The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity

New double‐ and triple‐hybrid Saccharomyces yeasts were characterized using PCR‐restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double‐hybrid S. cerevisiae × S. kudriavzevii in non‐fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in these hybrids revealed a high diversity of genome types. From these molecular characterizations, a reduction of the S. kudriavzevii fraction of the hybrid genomes is observed in most hybrids. Mitochondrial inheritance in hybrids was deduced from the analysis of mitochondrial COX2 gene sequences, which showed that most hybrids received the mitochondrial genome from the S. kudriavzevii parent. However, two strains inherited a S. cerevisiae COX2, being the first report of S. cerevisiae × S. kudriavzevii hybrids with S. cerevisiae mitochondrial genomes. These two strains are those showing a higher S. kudriavzevii nuclear genome reduction, especially in the wine hybrid AMH. This may be due to the release of selective pressures acting on the other hybrids to maintain kudriavzevii mitochondria‐interacting genes. Copyright © 2011 John Wiley & Sons, Ltd.     

Biochemistry,cell biology and molecular biology of lipids of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes. © 1998 John Wiley & Sons, Ltd.     

Alternative yeasts for winemaking: Saccharomyces non-cerevisiae and its hybrids

Wine fermentation has not significantly changed since ancient times and the most traditional aspects are seen by the market as elements that uplift wine nuances and quality. In recent years, new trends have emerged from the sector in line with consumer preferences, and due to the effects of global climate change on grape ripening. In the first cases, the consumers are looking for wines with less ethanol and fruitier aromas and in the second cases the wineries want to reduce the wine alcohol levels and/or astringency. New yeast starters of alternative Saccharomyces species and their hybrids can help to solve some problems that wineries face. In this article we review several physiological and genetic aspects of S. uvarum and S. kudriavzevii and the hybrids, which are especially relevant during the winemaking process, such as their good fermentative capabilities at low temperatures, resulting in wines with lower alcohol and higher glycerol amounts.     

The use of molecular biology techniques to study and manipulate the grapevine: Why and how?

This paper aims to give an overview of how molecular biology relates to grapevine research and viticulture. In it we explain some basic premises and techniques of molecular biology with the aim of introducing a wider readership to this rapidly developing field. Current and potential uses of molecular biology for research and practical viticulture are presented. These include molecular cloning, analysis of gene expression, grapevine transformation, marker-assisted breeding, cultivar identification and pest and disease diagnostics. A glossary of terms and a list of web sites for further information are also provided.     

A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth

Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell. In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations.     

国内生物分子学技术应用于酿酒酵母的研究进展

酿酒酵母的运用已广泛存在于人类社会的方方面面。相比于将其作为面包、馒头、酒类产品的发酵菌株,现今的酿酒酵母更是人类用以开发可再生资源而得到燃料乙醇的重要手段。分子生物学与遗传工程技术作为近代迅速发展起来的新兴学科,已大量应用于微生物的研究,如PCR技术、基因重组、原生质体技术等等,在微生物的鉴定筛选、调节产物的生产、基因工程菌的构建等方面发挥了推动作用。以酿酒酵母为参考对象,阐述了国内近年来运用分子生物学技术改造酿酒酵母的研究报道。     

Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae

The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences: Y', pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum.     

一种观察酿酒酵母原生质体的简易方法

酿酒酵母(Scerevisiae)原生质体不需任何染色剂染色，只需使用血球计数板，在光学显微镜下能清晰地观察原生质体形态，而且显微照相效果良好。该方法程序简单、操作方便、效果良好。     

Analysis of β-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography

We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)-β-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a ‘knr4 disrupted’ tetrad demonstrated that mutant cell walls contained much less (1→3)-β-glucan and (1→6)-β-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.     

发酵过程中啤酒酵母生长代谢活性的电化学分析

首次报道了电化学分析方法在 4 80t啤酒酵母发酵过程中对酵母细胞生长代谢的连续检测。从 0代到 4代 ,从满罐到降温结束 ,跟踪了 5代酵母发酵的全过程。研究表明 ,电化学方法可以描述细胞生长代谢的过程 ,反应细胞的代谢活性 ,在相应地细胞生长代谢阶段 ,细胞的电化学活性与细胞数有良好的线形关系。因此细胞电化学方法为酵母发酵过程提供了一个快速、准确的细胞代谢过程及代谢活性的分析方法     

Influence of sequential inoculum of Starmerella bacillaris and Saccharomyces cerevisiae on flavonoid composition of monovarietal Sangiovese wines

The selection of Starmerella bacillaris strains to be used with Saccharomyces cerevisiae as mixed cultures has been recently suggested in order to produce wines containing lower ethanol and higher glycerol concentrations and to promote fructose degradation due to their fructophilic character. However, studies about effects of such mixed starter cultures on phenolic compounds, which are responsible for the colour and health-enhancing properties in red wines, are currently lacking. Therefore, in this work, the influence of sequential inoculated fermentation (SIF) with Starm. bacillaris and S. cerevisiae on phenolic content of monovarietal Sangiovese wine was evaluated by fermentations at laboratory scale. Axenic fermentations (AXFs) with S. cerevisiae were performed as control. S. cerevisiae attained higher cell densities in AXF compared with SIF. The experimental wines obtained by SIF showed significant lower ethanol and higher glycerol concentrations, whereas no significant difference was detected in colour intensity. The total phenol index reached significantly lower values in SIF. Furthermore, the wines produced by SIF contained higher concentrations of vitisin A that has a greater colour stability than the anthocyanin monomer. Finally, a lower content of both free anthocyanins and flavan-3-ols, key compounds for wine quality possessing also health-enhancing properties, was found in wines obtained by SIF. On the contrary, no significant difference was detected on flavonol concentration between SIF and AXF. This study highlighted that the use of sequential inoculum of Starm. bacillaris and S. cerevisiae can contribute to increasing the colour stability of red wines, even if at the expense of compounds with health properties.     

高温高压下利用金刚石压腔实验技术对材料Raman光谱的研究

金刚石对顶砧装置（diamond anvil cell,DAC）是静态超高压装置的一种,由于金刚石具有高的硬度和良好的透光性,因此这种装置所实现的压力和能测量物性的种类都优于其他高压装置。Raman光谱是表征物质结构及其变化的一种重要手段,DAC超高压实验技术与激光Raman光谱测试方法相结合,为高压下物质结构相变研究提供了一种有效途径。实验利用金刚石压腔实验技术在高温高压条件下对氧化镓、硫酸锶固体材料进行了Raman光谱的测量。     

Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N- and C-termini of these proteins have distinctive and critical functions. They contain an N-terminal part that has a probable 2-dehydropantoate 2-reductase domain. In Svl3, this part can be replaced with the yeast 2-dehydropantoate 2-reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2-dehydropantoate 2-reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C-terminal part. One of such signals is the lysine-rich region located in the C-terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine-rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells.     

A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF.

In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S. servazzii. We find that the order of four genes surrounding the RE in S. cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii. However, there is an additional ORF in S. servazzii between PRD1 and KAR4 that is not homologous to any gene in S. cerevisiae or to genes in other organisms. Despite a 75-79% amino acid identity for KAR4 and SPB1, respectively, the S. servazzii sequence did not carry a well-conserved RE sequence and these sequences lacked RE function when introduced into S. cerevisiae. The S. servazzii region contains a sequence that supports autonomous DNA replication in S. cerevisiae and may represent a homologue of ARS304. The S. servazziii sequence has Genbank Accession No. BankIt359091 AF307954.     

Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae boulardii: a metabolomics approach

Weak acids inhibit the growth of probiotics, such as Saccharomyces boulardii. We explored the tolerance of S. boulardii to different weak acids. S. boulardii had better fermentation ability under lactic acid conditions compared with acetic and butyric acid conditions; however, the budding of S. boulardii was significantly stronger than that of Saccharomyces cerevisiae under acetic acid conditions. Although the surface structure of S. boulardii was destroyed, it produced more daughter cells. S. boulardii metabolites were also significantly different from S. cerevisiae under acidic stress. The growth of S. boulardii under weak acid conditions differed significantly from that of S. cerevisiae. S. boulardii-mediated fingerprints under weak acid conditions were identified as latent biomarkers, related to fructose and mannose metabolism, tricarboxylic acid cycle, and the glycolysis pathway. Identified biomarkers will aid in the genetic engineering of S. boulardii and other Saccharomyces strains for improved acid resistance and biomass yield.     

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale‐down cultivations

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large‐scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production‐scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two‐ and three‐compartment scale‐down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab‐scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale‐down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three‐compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single‐cell size distribution was observed under oscillatory growth conditions with three‐dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single‐cell‐based analysis provides useful information about the homogeneity of responses among the population.     

The yeast growth control gene GRC5 is highly homologous to the mammalian putative tumor suppressor gene QM

We isolated the Saccharomyces cerevisiae GRC5 (gr owth control) gene by functional complementation in vivo of a ts (t emperature s ensitive) mutation. Phenotypic analysis suggested involvement of GRC5 in cell growth and proliferation. Mutant cells arrest their cell cycles after one to three cell divisions predominantly as mother cells with a large bud. In the region of the septum, a massive accumulation of cell wall material is observed. The mother and daughter nuclei are well separated and spindles are no longer present, while the cytoskeleton is of aberrant appearance. Arrested cells do not perform protein synthesis and are unable to mate. Furthermore, grc5-1^ts cells rapidly lose viability at the restrictive temperature (37°C) only on full media, but not under nitrogen-starvation conditions, indicating that proper response to this nutrient limitation is still intact in mutant cells after cell cycle arrest. The sequence of GRC5 translates into a basic protein of 221 amino acids with a corresponding M_r of 25·4 kDa. GRC5 is a member of the highly conserved QM gene family, members of which have been reported from plants, invertebrates and vertebrates. The amino acid sequence of GRC5 over its entire length is more than 60% identical to the human QM protein, expression of which is associated with loss of the tumorigenic phenotype in a cell line derived from Wilms' tumor, a malignancy of the embyronic kidney. Here, we show that GRC5 is an essential yeast gene, the function of which as inferred from analysis of the grc5-1^ts mutant is crucial for establishment of proper cytoskeletal structure and regulation of growth in yeast cells.     

Scp160p,a new yeast protein associated with the nuclear membrane and the endoplasmic reticulum,is necessary for maintenance of exact ploidy

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER–nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER–nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.     

芽孢杆菌B15的分子生物学鉴定及高产蛋白酶菌株选育

利用16S rDNA序列分析法,对分离自土壤产蛋白酶和淀粉酶及拮抗物质的芽孢杆菌B15进行菌种鉴定.通过序列比对及构建系统发育树,表明菌株B15与Bacillus amyloliquefaciens subsp.plantarum AS43.3相似性达99％,鉴定为解淀粉芽孢杆菌植物亚种.菌株B15经过2次紫外线诱变,蛋白酶产量提高到初始菌株的147.1％.试验发现诱变株的淀粉酶产量与蛋白酶产量呈正相关.     

Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26S protease complex

There is accumulating evidence for a large, highly conserved gene family of putative ATPases. We have identified 12 different members of this novel gene family (the YTA family) in yeast and determined the nucleotide sequences of nine of these genes. All of the putative gene products are characterized by the presence of a highly conserved domain of 300 amino acids containing specialized forms of the A and B boxes of ATPases. YTA1, YTA2, YTA3 and YTA5 exhibit significant similarity to proteins involved in human immunodeficiency virus Tat-mediated gene expression but more significantly to subunits of the human 26S proteasome. YTA1 and YTA2 are essential genes in yeast. Remarkably, the cDNA of human TBP-1 can compensate for the loss of YTA1. Preliminary experiments indicate that YTA1 is a component of the 26S protease complex from yeast. Our findings lead us to propose that YTA1, YTA2, YTA3 and YTA5 function as regulatory subunits of the yeast 26S proteasome. YTA10, YTA11 and YTA12 share significant homology with the Escherichia coli FtsH protein, and together with YTA4 and YTA6 may constitute a separate subclass within this family of putative ATPases.