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.     

Variations in mRNA transcript levels of cell wall-associated genes of Saccharomyces cerevisiae following spheroplasting

A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids.     

Saccharomyces bayanus var. uvarum in Tokaj wine-making of Slovakia and Hungary

Using genetic hybridisation analysis and molecular karyotyping we revealed an association of Saccharomyces bayanus var. uvarum species with Tokaj wine-making. Along with identification of Saccharomyces strains isolated by E. Minárik in Slovakia, the composition of Tokaj populations in Hungary was studied. Twenty-eight Hungarian Saccharomyces strains were analysed in terms of karyotype. The majority of strains belong to S. bayanus var. uvarum. Two non-identified Saccharomyces strains were found to be polyploid according to their complex karyotype patterns.     

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

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

Saccharomyces bayanus var. Uvarum comb.nov., a new variety established by genetic analysis

Partial genetic isolation of two Saccharomyces bayanus varieties, S. bayanus var. bayanus and S. bayanus var. uvarum comb. nov., was established by hybridological analysis. The hybrids of these two varieties were semisterile: their ascospores were characterized by low survival. Earlier, the new variety was described as a group of cryophilic wine yeast cultivars capable of fermenting melibiose.     

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.     

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers

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.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.     

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.     

Molecular analysis of alpha-galactosidase MEL genes from Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the alpha-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 alpha-galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

Kinetics of citrate uptake in growing cells of Leuconostoc spp.

Abstract Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus , first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus . Enological strains classified as S., bayanus , S. chevalieri , and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus . Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.     

Comparative molecular-genetic analysis of the beta-fructosidases in yeast Saccharomyces

To infer the molecular evolution of polymeric beta-fructosidase SUC genes of the yeast Saccharomyces, we have cloned and sequenced a new SUC gene from S. cariocanus and determined the sequence similarity of beta-fructosidases within the genus Saccharomyces. The proteins of Saccharomyces cerevisiae and its five sibling species (S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus) have high degree of identity - 90-97%. The invertase of S. bayanus is the most divergent among the proteins studied. The data obtained indicated that the yeast invertases are highly conservative. In the coding regions of the SUC genes the pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. There is only one, probably, non-telomeric SUC gene in each of the Saccharomyces species. In S. cerevisiae, S. bayanus, S. kudriavzevii, S. mikatae and S. paradoxus the SUC gene have been mapped on chromosome IX, whereas in S. cariocanus this gene is located in chromosone XV, in the position of translocation.     

FTIR spectroscopic discrimination of Saccharomyces cerevisiae and Saccharomyces bayanus strains

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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.     

PCR–DGGE differentiation of strains of Saccharomyces sensu stricto

A quick molecular biology method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was developed for distinguishing strains belonging to the Saccharomyces sensu stricto group. Differentiation was obtained between S. cerevisiae, S. paradoxus and S. bayanus / S. pastorianus although no distinction was possible between S. bayanus and S. pastorianus using the amplification of the ITS regions. The ability to distinguish between different strains of the Saccharomyces sensu stricto group could allow for a better understanding of the ecology of these species on grapes as well as in musts and wines and the method developed can be useful for the quick identification of Saccharomyces sensu stricto strains from numerous isolates.     

Mitochondrial DNA polymorphism of the yeast Saccharomyces bayanus var. uvarum

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 (RFLP) 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.     

A molecular genetic study of natural strains of Saccharomyces isolated from Asturian cider fermentations

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.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

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.