Plasmids resembling 2-micrometers DNA in the osmotolerant yeasts Saccharomyces bailii and Saccharomyces bisporus

Five of eight strains of Saccharomyces bailii and one of 13 strains of S. bisporus were found to harbour DNA plasmids. pSB1 and pSB2 plasmids were isolated from S. bailii strains IFO 0488 and IFO 1047, respectively, and pSB3 and pSB4 from S. bisporus strain IFO 1730. All four plasmids resemble 2-micrometers DNA of S. cerevisiae in that their molecular sizes are about 6 kb, each molecule possesses a pair of inverted repeats, they exist as a mixture of two isomers and their copy numbers in the native host are similar. None of them showed homology with 2-micrometers DNA or with each other by Southern hybridization under moderately stringent conditions, but pSB4 hybridized with the pSR1 DNA, which was found previously in a strain of S. rouxii. Each of the pSB plasmids has DNA sequence(s) effective for autonomous replication in S. cerevisiae. In S. cerevisiae, pSB3 and pSB4 showed intramolecular recombination but neither supported isomerization of 2-micrometers DNA.     

Evaluation of the Biolog system for the identification of food and beverage yeasts

The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species. The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts. The system is based around a microtitre tray and includes assimilation and oxidation tests. This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin. Species correctly identified included Saccharomyces cerevisiae , Debaryomyces hansenii , Yarrowia lipolytica , Kluyveromyces marxianus , Kloeckera apiculata , Dekkera bruxellensis and Schizosaccharomyces pombe. Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time.     

Killer toxin-secreting double-stranded RNA mycoviruses in the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii.

Killer toxin-secreting strains of the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii were shown to contain linear double-stranded RNAs (dsRNAs) that persist within the cytoplasm of the infected host cell as encapsidated virus-like particles. In both yeasts, L- and M-dsRNAs were associated with 85-kDa major capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was shown to be encapsidated by a 35-kDa coat protein. Although Northern (RNA) blot hybridizations indicated that L-dsRNA from Z. bailii is a LA species, additional peptide maps of the purified 85-kDa capsid from Z. bailii and the 88- and 80-kDa major coat proteins from K1 and K28 killer viruses of Saccharomyces cerevisiae revealed distinctly different patterns of peptides. Electron microscopy of purified Z. bailii viruses (ZbV) identified icosahedral particles 40 nm in diameter which were undistinguishable from the S. cerevisiae killer viruses. We demonstrated that purified ZbVs are sufficient to confer the Z. bailii killer phenotype on transfected spheroplasts of a S. cerevisiae nonkiller strain and that the resulting transfectants secreted even more killer toxin that the original ZbV donor strain did. Curing experiments with ZbV-transfected S. cerevisiae strains indicated that the M-dsRNA satellite from Z. bailii contains the genetic information for toxin production, whereas expression of toxin immunity might be dependent on Z-dsRNA, which resembles a new dsRNA replicon in yeasts that is not dependent on an LA helper virus to be stably maintained and replicated within the cell.     

Amplification of a Zygosaccharomyces bailii DNA segment in wine yeast genomes by extrachromosomal circular DNA formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.     

The production of 2,3-butanediol as a differentiating character in wine yeasts

The capacity to produce 2,3-butanediol by 90 strains of four different species of wine yeasts (Kloeckera apiculata, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Zygosaccharomyces bailii) was tested in grape must by automated multiple development HPTLC. The total amount of 2,3-butanediol produced varied between 23mg l–1 and 857.7mg l–1 according to the yeast species. S. cerevisiae and Z. bailii behaved similarly, producing elevated amounts of 2,3-butanediol. K. apiculata and Sc. ludwigii, in contrast, were low producers. When considerable amounts of 2,3-butanediol were found, little acetoin was present; the amounts of butanediol and acetoin were characteristic of the individual species.     

Characterization of the yeast population from traditional corn and rye bread doughs

M.J. ALMEIDA AND C.S. PAIS. 1996. Yeasts were isolated from a variety of home-made bread doughs and identified. A pure culture of Saccharomyces cerevisiae was found in 18% of the doughs. The same species predominated in 80% of the doughs examined whereas Issatchenkia orientalis, Pichia membranaefaciens and Torulaspora delbrueckii were present in about 40% of the samples. About one quarter of the isolates displayed killer activity, strains of P. anomala showing the broadest spectra. Two isolates of S. cerevisiae and three of T. delbrueckii gave biomass values in sucrose medium similar to or higher than those obtained with commercial compressed baker's yeast strains.     

Physiological properties of some yeast strains

Twenty yeast strains have recently been isolated in pure cultures from natural and industrial sources and identified based mainly on physiological properties. The majority of the strains (15) are alcohologenic belonging to the genus Saccharomyces and comprise two brewer's (beer) yeast strains (S. carlsbergensis= S. uvarum A and B), two baker's yeast strains (S. cerevisiae CA and CP), one spirit yeast strain (S. cerevisiae CF) and ten wine yeast strains (S. cerevisiae var. ellipsoideus = S. ellipsoideus 1, 3, 4, 6, 8 and 9; S. oviformis 2, 5 and 7; and S. uvarum 10). The other 5 yeast strains belong to different species: Kloeckera apiculate, Candida mycoderma (Mycoderma vini), Pichia membranaefaciens, Rhodotorula glutinis and Torulopsis holmii, respectively.     

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2)

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.     

Polyphasic identification of wild yeast strains isolated from Greek sourdoughs

A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs. Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR). The latter methods confirmed the classical identification. According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica.     

Fatty acid patterns of film-forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens

P. NORONHA-DA-COSTA, C. RODRIGUES, I. SPENCER-MARTINS AND V. LOUREIRO. 1996. Total fatty acids (C14 : 0 to C18 : 3) of 50 strains assigned by classical identification methods to Pichia membranaefaciens (28), P. anomala (15), Dekkera anomala (2), D. bruxellensis (2) and Candida vini (3) were determined and data analysed by multivariate statistical procedures. Principal components cluster analysis defined six groups of strains. Thirteen strains of P. anomala formed a well-defined cluster, whereas P. membranaefaciens was split into three groups. The taxonomic status of the P. membranaefaciens strains was evaluated by determination of the G+C content and DNA-DNA hybridization. The results provided evidence in support of P. membranaefaciens encompassing distinct species. Only eight strains were certified as P. membranaefaciens and six were included in the same cluster, indicating that numerical analysis of fatty acid profiles points to genetic differences which remain undetectable by conventional phenotypic tests.     

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).     

The effect of hydroxycinnamic acids and potassium sorbate on the growth of 11 strains of spoilage yeasts

D. STEAD. 1995. Hydroxycinnamic acids and their derivatives occur widely in plants, fruits and wine. The effect of the common hydroxycinnamic acids (caffeic, coumaric and ferulic acids), at concentrations of 100 and 500 mg 1^-1, on growth of 11 strains of spoilage yeasts was measured spectrophotometrically and compared with that of potassium sorbate. Ferulic acid was the most generally inhibitory hydroxycinnamic acid. At 500 mg 1^-1 it appreciably inhibited Pichia anomala, Debaryomyces hansenii and Saccharomyces cerevisiae and prevented detectable growth of one strain each of P. anomala and D. hansenii. Caffeic acid was the least inhibitory compound and coumaric acid had an intermediate effect. The more resistant strains of yeast were P. membranaefaciens, Saccharomycodes ludwigii and Zygosaccharomyces bailii. Sensitivity to hydroxycinnamic acid was, in general, associated with sensitivity to potassium sorbate; at a given concentration potassium sorbate was more inhibitory than were any of the hydroxycinnamic acids.     

Whole-genome comparison reveals novel genetic elements that characterize the genome of industrial strains of Saccharomyces cerevisiae

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.     

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.     

新疆地区酸马奶中酵母菌的鉴定及其生物多样性分析

从新疆少数民族牧民家庭采集的28份传统工艺酿造酸马奶样品中分离出87株酵母菌,并对其进行了生理生化鉴定、分子生物学鉴定和生物多样性分析。生化试验结果表明,新疆地区酸马奶中的酵母菌为Saccharomyces unisporus(占总分离株的48.3%),Kluyveromyces marxianus(27.6%),Pichia membranaefaciens(15.0%)和Saccharomyces cerevisiae(9.2%)。选取其中的6株酵母菌和1株参考菌株,进行大亚基(26S)rDNA D1/D2区域(600bp左右)碱基序列分析,并通过GenBank进行同源序列搜索以确定各菌株的归属,进一步验证生理生化方法的正确性。从得到的结果中可以看出,S.unisporus和K.marxianus为新疆地区酸马奶中的优势菌。     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Killer-yeast strains with a broad spectrum of action: a search among collection strains and preliminary classification

Killer strains were looked for among certain yeast species belonging to five spore-forming genera (Hansenula, Pichia, Kluyveromyces, Williopsis and Zygowilliopsis) and one imperfect genus (Candida). 171 killers were found among 272 strains, and many of them had a high activity and a broad action spectrum. These as well as S. cerevisiae K1 and K2 killers (173 killers all in ail) were classified according to differences in their activity and the spectra of action on susceptible strains. Thirty-four groups belonging to 13 classes were isolated.     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).     

Contribution a l'étude des levures des chais- taxonomie,répartition des levures

Résumé Les populations levuriennes de deux chais de Bourgogne sont étudiées sur deux années d'un point de vue taxonomique et biologique. 122 souches se rapportant à 26 espèces, ont été isolées à partir de la voûte et du sol de la cave, sur les madriers supportant les tonneaux, dans la cuve, sur le pressoir et sur les tonneaux. En dehors d'un certain nombre d'espèces classiques dans ce type d'habitat, telles que Saccharomyces cerevisiae, Pichia membranaefaciens, Hansenula anomala et Kloeckera apiculata, nous avons pu mettre en évidence notamment: Debaryomyces hansenii, Torulopsis Candida, Saccharomyces italicus, ainsi qu'un certain nombre d'espèces appartenant aux genres Pichia, Candida et Trichosporon. La répartition qualitative et quantitative des espèces est également étudiée.

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The yeast populations of two burgundy cellars were studied during two years from a taxonomical and biological point of view. 122 strains distributed among 26 species have been isolated from the vault and ground of the cellar, on the planks supporting the barrels, in the tank, on the winepress and on the barrels. Apart from a number of standard species in this type of habitat such Saccharomy cescerevisiae, Pichia membranaefaciens, Hansenula anomala, Kloeckera apiculata we have encountered the more rare Debaryomyces hansenii, Torulopsis Candida, Saccharomyces italicus as also species of the genus Pichia, Candida, and Trichosporon.