only recently, however, have similar hybridization and speciation mechanisms been documented to occur frequently among the closely related group of sensu stricto Saccharomyces yeasts. The economically important lager beer yeast Saccharomyces pastorianus is such a hybrid, formed by the union of Saccharomyces cerevisiae and Saccharomyces bayanus-related yeasts; efforts to understand its complex genome, searching for both biological and brewing-related insights, have been underway since its hybrid nature was first discovered. It had been generally thought that a single hybridization event resulted in a unique S. pastorianus species, but it has been recently postulated that there have been two or more hybridization events. Here, we show that there may have been two independent origins of S. pastorianus strains, and that each independent group--defined by characteristic genome rearrangements, copy number variations, ploidy differences, and DNA sequence polymorphisms--is correlated with specific breweries and/or geographic locations. Finally, by reconstructing common ancestral genomes via array-CGH data analysis and by comparing representative DNA sequences of the S. pastorianus strains with those of many different S. cerevisiae isolates, we have determined that the most likely S. cerevisiae ancestral parent for each of the independent S. pastorianus groups was an ale yeast, with different, but closely related ale strains contributing to each group's parentage.

Baker's yeast, Saccharomyces cerevisiae, is not only an extensively used model system in genetics and molecular biology, it is an upcoming model for research in ecology and evolution. The available body of knowledge and molecular techniques make yeast ideal for work in areas such as evolutionary and ecological genomics, population genetics, microbial biogeography, community ecology and speciation. As long as ecological information remains scarce for this species, the vast amount of data that is being generated using S. cerevisiae as a model system will remain difficult to interpret in an evolutionary context. Here we review the current knowledge of the evolution and ecology of S. cerevisiae and closely related species in the Saccharomyces sensu stricto group, and suggest future research directions.

Although protein evolution can be approximated as a "molecular evolutionary clock," it is well known that sequence change departs from a clock-like Poisson expectation. Through studying the deviations from a molecular clock, insight can be gained into the forces shaping evolution at the level of proteins. Generally, substitution patterns that show greater variance than the Poisson expectation are said to be "overdispersed." Overdispersion of sequence change may result from temporal variation in the rate at which amino acid substitutions occur on a phylogeny. By comparing the genomes of four species of yeast, five species of Drosophila, and five species of mammals, we show that the extent of overdispersion shows a strong negative correlation with the effective population size of these organisms. Yeast proteins show very little overdispersion, while mammalian proteins show substantial overdispersion. Additionally, X-linked genes, which have reduced effective population size, have gene products that show increased overdispersion in both Drosophila and mammals. Our research suggests that mutational robustness is more pervasive in organisms with large population sizes and that robustness acts to stabilize the molecular evolutionary clock of sequence change.

The purpose of this study was to assess the retention ability of 12 different Saccharomyces sp. yeast strains with flocculent characteristics when inoculated in a continuous ethanol fermentation process. The system was comprised of two reactors connected in series with no cell recycling. The feeding substrate used was a synthetic medium containing glucose. The parameters assessed were total reducing sugars of the feeding substrate, total reducing sugars and ethanol at the outlet of the first and second reactors and quantification and classification of yeast population in the two reactors. The system reached yield levels of 83.53% of theoretical yield with a maximum total reducing sugars conversion of 92.68%. The conversion in this system was lower than expected. The dominant yeast in the process in both reactors, contrary to expectation, was the Saccharomyces CP6 strain which was unable to form pellets in spite of its flocculate growth.

Three ascomycetous yeast strains, H-6(T), ZX-15 and ZX-20, isolated from the bark of two tree species of the family Fagaceae collected from different regions of China, formed unconjugated and persistent asci containing two to four globose ascospores. 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region (including 5.8S rDNA) sequence analysis showed that they were closely related to the currently accepted Saccharomyces species with strong support. Comparisons of the rDNA sequences, electrophoretic karyotypes and physiological characters indicated that the three strains represent a novel species in the genus Saccharomyces. The name Saccharomyces arboricolus sp. nov. was proposed for the novel species, with H-6(T) (=AS 2.3317(T)=CBS 10644(T)) isolated from the bark of Quercus fabri as the type strain.

The results of fluorescence lifetime imaging microscopy of selected yeast strains were presented and the fact that the lifetime distributions can be successfully used for strain characterization and differentiation was demonstrated. Four strains of industrially relevant yeast Saccharomyces were excited at 405 nm and the autofluorescence observed within 440-540 nm. Using statistical tools such as empirical cumulative distribution functions with Kolmogorov-Smirnov testing, the four studied strains were categorized into three different groups for normal sample size of 70 cells slide(-1) at a significance level of 5%. The differentiation of all of the examined strains from one another was shown to be possible by increasing the sample size to 420 cells, which is achievable by taking the lifetime data at six different positions in the slide.

The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

Wild yeasts were isolated from process surfaces of two breweries. In total, 41 strains were obtained and differentiated by cultivation on CuSO(4) or crystal violet containing selective media, by fatty acid profiling and by a restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene. The restriction analysis showed the highest differentiating capacity and resulted in eleven groups. These groups were identified by the API ID 32 C kit or by sequencing the D1/D2 region of the 26S rRNA gene. Most of the wild yeasts were identified as Saccharomyces cerevisiae (46% of all isolates) and Candida pelliculosa (anamorph: Pichia anomala) (24%). No obvious differences were detected between the two breweries. While all of the S. cerevisiae isolates were able to grow in beer, only six out of 10 C. pelliculosa strains were able to tolerate this substrate. However, most of the C. pelliculosa strains showed biofilm formation in a microplate assay, but none of the S. cerevisiae isolates. Therefore, it is assumed that the former species is involved in attachment and primary biofilm formation on beer bottling plants, while S. cerevisiae is a late colonizer of a preformed biofilm but increased the beer spoiling potential of the biofilm.

Petite-positivity - the ability to tolerate the loss of mtDNA - was examined after the treatment with ethidium bromide (EB) in over hundred isolates from the Saccharomyces/Kluyveromyces complex. The identity of petite mutants was confirmed by the loss of specific mtDNA DAPI staining patterns. Besides unequivocal petite-positive and petite-negative phenotypes, a few species exhibited temperature sensitive petite positive phenotype and petiteness of a few other species could be observed only at the elevated EB concentrations. Several yeast species displayed a mixed 'moot' phenotype, where a major part of the population did not tolerate the loss of mtDNA but several cells did. The genera from postwhole-genome duplication lineages (Saccharomyces, Kazachstania, Naumovia, Nakaseomyces) were invariably petite-positive. However, petite-positive traits could also be observed among the prewhole-genome duplication species.

Aims: Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'., Methods and Results: A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity., Conclusions: It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles., Significance and Impact of the Study: Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.

There have been an increasing number of reports of yeast systemic infection involving Saccharomyces cerevisiae strains. The development of a rapid and reliable diagnostic tool is therefore warranted in order to explore the distribution of S. cerevisiae as an opportunistic pathogen in humans. In this study, we designed and validated five primer sets targeting the 26S rRNA gene of S. cerevisiae and the S. sensu stricto complex using 26 yeast strains. Among them, two sets of primers specifically amplified the 26S rRNA gene and the ITS region of S. cerevisiae strains, and three sets were specific for amplifying the same genes in the S. sensu stricto complex. After determining the optimal conditions of two primer pairs for quantitative real time PCR, human fecal samples were analyzed to examine the distribution of S. cerevisiae and the S. sensu stricto complex. It was possible to detect a single cell of S. cerevisiae in environmental sample. Qualitative PCR revealed that out of eleven fecal samples tested, one sample contained S. cerevisiae and four samples contained the S. sensu stricto complex. Quantitative real time PCR revealed that the target gene copy numbers of S. cerevisiae and the S. sensu stricto complex were 0.84 and 2.44 respectively, in 1 ng of DNA from the bulk fecal community.

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.

Aims: To analyse the genetic diversity and the dynamics of Saccharomyces strains in spontaneous fermentation in ciders. The effect of the cellar, harvest and cider-making technology were evaluated., Methods and Results: The ecology of spontaneous cider fermentations in the same cellar (Asturias) was studied for two consecutive harvests (2000 and 2001) by using mtDNA restriction analysis. Our results showed that there was a succession of genetically different strains of Saccharomyces during cider production. In general, strains of Saccharomyces bayanus species predominated at the early fermentation steps (begining and/or tumultuous fermentations), while Saccharomyces cerevisiae yeasts were the most abundant at the end of the fermentation. Five S. bayanus strains (patterns III, VII, VIII, XV and XVII) were present at significant frequencies in all the experimental tanks during the two consecutive years. The results of the cluster analysis (unweighted pair group method using average linkage) showed higher similarities for the patterns III, XV, VII and VIII. Therefore, these strains should be considered associated with the microbiota of this cellar., Conclusions: A high polymorphism within populations of Saccharomyces was found throughout the different stages of Asturian production of cider. In all the cider fermentations, a variable number of S. bayanus and S. cerevisiae strains was always present. Our results indicate, over the period of time studied, the existence of the natural microbiota in the cellar., Significance and Impact of the Study: This study has allowed us to gain a better understanding of the role of wild Saccharomyces yeast in Asturian cider fermentations.

To evaluate the influence of the genomic properties of yeasts on the formation of wine flavour, genotypic diversity among natural Saccharomyces cerevisiae strains originating from grapes collected in four localities of three Austrian vine-growing areas (Thermenregion: locations Perchtoldsdorf and Pfaffstätten, Neusiedlersee-Hügelland: location Eisenstadt, Neusiedlersee: location Halbturn) was investigated and the aroma compounds produced during fermentation of the grape must of 'Grüner Veltliner' were identified. Amplified fragment length polymorphism analysis (AFLP) showed that the yeast strains cluster in four groups corresponding to their geographical origin. The genotypic analysis and sequencing of the D1/D2 domain of 26S rRNA encoding gene and ITS1/ITS2 regions indicated that the Perchtoldsdorf strains were putative interspecies hybrids between S. cerevisiae and Saccharomyces kudriavzevii. Analysis of the aroma compounds by GS/MS indicated a region-specific influence of the yeasts on the chemical composition of the wines. The aroma compound profiles generated by the Perchtoldsdorf strains were more related to those produced by the Pfaffstätten strains than by the Eisenstadt and Halbturn strains. Similar to the Pfaffstätten yeasts, the putative hybrid strains were good ester producers, suggesting that they may influence the wine quality favourably.

Understanding the evolutionary forces that influence patterns of gene expression variation will provide insights into the mechanisms of evolutionary change and the molecular basis of phenotypic diversity. To date, studies of gene expression evolution have primarily been made by analyzing how gene expression levels vary within and between species. However, the fundamental unit of heritable variation in transcript abundance is the underlying regulatory allele, and as a result it is necessary to understand gene expression evolution at the level of DNA sequence variation. Here we describe the evolutionary forces shaping patterns of genetic variation for 1206 cis-regulatory QTL identified in a cross between two divergent strains of Saccharomyces cerevisiae. We demonstrate that purifying selection against mildly deleterious alleles is the dominant force governing cis-regulatory evolution in S. cerevisiae and estimate the strength of selection. We also find that essential genes and genes with larger codon bias are subject to slightly stronger cis-regulatory constraint and that positive selection has played a role in the evolution of major trans-acting QTL.

The budding yeast species Saccharomyces castellii has provided important new insights into molecular evolution when incorporated in comparative genomics studies and studies of mitochondrial inheritage. Although it shows some diversity in the specific molecular details, several analyses have shown that it contains many genetic pathways similar to those of S. cerevisiae. Here we have investigated the possibility of performing genetic analyses in S. castellii. We optimized the LiAc transformation protocol to achieve 200-300 transformants/microg plasmid DNA. We found that the commonly used plasmids for S. cerevisiae are stably maintained in S. castellii under selective conditions. Surprisingly, both 2micro and CEN/ARS plasmids are kept at a high copy number. Moreover, the kanMX cassette can be used as a resistance marker against the selective drug geneticin (G418). Finally, we determined that the S. cerevisiae GAL1 promoter can be used for the activation of transcription in S. castellii, thus enabling the controlled overexpression of genes when galactose is present in the medium. The availability of these tools provides the possibility of performing genetic analyses in S. castellii, and makes it a promising new model system in which hypotheses derived from bioinformatics studies can be experimentally tested.

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the alpha- galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79-100%) of their MEL genes. The phylogenetic analysis of all of the known alpha-galactosidases in the "Saccharomyces" clade showed that the MEL genes of the yeasts L. kluyveri. L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.

In plants and animals, new biological species clearly have arisen as a byproduct of genetic divergence in allopatry. However, our understanding of the processes that generate new microbial species remains limited [1] despite the large contribution of microbes to the world's biodiversity. A recent hypothesis claims that microbes lack biogeographical divergence because their population sizes are large and their migration rates are presumably high [2, 3]. In recapitulating the classic microbial-ecology dictum that "everything is everywhere, and the environment selects"[4, 5], this hypothesis casts doubt on whether geographic divergence promotes speciation in microbes. To date, its predictions have been tested primarily with data from eubacteria and archaebacteria [6-8]. However, this hypothesis's most important implication is in sexual eukaryotic microbes, where migration and genetic admixture are specifically predicted to inhibit allopatric divergence and speciation [9]. Here, we use nuclear-sequence data from globally distributed natural populations of the yeast Saccharomyces paradoxus to investigate the role of geography in generating diversity in sexual eukaryotic microbes. We show that these populations have undergone allopatric divergence and then secondary contact without genetic admixture. Our data thus support the occurrence of evolutionary processes necessary for allopatric speciation in sexual microbes.

Year-round studies of epiphytic yeast communities revealed that the number of ascosporogenous yeasts of the genus Saccharomyces inhabiting living and decaying leaves of some plants increased considerably in certain short periods (at the beginning of summer and in winter). Massive isolation of saccharomycetes was performed from 11 plant species; earlier, these yeasts had been revealed mainly in sugar-rich substrates. The isolates were identified as Saccharomyces paradoxus based on their physiological properties and the lengths of restriction fragments of 5.8S-ITS rDNA. Possible reasons for short-term increases in the number of saccharomycetes in plant phyllosphere are discussed.

The yeast Saccharomyces cerevisiae is characterized by its ability to: (a) degrade glucose or fructose to ethanol, even in the presence of oxygen (Crabtree effect); (b) grow in the absence of oxygen; and (c) generate respiratory-deficient mitochondrial mutants, so-called petites. How unique are these properties among yeasts in the Saccharomyces clade, and what is their origin? Recent progress in genome sequencing has elucidated the phylogenetic relationships among yeasts in the Saccharomyces complex, providing a framework for the understanding of the evolutionary history of several modern traits. In this study, we analyzed over 40 yeasts that reflect over 150 million years of evolutionary history for their ability to ferment, grow in the absence of oxygen, and generate petites. A great majority of isolates exhibited good fermentation ability, suggesting that this trait could already be an intrinsic property of the progenitor yeast. We found that lineages that underwent the whole-genome duplication, in general, exhibit a fermentative lifestyle, the Crabtree effect, and the ability to grow without oxygen, and can generate stable petite mutants. Some of the pre-genome duplication lineages also exhibit some of these traits, but a majority of the tested species are petite-negative, and show a reduced Crabtree effect and a reduced ability to grow in the absence of oxygen. It could be that the ability to accumulate ethanol in the presence of oxygen, a gradual independence from oxygen and/or the ability to generate petites were developed later in several lineages. However, these traits have been combined and developed to perfection only in the lineage that underwent the whole-genome duplication and led to the modern Saccharomyces cerevisiae yeast.

This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by non-Saccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations.

Patients with renal failure are more frequently at risk of fungal infections than the healthy individuals. The aim of the study was: (1) Evaluation of the prevalence of fungi in biological materials obtained from different ontocenoses from patients with end-stage and chronic renal failure undergoing haemodialysis and conservative treatment, respectively. (2) Species determining of isolated fungal strains and evaluation their morphological and biochemical features with regard to biotyping. (3) Examining the connection between intraspecies features of fungal strains isolated from different ontocenoses of the same patient. The study group comprised 136 persons, including 56 patients with end-stage renal failure dialysed for the mean period of 36.2 (+/- 1.62) months--all patients were on chronic haemodialysis therapy (4 hours sessions, 3 times per week), 50 patients with chronic renal failure undergoing conservative treatment and 30 persons with the negative history of any renal disease--control group. At the moment of the evaluation and collection of samples all patients were in good condition, none of the patients revealed symptoms suggesting possible fungal infection. Material for mycological examinations included washings from the oral cavity and samples of urine and faeces. In order to evaluate morphological and biochemical features of fungi the following methods were applied: (1) macrocultures in solid Sabouraud medium, (2) direct microscopic slides, (3) API 20 C AUX test and (4) API ZYM test (bioMérieux). Totally 385 samples for mycological examinations were collected from different ontocenoses, from which 161 fungal strains were isolated and classified to 17 species from 5 genera. Most strains belonged to the genus Candida 96.9% and the most frequently occurring species was C. albicans (60.3%). Other species from this genus composed 39.7%, among which in 11.8% of cases C. parapsilosis was determined, and the following were: C. guilliermondii and C. humicola--both species isolated in 5.59%, C. glabrata (4.35%), C. rugosa and C. tropicalis (both species were isolated in 2.48%). The remaining isolated fungal strains (3.11 +/- 1.37%) belonged to genera Cryptococcus, Geotrichum, Saccharomyces and Trichosporon. The prevalence of fungi in collected biological materials from chronic dialysed patients, treated conservatively and persons from control group was determined at the level of 96.4%, 64.0% and 63.3%, respectively. In all examined groups fungi were found in high percentage in the ontocenoses of oral cavity and gastrointestinal tract; in dialysed patients (40.7%), treated conservatively (34.4%) and in persons with negative history of any renal disease (42.1%). It should be expressed that in 9.37% of patients with chronic renal failure undergoing conservative treatment fungi were determined at the same time in materials obtained from three ontocenoses--oral cavity, gastrointestinal tract and urinary tract. The majority of C. albicans strains (34.0%) was described by the use of code AUX 2 576 174; code 2 566 174 was found in 23.4% of species. Strains with code AUX 2 576 174 in 96,9% were isolated from chronic haemodialysed patients whereas code 2 566 174 characterized strains (68.2%) obtained from patients treated conservatively. Examined fungal strains from genus Candida obtained from all persons revealed the activity of 10 to 17 enzymes in the API ZYM test. Strains of genus Candida albicans obtained from patients with chronic renal failure treated conservatively and undergoing haemodialysis in majority belonged to biotype A (58.8%) and D3 (40.4%), respectively.

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.

The white wine Chacolía de Vizcaya/Bizkaiko Txakolina is characteristic from The Basque Country region and regulated under Appellation Contrôlée standards (BOPV 14/6/94). The objective of this study was the identification and selection of autochthonous yeast strains, to improve the conditions used to maintain the typical characteristics of this region wines. Yeasts identified as Saccharomyces bayanus isolated around these fields from 1996 to 1998, were subjected to a selective procedure based on enological characteristics and fermentative behaviour. Three of the selected strains were used to inoculate, at winery scale, two grape juice varieties accepted by the Appellation Contrôlée (Hondarrabi Zuri and Folle Blanche). The inoculated strains on the respective vinifications was followed by restriction fragment length polymorphism of mitochondrial DNA (REAmt) method with AluI enzyme, due to their specificity, short outcome, and technological simplicity compared with other molecular typing methods such as: chromosomal karyotyping analyzed by pulsed field gel electrophoresis, Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and restriction fragment length polymorphism using the infrequently cutting enzyme SfiI (REA infrequent). This study demonstrated that strains with different phenotypic traits could show indistinguishable restriction patterns with REAmt, but could be discriminated using other typing methods such as RAPD-PCR, which although showing low reproducibility could be used as complementary to REAmt. Our results demonstrate that in spite of using autochthonous selected strains, the inoculation of musts with a particular strain do not guarantee its predominance and driving fermentation features. Of all yeast strains studied, strain no. 2 showed the best results in sensory testing and at the implantation process. Therefore, it could be used with commercial purposes for the production of Chacolí de Vizcaya/Bizkaiko Txakolina, especially when using musts from Folle Blanche.

Several wine isolates of Saccharomyces were analysed for six molecular markers, five nuclear and one mitochondrial, and new natural interspecific hybrids were identified. The molecular characterization of these Saccharomyces hybrids was performed based on the restriction analysis of five nuclear genes (CAT8, CYR1, GSY1, MET6 and OPY1, located in different chromosomes), the ribosomal region encompassing the 5.8S rRNA gene and the two internal transcribed spacers, and sequence analysis of the mitochondrial gene COX2. This method allowed us to identify and characterize new hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii, between S. cerevisiae and Saccharomyces bayanus, as well as a triple hybrid S. bayanusxS. cerevisiaexS. kudriavzevii. This is the first time that S. cerevisiaexS. kudriavzevii hybrids have been described which have been involved in wine fermentation.

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.

Essential genes are required for an organism's viability, and the ability to identify these genes in pathogens is crucial to directed drug development. Predicting essential genes through computational methods is appealing because it circumvents expensive and difficult experimental screens. Most such prediction is based on homology mapping to experimentally verified essential genes in model organisms. We present here a different approach, one that relies exclusively on sequence features of a gene to estimate essentiality and offers a promising way to identify essential genes in unstudied or uncultured organisms. We identified 14 characteristic sequence features potentially associated with essentiality, such as localization signals, codon adaptation, GC content, and overall hydrophobicity. Using the well-characterized baker's yeast Saccharomyces cerevisiae, we employed a simple Bayesian framework to measure the correlation of each of these features with essentiality. We then employed the 14 features to learn the parameters of a machine learning classifier capable of predicting essential genes. We trained our classifier on known essential genes in S. cerevisiae and applied it to the closely related and relatively unstudied yeast Saccharomyces mikatae. We assessed predictive success in two ways: First, we compared all of our predictions with those generated by homology mapping between these two species. Second, we verified a subset of our predictions with eight in vivo knockouts in S. mikatae, and we present here the first experimentally confirmed essential genes in this species.

Using molecular and genetic analyses, 28 Arthroascus strains were analysed, isolated from widely different geographical localities in Europe, North America, Far-East Asia and Hawaii. Most of the strains have been assigned to the species Arthroascus schoenii. PCR-RAPD revealed two Japanese Arthroascus strains (UCD 67-278 and IFO 10138) to have peculiar patterns. Comparative rDNA (D1/D2 26S, ITS1 and ITS2) sequence analysis showed that the two strains respectively represent a novel species and a novel variety. Based on the results of sequence analysis, genetic hybridization and DNA-DNA reassociation, two new members of the genus Arthroascus are formally described, Arthroascus babjevae sp. nov. (type strain UCD 67-278(T)=CBS 9167(T)) and Arthroascus fermentans var. arxii var. nov. (type strain IFO 10138(T)=CBS 9168(T)). These results show that A. schoenii has a worldwide distribution, while the species Arthroascus javanensis is represented only by the type culture CBS 2555(T), isolated in Indonesia. Cluster analysis revealed a correlation between PCR-RAPD fingerprints and geographical origin of the A. schoenii strains. Despite this molecular differentiation, A. schoenii strains collected in different regions of the world formed predominantly fertile hybrids, with normal recombination of control markers.

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.