Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.     

The role of galectin‐3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin‐3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro‐inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.     

Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

Background  

Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast.     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Anti-infectious potential of beta-mercapto-ethanol treated baker's yeast in gnotobiotic Artemia challenge test

Aim: To evaluate nutritional and anti‐infectious characteristics of the chemically treated baker’s yeast with 2‐mercapto‐ethanol (2ME) for gnotobiotically grown Artemia. Methods and Results: A selection of isogenic yeast strains was treated with 2ME and fed to gnotobiotically grown Artemia. In the first experiment the effect of the chemical treatment on the yeast nutritional value was studied. In most cases, 2ME‐treated yeast cells were better feed for Artemia than the untreated cells. In the second experiment, a small quantity of 2ME‐treated yeast cells was fed to Vibrio campbellii (VC) challenged Artemia. The 2ME‐treatment on some yeast strains (e.g. gas1, kre6 and chs3) significantly improved Artemia resistance against VC compared with the respective untreated yeast cells. Conclusion: Simple chemical treatment with 2ME could significantly improve the nutritional and anti‐infectious properties of some baker’s yeast strains for gnotobiotically grown Artemia. Significance and Impact of the Study: The gnotobiotic Artemia test system provides a unique opportunity (because of noninterference of other microbial compounds) to investigate how the yeast cell wall composition influences macro parameters (e.g. growth and survival) in an organism. In addition, gene expression studies in these gnotobiotically grown Artemia should provide further documentation on direct effects of yeast cells on the genes involved in immune functions.     

Enhancement of biocontrol activity of Torulaspora delbrueckii with methyl jasmonate against apple blue mould disease

Torulaspora delbrueckii alone and in combination with methyl jasmonate was applied to the control of Penicillium expansum. For evaluation of direct effect of Methyl jasmonate on mycelial growth of pathogen, it was added to potato dextrose agar culture at different concentrations. Effect of methyl jasmonate on population of yeast in nutrient yeast dextrose broth media was determined after 24 and 48 h. Results showed that methyl jasmonate had no significant direct effect on pathogen and yeast. Also, evaluation of methyl jasmonate effect on the population of yeast in apple wounds indicated that methyl jasmonate at different concentrations increased population growth of yeast at 20°C, 8 and 15 days after inoculation in toward the control and it had no significant effect on population dynamics of yeast at 4°C. In vivo, the results indicated that combination of methyl jasmonate with antagonistic yeast reduced the blue mould of apples better than methyl jasmonate and yeast alone.     

The effects of sterol mutants of the yeast Saccharomyces cerevisiae on the outcome of competition between Drosophila melanogaster and D. simulans

The effect of different yeast variants of the yeast Saccharomyces cerevisiae on the outcome of competition between Drosophila simulans and D. melanogaster was investigated. In all experiments differential birth rate was the effective mode of species competition. Addition of live yeast and especially mixtures of yeast strains improved the competition situation of a species, but could not prevent the extinction.     

Adhesive interactions between voice prosthetic yeast and bacteria on silicone rubber in the absence and presence of saliva

Biofilms on silicone rubber voice prostheses are the major cause for frequent failure and replacement of these devices. The presence of both bacterial strains and yeast has been suggested to be crucial for the development of voice prosthetic biofilms. Adhesive interactions between Candida albicans, Candida krusei, and Candida tropicalis with 14 bacterial strains, all isolated from explanted voice prostheses were investigated in a parallel plate flow chamber. Bacteria were first allowed to adhere to silicone rubber, after which the flow chamber was perfused with yeast, suspended either in saliva or buffer. Generally, when yeast were adhering from buffer and saliva, the presence of adhering bacteria suppressed adhesion of yeast. In saliva, Rothia dentocariosa and Staphylococcus aureus enhanced adhesion of yeast, especially of C. albicans. This study shows that bacterial adhesion mostly reduces subsequent adhesion of yeast, while only a few bacterial strains stimulate adhesion of yeast, provided salivary adhesion mediators are present. Interestingly, different clinical studies have identified R. dentocariosa and S. aureus in biofilms on explanted prostheses of patients needing most frequent replacement, while C. albicans is one of the yeast generally held responsible for silicone rubber deterioration.     

Production,purification, and characterization of a novel killer toxin from <Emphasis Type="BoldItalic">Kluyveromyces siamensis</Emphasis> against a pathogenic yeast in crab

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.     

Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.     

The use of marine yeast (Candida sp.) and bakers' yeast (Saccharomyces cerevisiae) in combination with Chlorella sp. for mass culture of the rotifer Brachionus plicatilis

Use of marine yeast and bakers' yeast in combination with Chlorella sp. for the large-scale production of the rotifer Brachionus plicatilis was investigated. The culture density of marine yeast fed rotifers was significantly higher than rotifers fed bakers' yeast. Rotifer production was significantly higher and the doubling time was lower for marine yeast fed rotifers than for bakers' yeast fed rotifers. It appears that the addition of marine yeast to the feed enhances the birth rate and overall production of rotifers in the culture system. The nutritional quality of rotifers is discussed.     

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Summary Two yeast DNA pools inserted in an hybridEscherichia coli-yeast vector pFL1 were used to transformE. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool — aBamHI yeast DNA digest — a 6.4 kbBamHI fragment was recovered that gave good complementation of theE. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool — a partialSau3A yeast DNA digest — five independent plasmids complementing eitherE. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kbBamHI fragment. One of these plasmids, which complemented the twoURA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the fullURA2 gene. A restriction map of theURA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization withURA2 messenger RNA, allowing us to estimate the limits of the gene.     

Yeast communities of the cactusPilosocereus arrabidae and associated insects in the Sandy Coastal Plains of Southeastern Brazil

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the mothSigelgaita sp. in the cactusPilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactusPilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent speciesPichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp.,Myxozyma mucilagina andSporopachydermia sp. A, representing about 80% of the total isolates.Pichia sp. A and aCandida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genusKloeckera, basidiomicetous anamorphs of the genusCryptococcus, the black yeastAureobasidium pullulans, Pichia sp. A, aCandida domercqii-like species, and some cactophilic yeasts, especiallyClavispora opuntiae. The feeding site ofSigelgaita sp. larvae hadClavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

Insertion of a genetic marker into the ribosomal DNA of yeast

Plasmid pBR322 carrying the yeast LEU2+ gene transforms leu⁻ yeast into LEU+ at a low frequency by integration at homologous chromosomal DNA. When one-half of the yeast rDNA repeat unit (BglII-A) is inserted into the plasmid, the frequency of yeast transformation increases 100- to 200-fold, in proportion to the increased amount of homologous repetitive rDNA available for integration. When the other half of the repeat unit (BglII-B) is inserted into the plasmid, the transformation frequency increases by a factor of 10⁴, and the transformants are very unstable. It is likely that this fragment of rDNA contains a yeast origin of replication. This plasmid is a useful vector for cloning fragments of yeast DNA in yeast. We have used the LEU2+ gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA, in a procedure analogous to the use of antibiotic resistance transposons in the mapping of bacterial genes. Yeast ribosomal DNA is on chromosome XII between asp5 and ura4 as determined by mitotic linkage. Genetic analysis of markers inserted at the rDNA locus should be a useful tool for studying the conservation of sequence homology and the conservation of copy number of repeated genes.     

Use of fluorescence spectroscopy to differentiate yeast and bacterial cells

This study focuses on the characterization of bacterial and yeast species through their autofluorescence spectra. Lactic acid bacteria (Lactobacillus sp.), and yeast (Saccharomyces sp.) were cultured under controlled conditions and studied for variations in their autofluorescence, particularly in the area representative of tryptophan residues of proteins. The emission and excitation spectra clearly reveal that bacterial and yeast species can be differentiated by their intrinsic fluorescence with UV excitation. The possibility of differentiation between different strains of Saccharomyces yeast was also studied, with clear differences observed for selected strains. The study shows that fluorescence can be successfully used to differentiate between yeast and bacteria and between different yeast species, through the identification of spectroscopic fingerprints, without the need for fluorescent staining.     

Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol

Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3–52 trp1Δ2 his ade 2–1 can1–100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.     

Yeast Extract from Express Five Serum-free Medium contains Factors at about 35 kDa,Essential for Growth of <Emphasis Type="Italic">Trichoplusia ni</Emphasis> Insect Cells

The yeast extract (of unknown origin) present in the commercially available serum-free medium ‘Express Five’ contains factors (‘yeast extract factors’) up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors. The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components, which seem to be generally present in yeast extracts, are also required for T. ni proliferation.     

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants

Cell lysis is induced in Schizosaccharomyces pombe ?ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ?ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ?ura4 cells.     

Specific fluorescent antiglobulins for the detection and identification of blastomyces dermatitidis yeast-phase cells

Summary Two lots of rabbit anti-Blastomyces dermatitidis globulins were conjugated with fluorescein isothiocyanate. These reagents stained brightly elements of the yeast and mycelial phases of 10 strains ofB. dermatitidis. In addition, the labeled antibodies cross-reacted with elements of the yeast and mycelial phases of 7 strains ofHistoplasma capsulatum and cells of numerous other heterologous fungi. Adsorption of one lot of labeled antibodies twice with yeast cells ofH. capsulatum and once with elements ofGeotrichum candidum rendered the conjugate specific for the yeast phase ofB. dermatitidis. Three adsorptions with yeast cells ofH. capsulatum followed by a single adsorption with elements ofG. candidum rendered the second conjugate specific for yeast-phase cells ofB. dermatitidis. The specific reagents did not react with the mycelial phase of this fungus.