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.     

Studies on immobilized Saccharomyces cerevisiae. III. Physiology of growth and metabolism on various supports

In earlier communications general analyses of rapid ethanol fermentation by Saccharomyces cerevisiae immobilized on inert supports were described. In this article physiology of growth and metabolism (parameters like rates of CO(2) evolution and O(2) uptake, respiratory quotient, and generation time) of Saccharomyces cerevisiae immobilized on different supports are reported. Values of the ratio of specific oxygen uptake rate for immobilized cells to free cells have been found to be 0.732, 0.781 and 0.785 for carrier A, carrier B, and covalently crosslinked controlled pore glass (CPG, specific surface area of 439 m(2) g(-1)), respectively. Rates of specific CO(2) evolution for immobilized cells to free cells for these supports are 0.784, 0.822, and 0.783, respectively. Marked reduction in generation time of Saccharomyces cerevisiae on all the supports has been observed. No change in size (4.8-5 mum) and specific growth rate (mu(m) = 0.275 h(-1)) of cells leaving the reactor has been observed.     

Comparative study of some parameters of energy metabolism in two strains of Saccharomyces cerevisiae

A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain N 73 and laser-irradiated mutant strain Y-503) was performed. In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain. The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase. The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical. There are two sites of energy coupling in the respiratory chain of mitochondria in S. cerevisiae N 73 and Y-503, and electron flow mainly is mainly mediated by cytochrome oxidase. The data suggest that a higher respiratory activity of S. cerevisiae Y-503 cells in comparison with N 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to a high physiological and biochemical activity of this strain.     

Harvesting recombinant microbial cells using crossflow filtration.

A contained, crossflow filtration (CFF) membrane system is described for harvesting Saccharomyces cerevisiae and Escherichia coli cells. This system is portable and can be cleaned and sanitized in place. Low- and high-cell density (LCD, HCD) fermentations of recombinant cells in 10- to 200-l volumes were used as the starting material. LCD fermentations, up to 8.3 g l-1 dry weight (dcw) of S. cerevisiae, with volumes of 10 to 200 l were harvested and diafiltered in 0.5 and 1.5 h, respectively. HCD 200-l fermentations of S. cerevisiae (47-63 g l-1 dcw) were harvested and diafiltered in approximately 2 h. E. coli fermentations, LCD and HCD (up to 16.2 g l-1 dcw), of 200-l volumes were harvested and diafiltered in 2.3 h while employing 14 and 75 ft2 of membrane area, respectively. Using hollow fiber or flat sheet membranes from different sources, cell harvesting times were less than 2.5 h. These studies demonstrate that CFF is an efficient method for harvesting and diafiltering recombinant S. cerevisiae and E. coli cells from fermentation broth.     

Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract The plasma membrane of Saccharomyces cerevisiae and Schizosaccharomyces pombe in stationary phase had abundant invaginations. A round uninvaginated area emerged before budding when S. cerevisiae cells were given fresh medium. Middle-sized buds had some invaginations, whereas the neck between the bud and mother had very few. S. pombe which has neither the neck nor the predetermined position to divide had no uninvaginated ring area even in long cells during elongation in fresh medium. However, an uninvaginated ring area emerged as the earliest noticeable stage of cytokinesis. The uninvaginated state of the plasma membrane appeared to be correlated with budding and cell division.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

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.     

Reassessment of antigenic determinant of Saccharomyces cerevisiae serotype Ia.

We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat-killed cells of Candida glabrata, or from anti-S. cerevisiae Ia (M 6001) serum by adsorption with heat-killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D-mannan gave five oligosaccharides. Signals of 1H-nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of alpha-1,3'-linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A-D. Agglutination-inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of alpha-1,3'-linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal alpha-(1-3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two alpha-(1-3) linkages is responsible for the specificity of S. cerevisiae Ia.     

5-溴脱氧尿嘧啶核苷标记酵母基因组DNA的方法

胸腺嘧啶类似物5-溴脱氧尿嘧啶核苷(BrdU)标记技术是一种研究DNA复制、修复等生命过程的有效手段。由于酿酒酵母(Saccharomyces cerevisiae)中缺少胸腺嘧啶核苷酸补救途径,胞外BrdU不能有效的渗入到基因组中,使该技术在酿酒酵母中的应用受到极大制约。通过在基因组中引入单纯疱疹病毒胞苷激酶(HSV-TK)和人类平衡核苷转运蛋白(hENT1)基因,工作建立了BrdU标记酵母基因组DNA的方法。在生长对数中期加入0.2mg/ml BrdU,离体检测法检测发现,标记3h的荧光信号较1h、5h时强;胞内检测法结果显示,标记3h时55.3%的基因组DNA中能够渗入BrdU。该工作为酿酒酵母DNA复制、修复等方面提供了直接有效的研究方法。     

The location and structure of double-strand DNA breaks induced during yeast meiosis: evidence for a covalently linked DNA-protein intermediate.

We have determined the precise location and structure of the double-strand DNA breaks (DSBs) formed during Saccharomyces cerevisiae meiosis. Breaks were examined at two recombination hot spots in both wild-type and rad50S mutant cells. At both loci, breaks occurred at multiple, irregularly spaced sites in a approximately 150 nucleotide interval contained within an area of nuclease-hypersensitive chromatin. No consensus sequence could be discerned at or around break sites. Patterns of cleavage observed on individual strands indicated that breaks initially form with a two nucleotide 5' overhang. Broken strands from rad50S mutant cells contained tightly bound protein at their 5' ends. We suggest that, in S.cerevisiae, meiotic recombination is initiated by a DSB-forming activity that creates a covalently linked protein-DNA intermediate.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Limitations on the use of polymerase chain reaction--restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.     

Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae

Glyoxylate biosynthesis in Saccharomyces cerevisiae is traditionally mainly ascribed to the reaction catalyzed by isocitrate lyase (Icl), which converts isocitrate to glyoxylate and succinate. However, Icl is generally reported to be repressed by glucose and yet glyoxylate is detected at high levels in S. cerevisiae extracts during cultivation on glucose. In bacteria there is an alternative pathway for glyoxylate biosynthesis that involves a direct oxidation of glycine. Therefore, we investigated the glycine metabolism in S. cerevisiae coupling metabolomics data and (13)C-isotope-labeling analysis of two reference strains and a mutant with a deletion in a gene encoding an alanine:glyoxylate aminotransferase. The strains were cultivated on minimal medium containing glucose or galactose, and (13)C-glycine as sole nitrogen source. Glyoxylate presented (13)C-labeling in all cultivation conditions. Furthermore, glyoxylate seemed to be converted to 2-oxovalerate, an unusual metabolite in S. cerevisiae. 2-Oxovalerate can possibly be converted to 2-oxoisovalerate, a key precursor in the biosynthesis of branched-chain amino acids. Hence, we propose a new pathway for glycine catabolism and glyoxylate biosynthesis in S. cerevisiae that seems not to be repressed by glucose and is active under both aerobic and anaerobic conditions. This work demonstrates the great potential of coupling metabolomics data and isotope-labeling analysis for pathway reconstructions.     

Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae.

A gene (FDH1) of Candida maltosa which confers resistance to formaldehyde in Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The gene has a single intron which possesses the highly conserved splicing signals found in S. cerevisiae introns. We demonstrated that processing of the pre-mRNA of the cloned gene occurred identically in both S. cerevisiae and C. maltosa. The predicted amino acid sequence from the cloned gene showed 65.5% identity to human alcohol dehydrogenase (ADH) class III and 23.9% identity to S. cerevisiae ADH1. The most probable mechanism of resistance to formaldehyde is thought to be the glutathione-dependent oxidation of formaldehyde which is characteristic for ADH class III. The cloned FDH1 gene was successfully employed as a dominant selectable marker in the transformation of S. cerevisiae.     

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.     

Amyloid of the Candida albicans Ure2p prion domain is infectious and has an in-register parallel β-sheet structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.     

Identification of an essential core element and stimulatory sequences in a Kluyveromyces lactis ARS element, KARS101

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient for replication in Saccharomyces cerevisiae and K. lactis . This fragment contains a 12 bp sequence 5'-ATTTATTGTTTT-3' that is related to the S. cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site-directed mutagenesis and the effect on K. lactis and S. cerevisiae ARS (autonomous replicating sequence) activity was determined. The dodecamer is essential for S. cerevisiae ARS function but only contributes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS.
A 103 bp subclone was found to retain ARS activity in both yeasts, but the plasmid was very unstable in S. cerevisiae . Deletion and linker substitution mutagenesis of this fragment was undertaken to define the DNA sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coincide. We found a 39 bp core region essential for K. lactis ARS function flanked by sequences that contribute to ARS efficiency. The instability of the plasmid in S. cerevisiae made a fine-structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high-frequency transformation in S. cerevisiae overlap the essential core of the K. lactis ARS element but have different end-points.     

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.