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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Chromosomal structures of bottom fermenting yeasts.

A genomic comparison of bottom fermenting yeasts was performed by pulsed-field gel electrophoresis and Southern blot analysis with some S. cerevisiae gene probes. We confirmed that strains of bottom fermenting yeast have four chromosomes originating from S. bayanus. Since the structures of these chromosomes were recombined with S. cerevisiae chromosomes, these S. bayanus chromosomes could be differentiated from S. cerevisiae chromosomes using Southern hybridization. Our Southern hybridization results indicate that bottom fermenting yeasts have both chromosomes originating from both S. cerevisiae and S. bayanus. It was reconfirmed that top fermenting yeast should be classified as S. cerevisiae, based on the chromosomal structure. The chromosomal structure of S. pastorianus CBS1538, the type stain of S. pastorianus, was also investigated. This strain has chromosomes originating only from S. bayanus. S. carlsbergensis CBS1513 has chromosomes originating from both S. cerevisiae and S. bayanus. From these results, we contend that bottom fermenting yeasts should be classified as S. carlsbergensis.     

Characterization of the metabolic shift of Saccharomyces bayanus var. uvarum by continuous aerobic culture

Saccharomyces bayanus, being of interest for wine-making, is not as well known as S. cerevisiae and, due to many changes in the yeast classification, accurate data concerning its metabolic activity are difficult to find. In order to produce this yeast as an active dry yeast to be used as a starter in wine-making, its sensitivity to glucose was determined as the objective of our work. Using the pulse technique in continuous culture, it was found that growth in a synthetic medium was not limited by vitamins or mineral salts. We determined the critical dilution rate of a continuous culture and performed an aerobic continuous culture, measuring the respiratory quotient on-line in order to observe the metabolic shift from respiratory to fermentative metabolism. The S. bayanus var. uvarum strain studied was Crabtree-positive (glucose-sensitive) but had a weaker respiratory capacity than S. cerevisiae since the dilution rate of the metabolic shift was only 0.15 h(-1). These new data provide essential information for the biomass production of this yeast strain for wine-making.     

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

Partial genetic isolation of twoSaccharomyces bayanus varieties, 5.bayanus var.bayanus andS. 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.     

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

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

<Emphasis Type="Italic">Actinomucor elegans</Emphasis> var. <Emphasis Type="Italic">kuwaitiensis</Emphasis> isolated from the wound of a diabetic patient

The new variety of Actinomucor elegans var. kuwaitiensis, isolated from an open necrotic wound of a diabetic patient is described. This strain differed from the two previously described varieties of A. elegans, A. elegans var. elegans and A. elegans var. meitauzae by nearly 1% or more sequence divergence within D1/D2 regions of 28S rRNA and the ITS region of rRNA genes. Like the other two varieties, no zygospores were observed, however, there was evidence suggesting intersexual diploidy, a feature not described previously in this species. Additionally, A. elegans var. kuwaitiensis was pathogenic to white mice causing 100% mortality within 5 days.     

牛尾蒿两变种精油的化学成分

本文以气相色谱质谱计算机联用的方法分析了牛尾蒿原变种Artemisia dubia var. dubia及无毛变种B A. dubia var. subdigitata精油化学成分,共鉴定出82种化合物,其中萜类50种,芳香类12种,脂肪族类20种。经比较研究表明,这两变种的精油中有39种化合物(27 种萜类,5种芳香类和7种脂肪族类化合物)相同,而它们的差别在于牛尾蒿无毛变种中二环、三环倍半萜、芳香类及脂肪族类化合物的类型比原变种多。从精油的萜类化学组成特点来看,这两个变种的萜类化合物环化程度比蒿亚属植物高,而与龙蒿亚属植物相近。因此,这两个变种归属于龙蒿亚属较合适。     

Chemical Constituents of Essential Oil of Two Varieties of Artemisia dubia Wall. ex Bess.

The essential oil of Artemisia dubia var. dubia and dubia var. subdigitata were analysed by GCMS and a tatal of 82 compounds including 50 terpenoids, 12 aromatics and 20 aliphatics were identified, among which variety dubia and var. subdigitata contained 54 and 67 compounds respectivey. Comparative studies indicate that 39 compounds (27 terpenoids, 5 aromatics and 7 aliphatics) were shared by the two varieties. The difference in constituents of essential oil between the two varieties is that more types of biocyclic and tricyclic sesquiterpenes, aromatics and aliphatics were shown inA. Dubia var. subdigitata than A. dubia var. dubia. Compositionally, the terpenoids in essential oil of the two varieties were endowed with a higher degree of cyclization than those of the species from Subgen. Artemisia, but similar to those of the species from Subgen. Dracunculus. Thus the assignment of the two varieties in Subgen. Dracunculus was consistent with the phylogeny of the genus Artemisia.     

Heterogeneity of the yeasts Zygowilliopsis californica: Z. californica var. dimennae comb. nov., stat. nov. and Z. californica var. fukushimae comb. nov., stat. nov

A significant heterogeneity of the species Zygowilliopsis californica was revealed using RFLP-analysis of the PCR-amplified rDNA fragment spanning the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. Phylogenetic analysis of the nucleotide sequences of ITS1 and ITS2 rDNA differentiated three varieties: Z. californica var. californica, Z. californica var. dimennae, and Z. californica var. fukushimae. The most variable was the ITS 2 region, where 7-26 nucleotide substitutions were revealed. The varieties formed semisterile hybrids with meiotic segregation of control markers. The limits of the phylogenetic species concept are discussed.     

Temperature adaptation markedly determines evolution within the genus Saccharomyces

The present study uses a mathematical-empirical approach to estimate the cardinal growth temperature parameters (T(min), the temperature below which growth is no longer observed; T(opt), the temperature at which the μ(max) equals its optimal value; μ(opt), the optimal value of μ(max); and T(max), the temperature above which no growth occurs) of 27 yeast strains belonging to different Saccharomyces and non-Saccharomyces species. S. cerevisiae was the yeast best adapted to grow at high temperatures within the Saccharomyces genus, with the highest optimum (32.3°C) and maximum (45.4°C) growth temperatures. On the other hand, S. kudriavzevii and S. bayanus var. uvarum showed the lowest optimum (23.6 and 26.2°C) and maximum (36.8 and 38.4°C) growth temperatures, respectively, confirming that both species are more psychrophilic than S. cerevisiae. The remaining Saccharomyces species (S. paradoxus, S. mikatae, S. arboricolus, and S. cariocanus) showed intermediate responses. With respect to the minimum temperature which supported growth, this parameter ranged from 1.3 (S. cariocanus) to 4.3°C (S. kudriavzevii). We also tested whether these physiological traits were correlated with the phylogeny, which was accomplished by means of a statistical orthogram method. The analysis suggested that the most important shift in the adaptation to grow at higher temperatures occurred in the Saccharomyces genus after the divergence of the S. arboricolus, S. mikatae, S. cariocanus, S. paradoxus, and S. cerevisiae lineages from the S. kudriavzevii and S. bayanus var. uvarum lineages. Finally, our mathematical models suggest that temperature may also play an important role in the imposition of S. cerevisiae versus non-Saccharomyces species during wine fermentation.     

Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen.

A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. bayanus should be reinstated as a separate taxon.