ALBERT

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Fission yeast is a powerful model organism that has provided insights on important cellular processes thanks to the easiness of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in S. pombe . In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe .

Description: CaGpi14 is the catalytic subunit of the first mannosyltransferase that is involved in the GPI biosynthetic pathway in Candida albicans . We show that CaGPI14 is able to rescue a conditionally lethal gpi14 mutant of S . cerevisiae unlike its mammalian homolog. The depletion of this enzyme in C . albicans leads to severe growth defects, besides causing deficiencies in GPI anchor levels. In addition, CaGpi14 depletion results in cell wall defects and upregulation of the cell wall integrity response pathway. This in turn appears to trigger the osmotic stress dependent activation of the HOG1 pathway and an upregulation of HOG1 as well as its downstream target, SKO1 , a known suppressor of expression of hyphae specific genes. Consistent with this, mutants of CaGPI14 are unable to undergo hyphal transformations in different hyphae inducing medium, under conditions that produce abundant hyphae in the wild type cells. Hyphal defects in the CaGPI14 mutants could not be attributed either to reduced PKC activation or to defective Ras signaling in these cells but appeared to be driven by perturbations in the HOG1 pathway.

Description: Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess if the strains found in wasp intestines are characterized by peculiar traits. We analyzed strains isolated from social wasps’ intestine and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to represent a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains: their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved to safeguard not only the natural variance of this microorganism, but also the interest of wine-makers, who could take advantages from the exploitation of their phenotypic variability.

Description: During the last decade, the use of innovative yeast cultures of both Saccharomyces cerevisiae and non- Saccharomyces yeasts as alternative tools to manage the winemaking process have turned the oenology industry. Although the contribution of different yeast species to wine quality during fermentation is increasingly understood, the information about their role in wine ageing over-lees is really scarce. This work aims to analyse the incidence of 3 non- Saccharomyces yeast species of oenological interest ( Torulaspora delbrueckii , Lachancea thermotolerans and Metschnikowia pulcherrima ) and of a commercial mannoprotein-overproducer S . cerevisiae strain compared with a conventional industrial yeast strain during wine ageing over-lees. To evaluate their incidence in mouthfeel properties of wine after 4 months of ageing, mannoprotein content of wines was evaluated, together with other wine analytic parameters such as colour and aroma, biogenic amines and amino acids profile. Some differences among the studied parameters were observed during the study, especially regarding the mannoprotein concentration of wines. Our results suggest that the use of T . delbrueckii lees in wine ageing is a useful tool for the improvement of overall wine quality by notably increasing mannoproteins, reaching values higher than obtained using a S . cerevisiae overproducer strain. This article is protected by copyright. All rights reserved.

Description: The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms’ viability in these conditions were revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to cell wall and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment. This phenomenon is explained by the compensatory synthesis of chitin. Results clearly indicate that the cell wall structure/composition belongs to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage of cell wall proteins during yeast desiccation can be an important factor leading to the cell death. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi . Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural was used for cultivations of L . starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% of hydrolysate, at pH 6. At pH 5, growth stopped already upon addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose/ acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH-increase. This phenomenon was utilised to establish a pH-stat fed batch cultivation, where, after an initial feeding hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under those conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield 0.1 g/ g carbon source was obtained. Lipid composition was similar in all cultivations with C18:1 and C16:0 being the most abundant fatty acids. This article is protected by copyright. All rights reserved.

Description: Metabolic effects induced by resveratrol have been associated mainly with consumption of high-calorie diets; however, its effects under standard or low-calorie diets remain unclear. To better understand the interactions between resveratrol and cellular energy levels, we used Saccharomyces cerevisiae as a model. Herein it is shown that resveratrol: i) decreased cell viability in energy dependent manner; ii) lessening of cell viability occurred specifically when cells are under cellular respiration; and iii) inhibition of oxygen consumption in state 4 occurs in low and standard energy levels, whereas in high energy levels promotes oxygen consumption. These findings indicate that the effects of resveratrol are dependent on the cellular energy status and linked to metabolic respiration. Importantly, our study also revealed that S. cerevisiae is a suitable and useful model to elucidate the molecular targets of resveratrol under different nutritional status. This article is protected by copyright. All rights reserved.

Description: In several grape varieties the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoids related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aroma after being hydrolyzed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora , are known to predominate during the first stages of alcoholic fermentation. Although non- Saccharomyces yeast strains have a well-recognized genetic diversity, the understanding of their impact on wine flavor richness is still incipient. In this work, eleven Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate, and 2- phenylethyl acetate are significantly synthetized by H . vineae strains. Levels of these compounds found in fermentations with eleven H . vineae different strains were 1-2 orders of magnitude higher than those measured in fermentations with a known S . cerevisiae wine strain. Implications on winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate, and 2- phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: The present research was undertaken to study the probiotic characteristics of Pichia kudriavzevii isolated from frozen idli batter. PCR amplification with 18S rRNA primers confirmed Pichia kudriavzevii , a xylose utilizing probiotic strain. It was resistant to physiological concentrations of bile salts, pepsin and pancreatic enzyme. It also showed efficient auto-aggregation as well as co-aggregation ability with 4 commercial probiotic yeasts and exhibited good hydrophobicity in xylene and toluene. The strain inhibited the growth of 13 enteropathogens and showed commensal relationship with 4 commercial probiotic yeast and bacteria. Moreover, it was resistant to 30 antibiotics with different modes of action. The yeast exhibited thermo-tolerance up to 95 °C for 2 h. The cell-free supernatants were also found to be heat-stable indicating the presence of thermo-stable secondary metabolites. Hence, it could be exploited as starter culture, co-culture or probiotic in the preparation of fermented products or incorporated in heat-able foods as well.

Description: Blastobotrys adeninivorans (syn. Arxula adeninivorans ) is a non-conventional, non-pathogenic, imperfect, haploid, yeast, belonging to the Subphylum Saccharomycotina , which has to date received comparatively little attention from researchers. It possesses unusual properties such as thermo- and osmotolerance, and a broad substrate spectrum. Depending on the cultivation temperature B. ( A. ) adeninivorans exhibits different morphological forms and various post-translational modifications and protein expression properties which are strongly correlated with the morphology. The genome has been completely sequenced and in addition, there is a well-developed transformation/expression platform, which makes rapid, simple gene manipulations possible. This yeast species is a very good host for homologous and heterologous gene expression and is also a useful gene donor. B. ( A. ) adeninivorans is able to use a very wide range of substrates as carbon and/or nitrogen sources and is an interesting organism due to the presence of many metabolic pathways, for example, degradation of n-butanol, purines, and tannin. In addition, its unusual properties and robustness make it a useful bio-component for whole cell biosensors. There are currently a number of products on the market produced by B. ( A. ) adeninivorans and further investigation may contribute further innovative solutions for current challenges that exist in the biotechnology industry. Additionally it may become a useful alternative to existing commercial yeast strains and as a model organism in research. In this review we present information relevant to the exploitation of B. ( A. ) adeninivorans in research and industrial settings. This article is protected by copyright. All rights reserved.

Description: Rhodotorula taiwanensis RS1 (Rt) is a high-aluminum (Al)-tolerant yeast that can survive Al at concentrations up to 200 mM. In this study, we compared Rt with an Al-sensitive congeneric strain, R. mucilaginosa AKU 4812 (Rm) and Al sensitive mutant 1 (alsm1) of Rt, to explore the Al tolerance mechanisms of Rt. The growth of Rm was completely inhibited by 1 mM Al, but that of Rt was not inhibited until Al concentration was more than 70 mM. The growth of alsm1 was inhibited much more by 70 mM and 100 mM Al than that of Rt. Compared with Rm cells, Rt cells accumulated less Al in the cell wall and cytoplasm. A time-course analysis showed that Al was absorbed by Rm cells much more rapidly than by Rt cells when exposed to the same Al concentration. Meanwhile, the Al content of alsm1 was higher than that of Rt. Although the cell wall of Rt was thicker than that of alsm1 and Rm under control and 0.1 mM Al, that of Rt was thinner than that of alsm1 under 70 mM Al despite that their cell walls were thickened. The alcian blue adsorption was lower and cell wall zeta-potential was higher in Rt and alsm1 than in Rm, indicating a less negative charge of cell wall of Rt and alsm1 than that of Rm. Taken together, the less negatively charged cell wall of Rt may restrict the adsorption of cationic Al in cells, potentially contributing to its high Al tolerance. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Conversion of byproduct from biodiesel production glycerol to high-value compounds is of great importance. Ethanol is considered as promising product of glycerol bioconversion. Methylotrophic thermotolerant yeast Ogataea (Hansenula) polymorpha is of great interest for this purpose as byproduct glycerol contains methanol and heavy metals as contaminants and this yeast utilizes methanol and is relatively resistant to heavy metals. Besides, O. polymorpha shows robust grows on glycerol and produces ethanol from different carbon sources. Thermotolerance of this yeast is an additional advantage that allows increasing the fermentation temperature to 45° - 48 °C leading to the increase of the rate of the fermentation process and to drop of the costs of distillation. Wild-type strain of O. polymorpha produces insignificant amounts of ethanol from glycerol (0.8 g/L). Overexpression of PDC1 coding for pyruvate decarboxylase enhanced ethanol production till 3.1 g/L whereas simultaneous overexpression of PDC1 and ADH1 (coding for alcohol dehydrogenase) led to further increase in ethanol production from glycerol. Moreover, the increased temperature of fermentation up to 45 °C stimulated the production of ethanol from glycerol used as the only carbon source up to 5.0 g/L which exceeds data obtained by methylotrophic yeast strains reported so far.

Description: Debaryomyces hansenii is a halotolerant yeast with a high biotechnological potential, particularly in food industry. However research in this yeast is limited by its molecular peculiarities. In this review we summarise the state of the art about research in this microorganisms, describing both pros and cons. We discuss (i) its halotolerance, (ii) the molecular factors involved in saline and osmotic stress, (iii) its high gene density and ambiguous CUG decoding, and (iv) its biotechnological and medical interests. We trust that all the bottlenecks in its study will be soon overcome and D. hansenii will become a fundamental organism for food biotechnological processes.

Description: The purpose of this review is to introduce Metschnikowia to the yeast researcher community and to convince readers that the genus is a worthwhile object of study in developmental biology, genetics, ecology, and biotechnology. Metschnikowia sits at the foundation of modern immunology, having been instrumental in the discovery of animal phagocytosis. Some 81 species form a monophyletic group within the Metschnikowiaceae, which also include the smaller genus Clavispora and a few clades of Candida species. The family stands out by the habit of forming, by meiosis, only two ascospores, which in Metschnikowia are needle shaped. In some cases, the spores can reach enormous proportions, exceeding 200 µm in length; in others, ascus formation is preceded by the development of chlamydospores. The adaptive value of such features remains to be elucidated. Extensive genetic studies are lacking, but attempts to apply methods developed for model species have been successful. Some species are found at the plant-insect interface whereas others are pathogens of aquatic animals and have served as model organisms in the exploration of host-parasite theory. Some species are globally distributed and others exhibit extreme endemism. Many species are remarkably easy to recover by sampling their known habitats. M. pulcherrima and close relatives may play an important role in wine quality and produce pulcherrimin, an iron-dipeptide complex that can interfere with the growth of other microorganisms. Some symbiotic species incapable of growth in culture media have been assigned to the genus, but their kinship with the group remains to be demonstrated.

Description: Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% comparing to that of HSA (〉1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 involved in protein folding and Sec1 and Sly1 involved in vesicle trafficking. We evaluated the possible effects of individual overexpressing these secretion helper factors on the secretion of IL2-HSA in P. pastoris . Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1, and Sly1 improved the secretion level of IL2-HSA to approximately 2.3, 1.9, 2.2, 2.5, and 1.9 folds of that in the control strain respectively in shake flasks. We evaluated the changes of mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2, and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris , which provided new candidates for combinatorial engineering in future study. This article is protected by copyright. All rights reserved.

Description: The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the S. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini-chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of nonessential gene deletions will add to the toolkits available for the study of biological processes in S. pombe .

Description: No abstract is available for this article.

Description: During the last decade, the molecular basis for gene expression noise was mostly deciphered, helping understanding how gene regulation is controlled and how the generation of cell-to-cell non-genetic heterogeneity is modulated through noise. In the same time, the functional importance of phenotypic heterogeneity among cell populations was recognized and widely involved in major biological phenomena. Surprisingly, only few works connect these two highly active research fields, most of them having been obtained with Saccharomyces cerevisiae . This organism has long been the preferred model for studying many aspects of gene expression noise, especially revealing that evolution seems to act to either increase or decrease gene expression noise depending on whether the associated phenotypic heterogeneity is beneficial or deleterious to the population. Nevertheless direct evidences of phenotypic consequences of noise differences are often lacking in spite of this evolutionary tendency. This rarity is probably due to the complex relationships between mean and noise levels, making difficult the study of the sole effect of noise, and also to the problems caused by the detection of cell-to-cell expression variability of native functional proteins allowing testing specific phenotypic effects. Despite these difficulties, the widespread use of gene expression noise as an experimental parameter at equal mean expression levels to test phenotypic consequences would often help changing the way to explain cell population behavior beyond the simple consideration of average expression levels, and constitute a major step towards single-cell biology.

Description: During fermentation processes, Saccharomyces cerevisiae cells are exposed to multiple stresses, including a high concentration of ethanol that represents toxicity through intracellular reactive oxygen species (ROS) generation. We previously reported that proline protected yeast cells from damage caused by various stresses, such as freezing and ethanol. As an antioxidant, proline is suggested to scavenge intracellular ROS. In this study, we examined the role of intracellular proline during ethanol treatment in S. cerevisiae strains that accumulate different concentrations of proline. When cultured in YPD medium, there was a significant accumulation of proline in the put1 mutant strain, which is deficient in proline oxidase, in the stationary phase. Expression of the mutant PRO1 gene, which encodes the γ-glutamyl kinase variant (Asp154Asn or Ile150Thr) with desensitization to feedback inhibition by proline, in the put1 mutant strain showed a prominent increase in proline content, as compared with that of the wild-type strain. The oxidation level was clearly increased in wild-type cells after exposure to ethanol, indicating that the generation of ROS occurred. Interestingly, proline accumulation significantly reduces the ROS level and increases the survival rate of yeast cells in the stationary phase under ethanol stress conditions. However, there was not a clear correlation between proline content and survival rate in yeast cells. An appropriate level of intracellular proline in yeast might be important for its stress-protective effect. Hence, the engineering of proline metabolism could be promising for breeding stress-tolerant industrial yeast strains. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: The proteolytic clipping of histone tails have recently emerged as a novel form of irreversible post-translational modifications (PTMs) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone specific proteases. Here, we employed various biochemical methods to report a histone H3 specific proteolytic activity from budding yeast. Our results demonstrate that the H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), the yeast H3 proteolytic activity was not inhibited by Stefin B. Together; our findings suggest the existence of novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity along with the physiological inhibitor in yeast suggests an interesting molecular mechanism that regulates the activity of histone proteases. This article is protected by copyright. All rights reserved.

Description: S. cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose, and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast Ogataea polymorpha ( Op ) in the light of this hypothesis. The MAL1 has extended substrate specificity and its properties are strikingly similar to resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between the Op MAL1 and ancMALS. The Op MAL1 represents an α-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides) confirming the requirement of Thr at respective position for this function. Differential scanning fluorimetry (DSF) of catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at -1 subsite. DSF assay was in good accordance with affinity (K m ) and inhibition (K i ) data of the enzyme for tested substrates indicating the power of the method to predict substrate binding. Deletion of either maltase ( MAL1 ) or α-glucoside permease ( MAL2 ) gene in Op abolished growth of yeast on MAL1 substrates confirming requirement of both proteins for usage of these sugars. This article is protected by copyright. All rights reserved.

Description: Acute tryptophan depletion is used to induce low levels of serotonin in the brain. This method has been widely used in psychiatric studies to evaluate the effect of low levels of serotonin, and is generally considered a safe and reversible procedure. Here we use the budding yeast Saccharomyces cerevisiae to study the effects of tryptophan depletion on growth rate upon exposure to DNA damaging agents. Surprisingly, we find that budding yeast undergoing tryptophan depletion are more sensitive to DNA damaging agents such as methyl methanesulfonate (MMS) and hydroxyurea (HU). We find that this defect is independent of several DNA repair pathways such as homologous recombination, base excision repair, and translesion synthesis, and that this damage sensitivity is not due to impaired S-phase signaling. Upon further analysis, we find that the DNA damage sensitivity of tryptophan depletion is likely due to impaired protein synthesis. These studies describe an important source of variance in budding yeast when using tryptophan as an auxotrophic marker particularly on studies focusing on DNA repair, and suggest that further testing of the effect of tryptophan depletion on DNA repair in mammalian cells is warranted.

Description: Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time novel primer sets were developed to determine the expression of ATG 1, ATG 17 and ATG 29 genes. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains, and were independent from the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production.

Description: The present work describes the behaviour of Lachancea thermotolerans and Saccharomyces cerevisiae in pure, co-cultured and sequential fermentations in cv. Emir grape must. Faster fermentation rates were observed in the wine made with pure culture of S. cerevisiae and the wine produced with simultaneously inoculated cultures of L. thermotolerans and S. cerevisiae . Both L. thermotolerans and S. cerevisiae gave high population numbers. L. thermotolerans usage in mixed and sequential cultures led to an increase in final total acidity content in the wines, varying in the range of 5.40-6.28 g/l as tartaric acid compared to pure culture S. cerevisiae that gave the lowest level of total acidity (5 g/l). The increase was in the order of 1.18-2.06 g/l total acidity. Increase in final acidity by the usage of L. thermotolerans might be useful to improve wines with low acidity due to global climate change. Volatile acidity levels as acetic acid ranged from 0.53 g/l - 0.73 g/l, while the concentration of ethyl alcohol varied between 10.76% v/v and 11.62% v/v. Sequential fermentations of wines and pure culture fermentation of L. thermotolerans resulted in reduction in the concentrations of acetaldehyde, higher alcohols with exception of n-propanol and esters. According to the sensory analysis, wine obtained with sequential inoculation of L. thermotolerans followed by inoculation of S. cerevisiae after 24 h, and simultaneous inoculation of these yeasts was the most preferred.

Description: No abstract is available for this article.

Description: Acetolactate synthase is a mitochondrial enzyme that catalyzes the conversion of two pyruvate molecules to an acetolactate molecule with release of carbon dioxide. The overexpression of the truncated version of the corresponding gene ILV2 that codes for presumably cytosolic acetolactate synthase in the yeast Saccharomyces cerevisiae, led to a decrease in intracellular pyruvate concentration. This recombinant strain was also characterized by a 4-fold increase in glycerol production with a concomitant 1.8-fold reduction in ethanol production when compared to that of the wild-type strain under anaerobic conditions in a glucose alcoholic fermentation. This article is protected by copyright. All rights reserved.

Description: Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation, antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R . mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. We applied 2D PAGE, 2D-DIGE, SDS-PAGE separation followed by gel slicing, and filter-aided sample preparation after optimization of protein extraction, and carried out protein identification and analysis by tandem mass spectrometry. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied not all spots returned a valid identification, and no carotenogenic enzymes were identified, even upon application of different database search strategies. The subsequent application of three proteomic analysis workflows combining sample preparation strategies, techniques, and data analysis approaches with varying levels of sensitivity, provided a picture of the data analysis depth that can be reached with different analytical resources, and resulted in a plethora of information on R . mucilaginosa metabolism. However also in these cases no proteins related to the carotenogenic pathway were identified thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. This article is protected by copyright. All rights reserved.

Description: The twenty-first century has brought new opportunities and challenges to yeast culture collections, whether they are long-standing or recently established. Basic functions such as archiving, characterizing and distributing yeasts continue, but with expanded responsibilities and emerging opportunities. In addition to a number of well-known, large public repositories, there are dozens of smaller public collections that differ in the range of species and strains preserved, field of emphasis and services offered. Several collections have converted their catalogs to comprehensive databases and synchronize them continuously through public services, making it easier for users worldwide to locate a suitable source for specific yeast strains and the data associated with these yeasts. In-house research such as yeast taxonomy continues to be important at culture collections. Because yeast culture collections preserve a broad diversity of species and strains within a species, they are able to make discoveries in many other areas as well, such as biotechnology, functional, comparative and evolution genomics, bioprocesses and novel products. Due to the implementation of the Convention of Biological Diversity (CBD) and the Nagoya Protocol (NP), there are new requirements for both depositors and users to ensure that yeasts were collected following proper procedures and to guarantee that the country of origin will be considered if benefits arise from its utilisation. Intellectual Property Rights (IPRs) are extremely relevant to the current Access and Benefit Sharing (ABS) mechanisms; most research and development that involve genetic resources and associated traditional knowledge will be subject to this topic. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: In this study, the yeast microbiota of naturally fermented black olives made from cv. Gemlik grown in three different districts of Çukurova region in Turkey was investigated. Fermentations were conducted for 180 days in three different brines including NaCl 10% (w/v), NaCl 8% (w/v) and NaCl 8% (w/v) added with glucose 0.5%. Totally 223 yeasts were isolated and then identified by PCR-RFLP analysis of the 5.8S ITS rRNA region and sequence information for the D1/D2 domains of 26S rRNA gene. A broad range of yeast biodiversity was identified, including 8 genera and 9 species. Candida boidinii (41%), Wickerhamomyces anomalus (32%) and Saccharomyces sp. (18%) were predominant yeasts throughout the fermentations. To a lesser extend, the other species Candida aaseri, Meyerozyma sp. , Zygoascus hellenicus, Pichia kudriavzevii, Schwanniomyces etchellsii and Candida atlantica were also members of the olive fermenting microbiota. In Tarsus and Bahçe districts Candida boidinii and in Serinyol district Saccharomyces sp. were the most frequently identified species. Wickerhamomyces anomalus was the most frequently isolated species (by 48% of total yeasts) in NaCl 10% brines. Candida boidinii was the most dominant species in the brines including NaCl 8% and NaCl 8% + glucose 0.5% with the frequency of 42 and 61%, respectively. At the end of the 180 days of fermentation, total acidity values of the brines were in the range of 1.04-8.1 g/l lactic acid. This article is protected by copyright. All rights reserved.

Description: Caciofiore della Sibilla is a specialty ewe's milk cheese traditionally manufactured in a foothill area of the Marche region (Central Italy) with a crude extract of fresh young leaves of Carlina acanthifolia All. subsp. acanthifolia as a coagulating agent . The fungal dynamics and diversity of this specialty cheese were investigated throughout the manufacturing and 20-day ripening process, using a combined PCR-DGGE approach. The fungal biota of a control ewe's milk cheese manufactured with the same batch of milk coagulated with a commercial animal rennet was also monitored by PCR-DGGE, in order to investigate the contribution of the peculiar vegetable coagulant to the fungal diversity and dynamics of the cheese. Based on the overall results collected, the raw milk and the dairy environment represented the main sources of fungal contamination, with a marginal or null contribution of thistle rennet to the fungal diversity and dynamics of Caciofiore della Sibilla cheese.

Description: Apoptosis-inducing factor (AIF) is a conserved flavoprotein localized in the mitochondria, inducing apoptosis after translocation into the nucleus. However, its role in the important fungal pathogen, Candida albicans , remains to be investigated. In this study, we find that the C. albicans AIF protein Aif1, similar to its homologs in other organisms, is localized at the mitochondria and translocated into the nucleus under apoptosis-inducing conditions. Moreover, deletion of AIF1 causes attenuated apoptosis in this pathogen under apoptosis-inducing conditions, such as the treatment of 2 mM H 2 O 2 , 10 mM acetic acid or 0.08 mg/L caspofungin, and its overexpression enhances this process. Interestingly, under the treatment of high levels of these agents lead to reversed sensitivity of aif1 Δ/Δ and the overexpression strain AIF1ov . In addition, the virulence of C. albicans is not affected by deletion or overexpression of AIF1 . Hence, C. albicans Aif1, as a mitochondria-localized protein, plays a dual role in regulation of cell death under different concentrations of the stress-caused agents.

Description: Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviors has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeast are endless. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Draft genomes of 55 strains representing all known large-spored Metschnikowia species were used to construct a robust phylogeny of these yeasts found in association with flower-visiting insects. The genomes were annotated with reference to Clavispora lusitaniae . From 3016 orthologs identified, 1061 were present in all strains with enough overlap to generate alignments of 500 bp or more. We constructed trees for all those alignments and evaluated their accuracy from their ability to resolve each of 22 sets of conspecifics correctly as sister taxa. Neighbour-joining better identified species membership compared to maximum likelihood as did trees based on larger gene alignments. However, correct species assignment was not predictive of a gene's ability to resolve deeper topologies, which were more reliably identified by maximum likelihood analyses of large concatenations. Specifically, 14 trees based on independent concatenations ca . 100 kb in length were topologically consistent with a tree based on a single, large concatenation (1,410,065 positions), lending a high degree of confidence to the stability of the phylogeny. A tree based on a concatenation of intergenic regions (112,136 positions) was also congruent. Again, the best predictor of phylogenetic signal quality of a gene was the size of the alignment. Bootstraps were not always good indicators of phylogenetic quality, as they were sometimes affected by clade size. A tree constructed from a presence-absence matrix of all annotated genes was remarkably congruent with sequence-based phylogenies, suggesting that gain or loss of genes is worth exploring further as phylogenetically significant events. This article is protected by copyright. All rights reserved.

Description: Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with outstanding dynamic range. Here we evaluate the usefulness of the 19 kDa luciferase Nanoluc (Nluc) derived from the deep sea shrimp Oplophorus gracilirostris as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair growth of yeast cells and exhibit half-lives of 40 and 5 minutes, respectively. Commercial substrate (Nano-Glo®) is compatible with detection of yNluc bioluminescence in less than 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter ( P CYC1-HSE ), we find a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrate that bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein report on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate.

Description: During fermentation S. cerevisiae releases secondary metabolic products into the medium such as acetaldehyde, ableto react withanthocyanins producingmore stable derived pigments. However, very limited reports are found about non- Saccharomyces effects on grape fermentation. In this work, six non- Saccharomyces yeast strains, belonging to the genera Metschnikowia and Hanseniaspora , were screened for their effect on red wine color and winemaking capacity in pure culture conditions and mixed with Saccharomyces . An artificial red grape must was prepared containing a phenolic extract of Tannat grapes that allows monitoring changes of key phenol parameters during fermentation but without skin solids in the medium. When fermented in pure cultures S. cerevisiae produced higher concentration of acetaldehyde and vitisin B (acetaldehyde reaction dependent) compared to M. pulcherrima M00/09G, Hanseniaspora guillermondii T06/09G, Hanseniaspora opuntiae T06/01G, Hanseniaspora vineae T02/05F, Hanseniaspora clermontiae ( A10/82Fand C10/54F). However, co-fermentation of H.vineae and H. clermontiae species with S. cerevisiae resulted in a significant higher concentration of acetaldehydewhen comparedwiththe pure S. cerevisiae control. HPLC-DAD-MSanalysis confirmed an increasedformation of vitisin B in co-fermentation treatments when compared to the pure Saccharomyces fermentation, suggesting the key role of acetaldehyde.

Description: No abstract is available for this article.

Description: The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants - mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap such as: mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, likely due to genetic divergence. Future studies in this area will benefit from the access to a massive array of well annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map.This article is protected by copyright. All rights reserved.

Description: The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii ) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type ( MATa or MATα ) was determined for each hoΔ mutant, and was stable under sporulation inducing conditions, showing that the switching system was totally non-functional. The hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii . These haploid strains containing genetic markers will be useful tools for doing genetic analyses in N. castellii . Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.

Description: The ERG6 gene encodes a S-adenosylmethionine dependent sterol C-24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation both in K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied with increased expression of genes encoding last steps of ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell´s susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to Sec14 family. Decreased activity of Kl Pdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells.

Description: Naa10 is a N α -terminal acetyltransferase that, in a complex with its auxiliary subunit Naa15, co-translationally acetylates the α-amino group of newly synthetized proteins as they emerge from the ribosome. Roughly 40-50% of the human proteome is acetylated by Naa10, rendering this an enzyme with one of the most broad substrate ranges known. Recently, we reported an X-linked disorder of infancy, Ogden syndrome, in two families harboring a c.109 T 〉 C (p.Ser37Pro) variant in NAA10. In the present study we performed in-depth characterization of a yeast model of Ogden syndrome. Stress tests and proteomic analyses suggest that the S37P mutation disrupts Naa10 function and reduces cellular fitness during heat shock, possibly due to dysregulation of chaperone expression and accumulation. Microarray and RNA-seq revealed a pseudo-diploid gene expression profile in ΔNaa10 cells, likely responsible for a mating defect. In conclusion, the data presented here further support the disruptive nature of the S37P/Ogden mutation and identify affected cellular processes potentially contributing to the severe phenotype seen in Ogden syndrome. Data are available via GEO under identifier GSE86482 or with ProteomeXchange under identifier PXD004923.

Description: Naumovozyma castellii ( Saccharomyces castellii ) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained an increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Due to the suitable phylogenetic distance to S. cerevisiae the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii , focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution.

Description: No abstract is available for this article.

Description: The fission yeast Schizosaccharomyces pombe is a useful experimental system for studying the organization of chromosomes within the cell nucleus. S. pombe has a small genome that is organized into three chromosomes. The small size of the genome and the small number of chromosomes are advantageous for cytological and genome-wide studies of chromosomes; however, the small size of the nucleus impedes microscopic observations owing to limits in spatial resolution during imaging. Recent advances in microscopy, such as super-resolution microscopy, have greatly expanded the use of S. pombe as a model organism in a wide range of studies. In addition, biochemical studies, such as chromatin immunoprecipitation and chromosome conformation capture, have provided complementary approaches. Here, we review the spatial organization of the S. pombe genome as determined by a combination of cytological and biochemical studies.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Structural genes of phospholipid biosynthesis in the yeast S. cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4 which binds to ICRE (inositol/choline-responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE-dependent gene activation is not only repressed by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10-fold. Repression of ICRE-dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE-dependent gene expression by phosphorylation-dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction.

Description: The genus Paracoccidioides is composed of thermal dimorphic fungi, causative agents of paracoccidioidomycosis, one of the most frequent systemic mycoses in Latin America. Mitochondria have sophisticated machinery for ATP production, which involves metabolic pathways such as citric acid and glyoxylate cycles, electron transport chain and oxidative phosphorylation. In addition, this organelle performs a variety of functions in the cell, working as an exceptional metabolic signaling center that contributes to cellular stress responses, as autophagy and apoptosis in eukaryotic organisms. The aim of this work was to perform a descriptive proteomic analysis of mitochondria in Paracoccidioides lutzii , yeast cells. After mitochondria fractionation, samples enriched in mitochondrial proteins were digested with trypsin and analyzed using a NanoUPLC-MS E system (Waters Corporation, Manchester, UK). Ours results revealed that the established protocol for purification of mitochondria was very effective for P. lutzii , and 298 proteins were identified as primarily mitochondrial, in our analysis. To our knowledge, this is the first compilation of mitochondrial proteins from P. lutzii , to date. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Wine is a complex beverage, comprising thousands of metabolites that are produced through the action of a plethora of yeasts and bacteria during fermentation of grape must. These microbial communities originate in the vineyard and the winery and reflect the influence of several factors including grape variety, geographical location, climate, vineyard spraying, technological practices, processing stage and season (pre-harvest, harvest, post-harvest). Vineyard and winery microbial communities have the potential to participate during fermentation and influence wine flavour and aroma. Therefore, there is an enormous interest in isolating and characterising these communities, particularly non- Saccharomyces yeast species to increase wine flavour diversity, while also exploting regional signature microbial populations to enhance regionality. In this review we describe the role and relevance of the main non- Saccharomyces yeast species found in vineyards and wineries. This includes the latest reports covering the application of these species for winemaking; and the biotechnological characteristics and potential applications of non- Saccharomyces species in other areas. In particular, we focus attention on the species for which molecular and genomic tools and resources are available for study.

Description: No abstract is available for this article.

Description: Phosphatidic acid phosphatase (PAP) catalyzes the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5 fold with the addition of Mg 2+ (1 mM) in the assay mixture, which means that most of the PAP activity was due to Mg 2+ -dependent PAP enzymes (e.g. Pah1, App1). In contrast, N-ethylmaleimide (NEM) potently inhibited PAP activity indicating the presence of NEM-sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbors only a small amount of activity. PAP activity was regulated in a growth-dependent manner being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica .

Description: Secretory defects cause transcriptional repression of ribosome biogenesis in Saccharomyces cerevisiae . However, the molecular mechanism underlying secretory defect-induced transcriptional repression of ribosome biogenesis remains to be fully elucidated. In this study, we demonstrated that the Arp2/3 complex was required for reduction of ribosome protein gene expression in response to defective secretion by addition of tunicamycin. Two cmd1 mutants, cmd1-228 and cmd1-239 that cause mislocalization of calmodulin and defective mitotic spindle formation, respectively, fail to interact with Arc35, a component of the Arp2/3 complex. These mutants also caused defects in the reduction of ribosome protein gene expression induced by secretory blockade. A mutation in TUB4 ( tub4-1 ), whose product has an essential function in microtubule organization, showed a similar response. In addition, we showed that the response to a secretory defect required SUN protein Mps3, which was localized at the nuclear envelope and involved in spindle pole body assembly. These results suggest that the Arp2/3 complex is required to transmit signals resulting from secretory blockade, and that the spindle pole body functions as a transit point from cytoplasm to Mps3 at the nuclear envelope.

Description: Cryptococcus is predominantly an AIDS-related pathogen that causes significant morbidity and mortality in immunocompromised patients. Research studies have historically focused on understanding how the organism causes human disease through the use of in vivo and in vitro model systems to identify virulence factors. Cryptococcus is not an obligate pathogen, however, as human-human transmission is either absent or rare. Selection in the environment must thus be invoked to shape the evolution of this taxa, and directly influences genotypic and trait diversity. Importantly, the evolution and maintenance of pathogenicity must also stem directly from environmental selection. To that end, here we examine abiotic and biotic stresses in the environment, and discuss how they could shape the factors that are commonly identified as important virulence traits. We identify a number of important unanswered questions about Cryptococcus diversity and evolution that are critical for understanding this deadly pathogen, and discuss how implementation of modern sampling and genomic tools could be utilized to answer these questions.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Trichosporon asahii is a pathogenic basidiomycetous yeast. Individual strains of T. asahii have different colony morphologies. However, it is not clear whether cell surface phenotypes differ among the colony morphologies. Here we characterized the cell surface hydrophobicity and analyzed the carbohydrate contents of the cell surface polysaccharides in T. asahii clinical isolates with various colony morphologies. Among the three distinctive colony morphologies obtained from one clinical isolate, the white-type morphology exhibited higher hydrophobicity. The hydrophobicity of heat-killed T. asahii cells was greatly reduced after periodate oxidation of the cell surface carbohydrates. Furthermore, the cell wall and extracellular polysaccharide components differed among the morphologies. Our results suggest that T. asahii cell surface hydrophobicity is affected by cell surface carbohydrate composition.

Description: DNA helicases are ATP-driven motor proteins which translocate along DNA capable of dismantling DNA-DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe , which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. This article is protected by copyright. All rights reserved.

Description: PCR-mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N-terminus or the C-terminus of targeted genes. Although strategies of C-terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C-terminal epitope tagging is not practical for all proteins. For example, a C-terminal tag may affect protein function or a protein may get cleaved or processed resulting in the loss of the epitope tag. Therefore, N-terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N-terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre-LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N-terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N-terminal tagging which combines two previous strategies for epitope tagging in a haploid strain. These “N-ICE” plasmids were constructed so that non-essential and essential genes can be N -terminally 3 × FLAG tagged and expressed from an I nducible promoter ( GAL1 ), C onstitutive promoters ( CYC1 or PYK1 ), or the E ndogenous promoter. We have validated the N-ICE plasmid system by N-terminal tagging two non-essential genes ( SET1 and SET2) and two essential genes ( ERG11 and PKC1 ).

Description: No abstract is available for this article.

Description: Here we describe a set of tools to facilitate the use of maltose and the MAL32 promoter for regulated gene expression in yeast, alone or in combination with the GAL1 promoter. Using fluorescent protein reporters we find that under non-inducing conditions the MAL32 promoter exhibits a low basal level of expression, similar to the GAL1 promoter, and that both promoters can be induced independently of each other using the respective sugars, maltose and galactose. While their repression upon glucose addition is immediate and complete, we found that the MAL32 and GAL1 promoter each exhibit distinct induction kinetics. A set of plasmids is available to facilitate the application of the MAL32 promoter for chromosomal modifications using PCR targeting and for plasmid based gene expression. This article is protected by copyright. All rights reserved.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: Mutations in adenine biosynthesis pathway genes ADE1 & ADE2 have been conventionally used to score for prion [ PSI + ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [ psi - ] yeast appear red on YPD medium due to accumulation of a red intermediate compound in vacuoles. In [ PSI + ] yeast, partial inactivation of the translation termination factor, Sup35 protein, due to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red color development in ade1 & ade2 mutant yeast requires reduced-glutathione which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate (CAIR) into vacuoles that develops the red color. Here, we hypothesized that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red color in ade1 or ade2 yeast. Indeed, when we over-expressed amyloid forming human proteins TDP-43, Aβ-42 & Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast, yielded some white color colonies. Further, the white color eventually reverted back to red upon turning-off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, & ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress & toxicity.

Description: Geotrichum candidum is an ubiquitous yeast, and an essential component in the production of many soft cheeses. We developed a Multi-Locus Sequence Typing (MLST) scheme with five retained loci ( NUP116 , URA1 , URA3 , SAPT4 , PLB3 ) which were sufficiently divergent to distinguish 40 Sequence Types (STs) among the 67  G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a PCR inter-LTR scheme, a fast and reproducible RAPD-like method for G. candidum , to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analyzed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species.