Schizosaccharomyces osmophilus sp. nov., an osmophilic fission yeast occurring in bee bread of different solitary bee species.

Eight yeast strains that asexually reproduce by cell fission were isolated from bee bread of different solitary bees in Germany. DNA sequence analysis revealed that the strains shared the same sequence in the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene with a strain that was previously isolated from a fig snack from Spain. The closest related type strain was that of Schizosaccharomyces octosporus, which showed 98.2% sequence similarity (11 substitutions) with the new strains. By clone sequence analysis of the internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, and ITS2) a total of nine different copy types were identified. The new strains differed from S. octosporus by approximately 31% in the ITS region. Sequence analysis of the RNAse P gene further supported the description of a new species. The strains isolated during this study show some phenotypic characteristics that separate them from the closest related species, S. octosporus and S. cryophilus. Since all strains showed true osmophily the name of the new species is S. osmophilus (holotype: CBS 15793T; isotype: CLIB 3267 T = NCAIM Y.02225 T, MycoBank no.: MB829586).

Selection of appropriate Schizosaccharomyces strains for winemaking.

This paper describes the selection of Schizosaccharomyces yeasts with adequate oenological suitability and high capacity for the degradation of malic acid. Despite the almost non-existent number of commercial strains, the use of this yeast genus has recently been recommended by the International Organisation of Vine and Wine (OIV, in French). Thus, in the present study, a large number of Schizosaccharomyces strains were isolated using a selective differential medium. Initially, classic parameters of oenological interest for the use of fermentative strains of Saccharomyces cerevisiae (the most frequently used type of yeast) were assessed. Only five strains of moderate acetic acid production lower than 0.4 g/L were obtained at the end of fermentation. Other, more specific features of this yeast genus' physiology were also studied, including urease activity and the production of pyruvic acid and glycerol. Finally, oenological suitability was determined by comparing selected strains with other Schizosaccharomyces reference and S cerevisiae control strains. Schizosaccharomyces strains produced 80% less urea content, four times higher pyruvic acid levels and 1 g higher glycerol contents than the Saccharomyces reference strains. The results confirmed that it is possible to perform selective processes on microorganisms from the genus Schizosaccharomyces using methodology developed in this work to obtain strains of industrial interest.

A fast and inexpensive method for random spore analysis in Schizosaccharomyces pombe.

Random spore analysis is a fundamental tool of yeast genetics for determining gene linkage and the generation of recombinant progeny by genetic crosses. Experimentally it involves treatment of a mating mix with enzymes, such as zymolyase or lyticase, that selectively lyse the cell wall of vegetative cells rather than the spores. Here, we describe a method whereby the relative refractory nature of the spores to treatment with elevated temperature and repeated freeze-thawing facilitates random spore analysis at low cost in fission yeast Schizosaccharomyces pombe. Because of similar properties of spores in budding yeast, this method should prove to be useful for random spore analysis in both budding and fission yeasts.

Complete sequences of Schizosaccharomyces pombe subtelomeres reveal multiple patterns of genome variation.

Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions of a standard laboratory strain. In addition, some natural isolates of S. pombe were analyzed using previous sequencing data. Whole sequences of SH regions revealed that each SH region consists of two distinct parts with mosaics of multiple common segments or blocks showing high variation among subtelomeres and strains. Subtelomere regions show relatively high frequency of nucleotide variations among strains compared with the other chromosomal regions. Furthermore, we identified subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation with missense mutations, insertions, and deletions but no severe effects on their RNA expression. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.

Isolation and characterization of ethanol-producing Schizosaccharomyces pombe CHFY0201.

Ethanol-producing yeast strain, CHFY0201 was isolated from soil in South Korea using an enrichment technique in a yeast peptone dextrose medium supplemented with 5% (w/v) ethanol at 30 degrees C. The phenotypic and physiological characteristics, as well as molecular phylogenetic analysis based on the D1/D2 domains of the large subunit (26S) rDNA gene and the internally transcribed spacer (ITS) 1+2 regions suggested that the CHFY0201 was novel strain of Schizosaccharomyces pombe. During shaking flask cultivation, the highest ethanol productivity and theoretical yield of S. pombe CHFY0201 in YPD media containing 9.5% total sugars was 0.59 +/- 0.01 g/l/h and 88.4 +/- 0.91%, respectively. Simultaneous saccharification and fermentation for ethanol production was carried out using liquefied cassava (Manihot esculenta) powder in a 5 l lab-scale jar fermenter at 32 degrees C for 66 h with an agitation speed of 120 rpm. Under these conditions, S. pombe CHFY0201 yielded a final ethanol concentration of 72.1 +/- 0.27 g/l and a theoretical yield of 82.7 +/- 1.52% at a maximum ethanol productivity of 1.16 +/- 0.07 g/l/h. These results suggest that S. pombe CHFY0201 is a potential producer for industrial bioethanol production.

Yeast identification in grape juice concentrates from Argentina.

AIMS: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. METHODS AND RESULTS: Osmophilic and osmotolerant yeasts were isolated from spoiled--visually effervescent--and unspoiled--without any visible damage--grape juice concentrates by the spread-plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. CONCLUSIONS: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.

Schizosaccharomyces pombe Isolation Protocol.

This chapter describes a methodology to isolate yeast strains from Schizosaccharomyces pombe species. The method is based on a selective-differential medium that notably facilitates the isolation of S. pombe. The main difficulty in isolating microorganisms from this genus is their extremely low incidence in nature when they are compared to other microorganisms. The proposed methodology allows isolating and selecting strains from this species for industrial purposes. Methodologies allows detecting the presence of those yeasts when they are considered spoilage microorganisms. Several selective-differential agents based on the basic physiological characteristics of S. pombe species are exposed during the chapter introduction and the use is properly justified. Some of those representative characteristics are its extraordinary resistance to high sugar concentrations, sulfur dioxide, sorbic acid, benzoic acid, acetic acid, or their unique malo-ethanolic fermentation ability. The proposed selective medium is mainly based on S. pombe resistance to the antibiotic actidione and the unusual tolerance to the inhibitory agent benzoic acid compared to possible microorganisms that could produce false-positive results during an isolation process. In addition, malic acid is proposed as the main differential factor due to the exclusive ability of this species to metabolize malic acid into ethanol. This fact allows the detection of malic acid degradation. Cloramphenicol is used to inhibit bacteria growth and liquid media to avoid fungi development.

Identification of novel temperature-sensitive lethal alleles in essential beta-tubulin and nonessential alpha 2-tubulin genes as fission yeast polarity mutants.

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1-15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two alpha-tubulin genes) and alp12 to nda3 (the single beta-tubulin gene). atb2(+) is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between alpha/beta-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2(+); overexpression of atb2(+) lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Selecting Schizosaccharomyces pombe Diploids.

Here we describe procedures for the selection of diploid Schizosaccharomyces pombeade6-M210/ade6-M216 heteroallelic complementation is widely used to select for Ade+ diploids. Such diploids will readily sporulate when starved of nitrogen. For some investigations, stable diploids are preferable (e.g., for genetic complementation tests), and in these cases mating an h- strain with an h90 mat2-Pi-102 strain can be used to prevent sporulation. When ade6-M210/ade6-M216 mutations impact on, or show synthetic interactions with, the gene of interest, two different auxotrophic markers can be used to select complementation.

Spore Analysis and Tetrad Dissection of Schizosaccharomyces pombe.

Here we describe the processing of Schizosaccharomyces pombe spores in batches (random spore analysis) or through tetrad dissections. Spores are usually prepared from matings between haploid strains (producing zygotic asci) or from sporulating diploids (producing azygotic asci). In random spore analysis, a snail enzyme preparation is used to digest the walls of asci to release free spores that are diluted and plated to form colonies. In tetrad dissection, a needle attached to a micromanipulator is used to pick asci and separate spores. Tetrad dissection has traditionally been the method of choice for genetic mapping and is very useful in the study of genetic interactions (e.g., suppressor analysis). It is also the preferred method for routine crosses because it ensures that every colony stems from a single spore. This can never be certain in random spore analysis.

Construction, Growth, and Harvesting of Fission Yeast Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Strains.

Stable isotope labeling by amino acids in cell culture (SILAC) enables the relative quantification of protein amounts and posttranslational modifications in complex biological samples through the use of stable heavy isotope-labeled amino acids. Here we describe methods for the application of SILAC to fission yeast Schizosaccharomyces pombe using either labeled lysine or a combination of labeled lysine and labeled arginine. The latter approach is more complicated than the use of labeled lysine alone but may yield a more comprehensive (phospho)proteomic analysis. The protocol includes methods for construction of SILAC-compatible strains, growth of cultures in labeled medium, cell harvesting, and protein extraction.

Cold-sensitive nuclear division arrest mutants of the fission yeast Schizosaccharomyces pombe.

Thirteen recessive cold sensitive nuclear division arrest mutants were isolated from the fission yeast Schizosaccharomyces pombe. Twelve unlinked genes were defined; six in chromosome I, three in chromosome II and two in chromosome III. The map positions of three nuclear division arrest genes (nda1, nda2 and nda3) in chromosome II were determined precisely. Together with the previously obtained temperature-sensitive cell division cycle mutations, at least 20 genes appear to control the nuclear division of the fission yeast. Physiological studies indicated that most cold sensitive nda mutants incubated previously at 22 degrees C proceeded with a synchronously normal cell-cycle after temperature shift-up. The morphology of the nuclei and nuclear chromatin region was studied by the 4',6-diamidino-2-phenylindole staining method and by electron microscopy. Each mutant exhibited characteristic nuclear morphology at 22 degrees C, showing the specific blockages. The nda genes seem to control a pathway of structural alterations in the nuclear chromatin region with the order hemisphere, condensed ellipsoid, segregating U-form and separating hemispheres. Two genes, nda2 and nda3, pleiotropically control nuclear division, nuclear location and cell shape. The terminal phenotype of nda2-KM52 is characterized by the nuclear displacement, the absence of a spindle and abnormal locations of spindle pole bodies. The cells of nda3-KM311 were aberrant in shape and contained a partially separated chromatin region with a long spindle. Together with the results of the accompanying paper, we conclude that nda2 and nda3 genes control nuclear and cytoplasmic microtubular organization.

Isolation and characterization of Schizosaccharomyces pombe mutants affected in mitotic recombination.

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.

Isolation and characterization of fission yeast sns mutants defective at the mitosis-to-interphase transition.

pim1-d1ts was previously identified in a visual screen for fission yeast mutants unable to complete the mitosis-to-interphase transition. pim1+ encodes the guanine nucleotide exchange factor (GEF) for the spi1 GTPase. Perturbations of this GTPase system by either mutation or overproduction of its regulatory proteins cause cells to arrest with postmitotic condensed chromosomes, an unreplicated genome, and a wide medial septum. The septation phenotype of pim1-d1ts was used as the basis for a more extensive screen for this novel class of sns (septated, not in S-phase) mutants. Seventeen mutants representing 14 complementation groups were isolated. Three strains, sns-A3, sns-A5, and sns-A6, representing two different alleles, are mutated in the pim1+ gene. Of the 13 non-pim1ts sns complementation groups, 11 showed genetic interactions with the spi1 GTPase system. The genes mutated in 10 sns strains were synthetically lethal with pim1-d1, and six sns strains were hypersensitive to overexpression of one or more of the known components of the spil GTPase system. Epistasis analysis places the action of the genes mutated in nine of these strains downstream of pim1+ and the action of one gene upstream of pim1+. Three strains, sns-A2, sns-B1, and sns-B9, showed genetic interaction with the spil GTPase system in every test performed. sns-B1 and sns-B9 are likely to identify downstream targets, whereas sns-A2 is likely to identify upstream regulators of the spi1 GTPase system that are required for the mitosis-to-interphase transition.

Isolation of a Schizosaccharomyces pombe rad21ts mutant that is aberrant in chromosome segregation, microtubule function, DNA repair and sensitive to hydroxyurea: possible involvement of Rad21 in ubiquitin-mediated proteolysis.

The fission yeast DNA repair gene rad21+ is essential for cell growth. To investigate the function essential for cell proliferation, we have isolated a temperature-sensitive mutant of the rad21+ gene. The mutant, rad21-K1, showed abnormal mitosis at the nonpermissive temperature. Some cells contained abnormal nuclear structures, such as condensed chromosomes with short spindles, or chromosomes stretched or unequally separated by elongating spindles. Other cells exhibited the displaced nucleus or a cut-like phenotype. Similar abnormalities were observed when the Rad21 protein was depleted from cells. We therefore concluded that Rad21 is essential for proper segregation of chromosomes. Moreover, the rad21-K1 mutant is sensitive not only to UV and gamma-ray irradiation but to thiabendazole and hydroxyurea, indicating that Rad21 plays important roles in microtubule function, DNA repair, and S phase function. The relation to the microtubule function was further confirmed by the fact that rad21+ genetically interacts with tubulin genes, nda2+ and nda3+. Finally, the growth of the rad21-K1 mutant was inhibited at the permissive temperature by introduction of another mutation in the cut9+ gene, coding for a component of the 20S cyclosome/anaphase promoting complex, which is involved in ubiquitin-mediated proteolysis. The results suggest that these diverse functions of Rad21 may be facilitated through ubiquitin-mediated proteolysis.

Optimisation of methodology for enumeration of xerophilic yeasts from foods.

Xerophilic yeasts grow in intermediate moisture foods (aw, 0.65-0.85) such as sugar syrups, fruit concentrates, jams and brines. Non-osmophilic yeasts are enumerated by diluting in 0.1% peptone and then plated onto media such as malt extract or glucose yeast extract agar. In the presence of moulds the yeasts are enumerated in dichloran rose bengal chloramphenicol agar (DRBC). These procedures were demonstrated to be unsatisfactory for the enumeration of xerophilic yeasts in low aw foods. Investigations using pure cultures of xerophilic yeasts as well as naturally contaminated apple juice concentrates and glacé cherries have shown that a reduced aw diluent, in particular 30% w/w glycerol in combination with tryptone 10% glucose yeast extract agar (TGY) optimises the recovery of the yeasts, especially sublethally injured cells. The inclusion of sodium chloride in either the diluents or the culture media was not necessary to optimise the recovery of D. hansenii growing in 20% sodium chloride broths.

Effect of diluent type on viability of yeasts enumerated from foods or pure culture.

The effects of seven diluent types on the viability of yeasts enumerated from foods and in pure culture were studied. The diluents were laboratory glass distilled water; saline water (0.85% NaCl), sodium phosphate buffer (0.1 M, pH 7.0), 0.1% peptone, 0.1% yeast extract, 0.1% peptone in 0.1 M sodium phosphate buffer, pH 7.0, and 0.1% malt extract. For all foods studied, dilution in 0.1% peptone gave the highest counts, with saline and phosphate buffer diluents giving lower counts than those obtained with distilled water. When seven species of yeast were enumerated in pure culture, highest counts were obtained using 0.1% peptone as the diluent and, with three exceptions, all species gave higher counts when diluted in diluents other than distilled water. When yeast suspensions were held in diluents for up to 2 h before plating, cell death occurred. The extent of death was highest in distilled water, saline and phosphate buffer diluents. Cell death also occurred in 0.1% peptone, yeast extract and malt extract, but to a lesser degree.

Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe.

In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.

The isolation of protoplasts of the fission yeast Schizosaccharomyces by Trichoderma viride and snail enzymes.

The formation of protoplasts of the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces versatilis after the combined application of snail enzymes and Trichoderma viride enzymes in an osmotic stabilizer (0.4M KCl, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.

gitter: a robust and accurate method for quantification of colony sizes from plate images.

Colony-based screens that quantify the fitness of clonal populations on solid agar plates are perhaps the most important source of genome-scale functional information in microorganisms. The images of ordered arrays of mutants produced by such experiments can be difficult to process because of laboratory-specific plate features, morphed colonies, plate edges, noise, and other artifacts. Most of the tools developed to address this problem are optimized to handle a single setup and do not work out of the box in other settings. We present gitter, an image analysis tool for robust and accurate processing of images from colony-based screens. gitter works by first finding the grid of colonies from a preprocessed image and then locating the bounds of each colony separately. We show that gitter produces comparable colony sizes to other tools in simple cases but outperforms them by being able to handle a wider variety of screens and more accurately quantify colony sizes from difficult images. gitter is freely available as an R package from http://cran.r-project.org/web/packages/gitter under the LGPL. Tutorials and demos can be found at http://omarwagih.github.io/gitter.