Showing papers in "Fems Yeast Research in 2001"
TL;DR: Specific yeast communities vectored by beetles, drosophilids, and bees that visit ephemeral flowers, mostly in the genus Hibiscus and in the families Convolvulaceae and Cactaceae, in the Neotropical, Nearctic, and Australian biogeographic regions are studied.
Abstract: We studied specific yeast communities vectored by beetles, drosophilids, and bees that visit ephemeral flowers, mostly in the genus Hibiscus and in the families Convolvulaceae and Cactaceae, in the Neotropical, Nearctic, and Australian biogeographic regions. The communities consist mostly of yeasts in four clades centered around the genera Metschnikowia, Kodamaea, Wickerhamiella, and Starmerella. The largest geographic discontinuity occurs as a function of the nitidulid beetle species that dominate the non-pollinator insect visitors of the flowers. This partitions the New World, where the dominant beetle is in the genus Conotelus, from the Australian biogeographic region, dominated by species of Aethina. Distinct but sympatric insects may also carry radically different yeast communities.
261 citations
TL;DR: DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions, suggesting that respiration is more nitrogen-conserving than fermentation.
Abstract: DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions. The high nitrogen condition displayed elevated levels of expression of genes involved in biosynthesis of macromolecular precursors across the time course as compared to low-nitrogen. In contrast, expression of genes involved in translation and oxidative carbon metabolism were increased in the low-nitrogen condition, suggesting that respiration is more nitrogen-conserving than fermentation. Several genes under glucose repression control were induced in low-nitrogen in spite of very high (17%) external glucose concentrations, but there was no general relief of glucose repression. Expression of many stress response genes was elevated in stationary phase. Some of these genes were expressed regardless of the nitrogen concentration while others were found at higher levels only under high nitrogen conditions. A few genes, FSP2, RGS2, AQY1, YFL030W, were expressed more strongly with nitrogen limitation as compared to other conditions.
135 citations
TL;DR: Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts.
Abstract: Three molecular tools, amplified fragment length polymorphism (AFLPTm), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.
104 citations
TL;DR: Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods and can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule.
Abstract: Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (109 cells), RNA appears undegraded, making it suitable for molecular manipulations.
104 citations
TL;DR: In this article, the evolution of the yeast population during manufacturing and ripening of salami of the Lucania region (southern Italy) was investigated, and the most frequently isolated yeast species were Debaryomyces hansenii and its anamorph Candida famata, and Rhodotorula mucilaginosa.
Abstract: The evolution of the yeast population during manufacturing and ripening of ‘salsiccia sotto sugna’, a typical salami of the Lucania region (southern Italy), was investigated. Four different batches, produced in four farms in Lucania, were studied. Each batch showed a specific yeast population, and the most frequently isolated yeasts belonged to Debaryomyces hansenii and its anamorph Candida famata, and Rhodotorula mucilaginosa. Yarrowia lipolytica was isolated from three sausage batches. The Y. lipolytica isolates were further characterised, in particular for their lipolytic activity on pork fat. Lipolytic activity was maximal at pH 5.5, with oleic and palmitic acids as major free fatty acids produced. The use of randomly amplified polymorphic DNA-polymerase chain reaction allowed the detection of a high genetic heterogeneity among the isolates phenotypically assigned to the species Y. lipolytica.
103 citations
TL;DR: A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp.
Abstract: A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp n (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z kombuchaensis near Zygosaccharomyces lentus The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI
94 citations
TL;DR: The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence.
Abstract: The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l−1 were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.
80 citations
TL;DR: The effects of Hap4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN.PK 113-7D under a variety of growth conditions and it is found that the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain.
Abstract: The tendency of Saccharomyces cerevisiae to favor alcoholic fermentation over respiration is a complication in aerobic, biomass-directed applications of this yeast. Overproduction of Hap4p, a positive transcriptional regulator of genes involved in respiratory metabolism, has been reported to positively affect the balance between respiration and fermentation in aerobic glucose-grown batch cultures. In this study, the effects of HAP4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN.PK 113-7D under a variety of growth conditions. In aerobic glucose-limited chemostat cultures, overexpression of HAP4 increased the specific growth rate at which aerobic fermentation set in by about 10% relative to the isogenic wild-type. Upon relief of glucose-limited conditions, the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain. The effect of Hap4p overproduction was most drastic in aerobic, glucose-grown chemostat cultures in which ammonium was limiting. In such cultures, the biomass yield on glucose was double that of the wild-type.
71 citations
TL;DR: The results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and α-methylglucoside.
Abstract: α-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active α-glucoside transport system(s) present in a wild-type strain, and in strains with defined α-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and α-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (Km∼5 mM) for maltose, and a low affinity (Km∼90 mM) for trehalose. On the other hand, the α-glucoside permease encoded by the AGT1 gene had a high affinity (Km∼7 mM) for trehalose, a low affinity (Km∼18 mM) for maltose and maltotriose, and a very low affinity (Km∼35 mM) for α-methylglucoside.
68 citations
TL;DR: A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome.
Abstract: A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.
66 citations
TL;DR: Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling, which indicates that a regulatory function may be associated with the hexose kinases.
Abstract: Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.
TL;DR: Molecular sequence analysis confirmed that several isolates representing the genus Trichosporon were identical, yet distinct from other members of the genus, and suggested that T. porosum may be related to T. sporotrichoides, within a weakly related clade that includes species such as TrichOSporon laibachii and Trichsporon loubieri.
Abstract: Several isolates representing the genus Trichosporon were collected over a 6-year period from soils in The Netherlands. Based on classical growth tests with carbon and nitrogen compounds these were identical. Three of these (CBS 8396, CBS 8397 and CBS 8522) were subjected to molecular analysis of the D1/D2 region of the large subunit of rDNA. This confirmed that the three strains were identical, yet distinct from other members of the genus. Conspecificity was demonstrated with the type strain (CBS 2040) of Apiotrichum porosum Stautz (1931), with the exception that A. porosum, which had been isolated from exudate of a yew tree, differed morphologically from the soil strains. Based on the identity of DNA base sequences, morphology was not considered to be an adequate parameter to separate otherwise identical strains into two genera. Therefore, the new combination Trichosporon porosum is presented. Based on molecular sequence analysis, T. porosum may be related to T. sporotrichoides, within a weakly related clade that includes species such as Trichosporon laibachii and Trichosporon loubieri. The strains of T. porosum degrade phenolic compounds and hemicelluloses, which are characteristics with potential ecological importance in soil habitats. Characters distinguishing the nine species of the laibachii/loubieri group of species were listed. These include traditionally used tests as well as assimilation patterns of some aliphatic and phenolic compounds. Based on these tests, species such as Trichosporon multisporum and T. laibachii could be separated.
TL;DR: Deletion of GAL6 was shown to have a major impact on biomass and ethanol formation when cells were grown on galactose, and from the data obtained it is speculated that Gal6 may be involved in mRNA degradation of the GAL gene transcripts.
Abstract: The role of the proteins encoded by the GAL80 gene, the MIG1 gene and the GAL6 gene in glucose control of galactose consumption by Saccharomyces cerevisiae was studied by physiological characterisation of various GAL mutant strains. Dynamic experiments with the CEN.PK 113-7D wild-type strain and a Δgal80Δmig1 double-mutant strain in aerobic nitrogen-limited continuous cultivations at a dilution rate of 0.1 h−1, showed simultaneous glucose and galactose consumption by the Δgal80Δmig1 strain. The wild-type strain did not consume galactose in the presence of glucose. Aerobic batch cultivations on glucose–galactose mixtures with the wild-type strain and with recombinant strains with a de-regulated GAL system (the Δgal80Δmig1 strain, a Δgal6 deleted strain, a Δgal6Δgal80Δmig1 triple mutant, and a Δgal6Δgal80Δmig1 triple mutant harbouring a GAL4 high-copy vector) were carried out. Generally, a reduction of glucose control lowered the maximum specific growth rate on glucose and increased the ethanol yield obtained on galactose with more than 100%. In contrast to the wild-type strain, the Δgal6Δgal80Δmig1 triple mutant strain consumed glucose and galactose simultaneously, and this strain also showed the highest ethanol production with an overall ethanol yield of 0.35 g g−1 sugar, which is 17% higher than the yield on glucose obtained with the wild-type strain. GAL80 and MIG1 were demonstrated to be responsible for the majority of the glucose control on the GAL system, whereas GAL6 has a minor role in glucose control. Deletion of GAL6 was shown to have a major impact on biomass and ethanol formation when cells were grown on galactose, and from the data obtained we speculate that Gal6 may be involved in mRNA degradation of the GAL gene transcripts.
TL;DR: Ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival, and the results obtained suggest that a higher ergosterol content facilitates the flux of Glycerol across the plasma membrane of S. cerevisiae cells.
Abstract: The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1Δ strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1Δ strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.
TL;DR: Evidence that Hsp150/Pir2, a member of the Pir family of cell wall proteins, can be extracted from the purified cell walls of Saccharomyces cerevisiae by treatment with beta-mercaptoethanol is presented, demonstrating that at least part of this protein is attached to the cell wall through disulfide bridges.
Abstract: Here we present evidence that Hsp150/Pir2, a member of the Pir family of cell wall proteins, can be extracted from the purified cell walls of Saccharomyces cerevisiae by treatment with β-mercaptoethanol, demonstrating that at least part of this protein is attached to the cell wall through disulfide bridges. We also present evidence that Pir4, another member of this family, is partly secreted to the growth medium. Finally we propose a hypothesis to explain the relationship between the differently localized forms of particular members of the Pir family of cell wall proteins.
TL;DR: This review will focus on recent discoveries concerning the role of phosphoinositides in yeast, a rapidly growing, genetically modifiable and tractable organism of which the genome is fully sequenced.
Abstract: Research on signalling through phosphoinositides has made tremendous advances over the last few years. Studies with budding yeast (Saccharomyces cerevisiae) combine the advantage of a eukaryotic system with those of a rapidly growing, genetically modifiable and tractable organism of which the genome is fully sequenced. Hence, despite some differences in phosphoinositide signalling between mammals and yeast (e.g. the absence of PtdIns(3,4,5)P3), this model organism is at the forefront of phosphoinositide research. In this review we will focus on recent discoveries concerning the role of phosphoinositides in yeast.
TL;DR: It is shown that the uptake of cytoplasmic components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast.
Abstract: In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.
TL;DR: Six new species of the yeast genus Candida are described from their unique nucleotide sequences in the D1/D2 domain of 26S rDNA.
Abstract: Six new species of the yeast genus Candida are described from their unique nucleotide sequences in the D1/D2 domain of 26S rDNA. Five of these species form a clade with Candida tanzawaensis, and the sixth is basal to this group. The new species and their sources of isolation are the following: Candida ambrosiae (type strain NRRL YB-1316, CBS 8844), from insect frass, rotted wood and mushroom fruiting bodies; Candida canberraensis (type strain NRRL YB-2417, CBS 8846), from soil; Candida caryicola (type strain NRRL YB-1499, CBS 8847), from a pignut hickory tree; Candida prunicola (type strain NRRL YB-869, CBS 8848), from exuded gum of a black cherry tree; Candida pyralidae (type strain NRRL Y-27085, CBS 5035), from insect frass; Candida xylopsoci (type strain NRRL Y-27066, CBS 6037), from insect frass.
TL;DR: In this paper, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures.
Abstract: Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source. GOGAT's importance for cellular metabolism is still unclear. For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures. As reference, we did the same experiments with a wild-type strain (VWk43). Intracellular and extracellular metabolites were analysed during different steady states in both strains. The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively). The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria.
TL;DR: The results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.
Abstract: Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the β-1,3-glucan network via a β-1,6-glucan moiety. Addition of gentiobiose or β-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.
TL;DR: The YlODC gene is cloned and disrupted in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus, andstrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence.
Abstract: Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.
TL;DR: A genomic library of the yeast Z. bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli, and the nucleotide sequence and deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species.
Abstract: A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the β-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of β-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.
TL;DR: The results establish that the ERG26 gene is essential for growth and that inhibitors of the Erg26p may represent a new and highly effective class of antifungal agents.
Abstract: The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) was cloned by complementing a Saccharomyces cerevisiae erg26 mutant with a C. albicans genomic library. Sequence analysis showed a 70% identity between the C. albicans and S. cerevisiae ERG26 genes at the amino acid level. Sequential disruption of both copies of the ERG26 gene in the presence of an integrated rescue cassette containing a third copy of the ERG26 gene under the control of the inducible pMAL2 promoter, resulted in cells capable of growing only in the presence of the inducer. The results establish that the ERG26 gene is essential for growth and that inhibitors of the Erg26p may represent a new and highly effective class of antifungal agents.
TL;DR: In this article, a ballistoconidium-forming yeast strain, CH 2.506, isolated from a semi-dried leaf of Parthenocissus sp. collected near Kunming City in Yunnan, China, was shown to be closely related to the non-ballistoconium-forming species Cryptococcus luteolus (Saito) C.E. Skinner and the Bacillus sinensis Li by molecular phylogenetic analysis.
Abstract: A ballistoconidium-forming yeast strain, CH 2.506, isolated from a semi-dried leaf of Parthenocissus sp. collected near Kunming City in Yunnan, China, was shown to be closely related to the non-ballistoconidium-forming species Cryptococcus luteolus (Saito) C.E. Skinner and the ballistoconidium-forming species Bullera sinensis Li by molecular phylogenetic analysis based on 18S rDNA sequencing. This strain was demonstrated to represent a distinct undescribed yeast species by internal transcribed spacer (ITS) region sequence and G+C content comparison and DNA–DNA relatedness, for which the name Bullera kunmingensis sp. nov. is proposed. Meanwhile, the taxonomic relationships among Bullera sinensis and its synonyms B. derxii Nakase & Suzuki and B. alba (Hanna) Derx var. lactis Li, were clarified on the basis of molecular phylogenetic analysis and DNA–DNA reassociation. B. derxii was confirmed to be conspecific with B. sinensis, while B. alba var. lactis was shown to represent a variety of B. sinensis. A new combination, Bullera sinensis Li var. lactis (Li) Bai, Takashima et Nakase, is therefore proposed. Comparative analysis of different types of molecular criteria employed in the present study suggested that when inferring phylogenetic relationships among sibling taxa, sequence data from ITS regions should be interpreted with caution.
TL;DR: Analysis of rDNA sequences suggests that Candida galis occupies a basal position with respect to the Pichia anomala clade, with no obvious sister species, and growth is generally very slow.
Abstract: We report on the yeast community associated with sap fluxes of Maclura tinctoria, family Moraceae, in the dry forest of the Area de Conservacion Guanacaste, Costa Rica. Eleven samples yielded seven hitherto undescribed ascomycetous yeasts in the genera Candida and Myxozyma. We describe the two most abundant as new species. Candida galis utilizes very few carbon compounds limited to some alcohols and acids. Analysis of rDNA sequences suggests that it occupies a basal position with respect to the Pichia anomala clade, with no obvious sister species. Candida ortonii is also restricted in nutritional breadth, and growth is generally very slow. It is a sister species to Candida nemodendra. The type cultures are: C. galis, strain UWO(PS)00-159.2=CBS 8842; and C. ortonii, strain UWO(PS)00-159.3=CBS 8843.
TL;DR: On the basis of the phylogenetic analyses the type strain CBS 4214(T) together with four other C. hungaricus isolates were closely related to Bullera armeniaca and Bullera crocea, while strain CBS 6569 was much more similar to Cystofilobasidium than to the other
Abstract: Cryptococcus hungaricus is a basidiomycetous yeast with the abilities to synthesize carotenoid pigments and to grow under psychrophile conditions. Six C. hungaricus strains have been isolated so far from different habitats. In this study we wished to clarify the relationships amongst them. Morphological and physiological characters, mitochondrial DNA restriction profiles, and the presence of mycoviruses were examined. Internal transcribed spacers together with the 5.8S rDNA, the D1/D2 region of 26S rDNA, and partial sequences of the 18S rRNA gene were also analysed. On the basis of the phylogenetic analyses the type strain CBS 4214T together with four other C. hungaricus isolates were closely related to Bullera armeniaca and Bullera crocea, while strain CBS 6569 was much more similar to Cystofilobasidium than to the other C. hungaricus isolates.
TL;DR: Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed.
Abstract: Differences in glycosylation between the natural α-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH2-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man5GlcNAc2 to Man9GlcNAc2, of which Man8GlcNAc2 was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man7GlcNAc2 up to Man14GlcNAc2, with Man8GlcNAc2 and Man9GlcNAc2 being the most abundant structures. In both enzymes the first glycosylation site (Asn5) is always glycosylated. However, Asn537 and Asn540 are only partially glycosylated in an alternate manner.
TL;DR: The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.
Abstract: Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.