Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.     

SED1 gene length and sequence polymorphisms in feral strains of Saccharomyces cerevisiae

The SED1 gene (YDR077W), coding for the major cell wall glycoprotein of Saccharomyces cerevisiae stationary-phase cells, contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence. A PCR survey of the SED1 open reading frames (ORFs) of 186 previously uncharacterized grape must isolates of S. cerevisiae yielded 13 PCR profiles arising from different combinations of seven SED1 length variants in individuals homozygous or heterozygous for the gene. Comparison of the nucleotide sequences of a group of representatives of each of the seven length variants with those of S288C and the type strain, CBS1171, unequivocally identified them as SED1 alleles and provided evidence for the presence of two minisatellite-like sequences, variable in length, within the ORF of an S. cerevisiae gene. The segregation analyses of the SED1 length variants and other genetic markers in 13 isolates representative of each PCR profile suggested that molecular mechanisms involved in minisatellite expansion and contraction may be responsible for SED1 heterozygosities within a population of homothallic must isolates of S. cerevisiae.     

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.     

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques

AIMS: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. METHODS AND RESULTS: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. CONCLUSIONS: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.     

Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.     

Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS 380

PCR/RFLP of the NTS2 sequence of rDNA was shown to be suitable for differentiating Saccharomyces sensu stricto species. We previously showed that, within the presently accepted S. bayanus taxon, strains formerly classified as S. uvarum represented a distinct subgroup (Nguyen and Gaillardin, 1997). In this study, we reidentified 43 more strains isolated recently from wine, cider and various fermentation habitats, and confirmed by karyotyping, hybridization and mtDNA analysis the homogeneity of strains from the S. uvarum subspecies. Molecular typing of nuclear and mitochondrial genomes of strains preserved in collections, and often originating from beer like S. pastorianusNT, revealed the existence of hybrids between S. uvarum and S. cerevisiae. Surprisingly, S. bayanusT CBS380 appeared itself to be a hybrid between S. uvarum and S. cerevisiae. This strain has a mitochondrial genome identical to that of S. uvarum, and a very similar karyotype with 13 isomorphic chromosomes, six of which at least hybridize strongly with S. uvarum chromosomes or with a S. uvarum specific sequence. However, four of the chromosome bands of S. bayanusT bear Y' sequences indistinguishable from those of S. cerevisiae, a feature that is not observed among presently isolated S. uvarum strains. Because of the hybrid nature of S. bayanus(T) and of the scarcity of similar hybrids among present days isolates, we propose to reinstate S. uvarum as a proper species among the Saccharomyces sensu stricto complex.     

利用26S rDNAD1/D2区序列和微卫星标记分析各种工业酿酒酵母的种内遗传差异

选取30株传统发酵工业中不同来源的酿酒酵母菌株和实验室常用酿酒酵母菌株,利用大亚基(26S)rDNA D1/D2区序列和微卫星标记分析酿酒酵母种内菌株间的遗传多样性和系统发育关系,以揭示酿酒酵母在长期的各种工业发酵环境下发生的遗传变异。结果表明:30株酿酒酵母的26S rDNA D1/D2区序列(591bp)比较保守,与测序菌株S288C相比序列相似度在99.8%–100%,说明种内菌株间存在一定的多态性,但序列差异并不十分明显,其变异情况表现在个别碱基的差异(大多由转换突变引起);通过扩增11个微卫星位点,每个菌株均有其独特的基因型,即30种基因型,共得到188个等位基因,观测杂合度平均值和期望杂合度平均值分别为0.576、0.886,多态信息含量平均值高达0.858,说明酿酒酵母种内菌株间具有较高的遗传多样性,而聚类分析表明30株酿酒酵母可以得到很好地区分,但是没有呈现与其工业来源相关的聚类。     

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.     

SED1 Gene Length and Sequence Polymorphisms in Feral Strains of Saccharomyces cerevisiae

The SED1 gene (YDR077W), coding for the major cell wall glycoprotein of Saccharomyces cerevisiae stationary-phase cells, contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence. A PCR survey of the SED1 open reading frames (ORFs) of 186 previously uncharacterized grape must isolates of S. cerevisiae yielded 13 PCR profiles arising from different combinations of seven SED1 length variants in individuals homozygous or heterozygous for the gene. Comparison of the nucleotide sequences of a group of representatives of each of the seven length variants with those of S288C and the type strain, CBS1171, unequivocally identified them as SED1 alleles and provided evidence for the presence of two minisatellite-like sequences, variable in length, within the ORF of an S. cerevisiae gene. The segregation analyses of the SED1 length variants and other genetic markers in 13 isolates representative of each PCR profile suggested that molecular mechanisms involved in minisatellite expansion and contraction may be responsible for SED1 heterozygosities within a population of homothallic must isolates of S. cerevisiae.     

Protein fingerprinting of Saccharomyces isolates with therapeutic relevance using one- and two-dimensional electrophoresis

In therapeutic products and preparations Saccharomyces cerevisiae is used because of its nutritive properties. Moreover, so called Saccharomyces boulardii yeasts are used in the prevention and treatment of several types of diarrhea. Taxonomically however, S. boulardii is not accepted as a distinct species. The protein fingerprint obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for all isolates and therefore confirmed the designation of S. boulardii to the species S. cerevisiae. In contrast, using native polyacrylamide gel electrophoresis, 12 different protein fingerprints were detected, and allowed grouping of the product isolates. The spot patterns obtained by two-dimensional electrophoresis revealed a large degree of resemblance, however, small qualitative expression differences could be detected as well. Firstly, a spot having an isoelectric point of approximately 6 and 30 kDa could not be detected in S. boulardii yeasts. Secondly, nine different formations of spots occurred in the region around 16 kDa and pH 6. Therefore, on the one hand, it could be demonstrated that all of the product isolates belong to the same species, and on the other hand, it was possible to extensively subdivide the strains. In particular, two-dimensional electrophoresis allowed clustering of so called S. boulardii strains within the species S. cerevisiae.     

Molecular characterization of clinical Saccharomyces cerevisiae isolates and their association with non-clinical strains

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two baker's yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both baker's yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.     

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.     

Mitochondria--tool for taxonomic identification of yeasts from Saccharomyces sensu stricto complex

Mitochondrial genomes of Saccharomyces and close relatives previously used for transplacement of mitochondria to S. cerevisiae were examined. The origins of replication in mitochondrial DNA, the presence of nuclear and mitochondrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassify some collection yeasts and to assign others into four separate subgroups. The first included isolates identical to Saccharomyces cerevisiae (S. italicus, S. oviformis, S. chevalieri and S. capensis) which possess 5 or more replication origins. The second group consists of S paradoxus (var douglasii) mitochondrial genome with the equal number of ori sequences but incompatible mitochondria. The third group represents Saccharomyces sensu stricto petite-positive species (S. carlsbergensis, S. heterogenicus, S. uvarum, S. willianus) with 1-2 origins of replication significantly different from S. cerevisiae. In addition, the locus between tRNA(fMet) and tRNA(Pro) is about one-half of the 1400 bp members of S. cerevisiae complex. The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S. cerevisiae-like replication origins.     

Phylogenetic Relationships of Species of the Genus Kluyveromyces van der Walt (Saccharomycetaceae) Deduced from the Partial Sequences of 18S and 26S Ribosomal RNAs

In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined. The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae. The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S. cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces. From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K. polysporus, the type species of the genus. Kluyveromyces phaffii was also distinct enough to deserve another genus. Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus. Most of the strains of the species examined were fairly close to the strain of S. cerevisiae.     

Genotypic and physiological characterization of Saccharomyces boulardii, the probiotic strain of Saccharomyces cerevisiae

Saccharomyces boulardii, a yeast that was isolated from fruit in Indochina, has been used as a remedy for diarrhea since 1950 and is now a commercially available treatment throughout Europe, Africa, and South America. Though initially classified as a separate species of Saccharomyces, recent publications have shown that the genome of S. boulardii is so similar to Saccharomyces cerevisiae that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in S. boulardii that make it perform more effectively as a probiotic organism compared to other strains of S. cerevisiae. This investigation reports some of these distinguishing characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX, altered copy number of a number of individual genes, and sporulation deficiency have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of S. boulardii to its probiotic nature are discussed.     

Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species

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

Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays

To analyse the reliability and accuracy of genotype analysis with high-density oligonucleotide microarrays, this method and other experimental approaches were used to analyse genomic DNA of two popular Saccharomyces cerevisiae laboratory strains. S288C was used for systematic sequencing of 'the' S. cerevisiae genome; CEN.PK113-7D is a popular strain for physiological studies and functional genomics. Random amplified polymorphic DNA, electrophoretic karyotyping and microarray analysis all indicated a high level of sequence similarity between the two strains. In the microarray analysis, as few as 288 (4.5%) of the ca. 6300 represented yeast genes were identified that yielded significantly different hybridisation intensities between the two strains. These could be classified as amplified, absent, or with sequence polymorphism in CEN.PK113-7D compared to S288C. A detailed analysis focused on the subset of 25 genes called absent in CEN.PK113-7D. Among these absent genes, 17 were clustered together on five chromosomes, mainly in subtelomeric regions. Thorough analysis of these regions by polymerase chain reaction (PCR) and restriction fragment length polymorphism confirmed the absence of these genes in CEN.PK113-7D. Surprisingly, three of these regions were not smaller in CEN.PK113-7D chromosomes, indicating that they may harbour unidentified and potentially new sequences. In addition, eight genes called absent by the microarrays were scattered over the chromosomes. Using diagnostic PCR most of these genes were actually found to be present in CEN.PK113-7D, but after sequencing were found to differ significantly at the DNA level from S288C, explaining the poor hybridisation to the arrays. Our results indicate that DNA microarrays are a powerful tool for determining genotypic similarity between different yeast strains. However, to obtain meaningful information at the individual gene level, this method should be backed up by additional techniques.     

The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes

The osmotolerant yeast Zygosaccharomyces rouxii CBS732 contains only one copy of the ZrHOG1 and ZrSOD2-22 genes. Both genes were cloned and sequenced (Acc. Nos. AJ132606 and AJ252273, respectively) and their sequences were compared to homologous pairs of genes from Z. rouxii ATCC42981 (genes Z-HOG1, Z-HOG2, Z-SOD2, Z-SOD22). The CBS732 ZrHog1p is shorter than its ATCC42981 counterparts (380 aa residues vs. 407 and 420 aa, respectively) and is more similar to ATCC42981 Z-Hog2p than to Z-Hog1p. Also its promoter region corresponds to that one of Z-HOG2. The CBS732 ZrHOG1 promoter region is recognised by Saccharomyces cerevisiae, and the gene product (MAP kinase ZrHog1p) presence fully complements the osmosensitivity of a S. cerevisiae hog1 mutant strain. The CBS ZrSOD2-22 gene is highly similar to ATCC42981 Z-SOD2 but it contains also a segment of 15 aa residues specific for Z-SOD22. Z. rouxii ZrSod2-22 Na(+)/H(+) antiporter expressed in S. cerevisiae shows better activity toward toxic Na(+) and Li(+) cations than does S. cerevisiae's own Nha1 antiporter, and is efficient in improving the halotolerance of some S. cerevisiae wild types.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

中国酱香型白酒酿造酿酒酵母的独特生理代谢特征

【目的】酿酒酵母是白酒发酵的主要微生物,对白酒的产量及质量都有重要影响。而酱香酒酿造环境具有高温、酸性、高乙醇等胁迫因素,研究其中酿酒酵母的生理代谢特征并有目的地应用于白酒实际生产中的意义。【方法】从酱香酒酿造环境中筛选一株性能优良的酿酒酵母菌株,比较其与酿酒酵母模式菌株S288c和商业酵母的生理代谢特征。【结果】从酱香型酒醅中筛选得到性能优良的Saccharomyces cerevisiae MT1,该菌株可耐受高温42 °C,高浓度乙醇(16%,体积比),低pH (2.0),其最大比生长速率和最大比产乙醇速率分别达到了S288c的125%和114%,其乙醇转化率也要高于其他菌株。一些挥发性物质只有在MT1的发酵液中可检测到,包括苯并噻唑、2,3-二氢苯并呋喃、4-乙烯基愈创木酚以及丁羟甲苯等,MT1的苯乙醇、法呢醇、橙花叔醇、乙偶姻和大马酮量也要高于S288c。另外,MT1可以利用多种碳源发酵产乙醇,如半乳糖、麦芽糖、蜜二糖、松二糖、海藻糖和棉子糖等。【结论】来源于酱香酒酿造的S. cerevisiae MT1具有高耐受力、高效的发酵性能及更广泛的碳源利用图谱,并能生成多种挥发性物质。