SciCombinator

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Concept: Yeasts

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In order to better understand the differences in xylose metabolism between natural xylose-utilizing Pichia stipitis and metabolically engineered Saccharomyces cerevisiae, we constructed a series of recombinant S. cerevisiae strains with different xylose reductase/xylitol dehydrogenase/xylulokinase activity ratios by integrating xylitol dehydrogenase gene (XYL2) into the chromosome with variable copies and heterogeneously expressing xylose reductase gene (XYL1) and endogenous xylulokinase gene (XKS1). The strain with the highest specific xylose uptake rate and ethanol productivity on pure xylose fermentation was selected to compare to P. stipitis under oxygen-limited condition. Physiological and enzymatic comparison showed that they have different patterns of xylose metabolism and NADPH generation.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Brewing, Beer, Yeasts, Saccharomycetes, Saccharomycetaceae

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The search for new compounds with antifungal activity is accelerating due to rising yeast and fungal resistance to commonly prescribed drugs. Among the molecules being investigated, plant lectins can be highlighted. The present work shows the potential of six plant lectins which were tested in vitro against yeasts of medical importance, Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus gattii, Cryptococcus neoformans, Malassezia pachydermatis, Rhodotorula sp. and Trichosporon sp. Broth microdilution susceptibility testing was performed in accordance with standard protocols to evaluate antifungal activity. Minimum inhibitory concentration (MIC) was determined at 80 % yeast growth inhibition, whereas the minimum fungicidal concentration (MFC) was evaluated after making the subcultures of each dilution. Only C. parapsilosis growth was inhibited by the lectins tested. Abelmoschus esculentus lectin showed the highest MIC (0.97 μg ml(-1)). Lectins from Canavalia brasiliensis, Mucuna pruriens and Clitoria fairchildiana presented the highest MFC at (3.90 μg ml(-1)). These results encourage further studies with wider yeast strain selections, and open new perspectives for the development of pharmacological molecules.

Concepts: Fungus, Yeast, Cryptococcus neoformans, Candida albicans, Candida, Yeasts, Concanavalin A, Candida parapsilosis

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Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2- MCA) as an identification tool for distinguishing between 16 Candida spp., i.e. C. albicans, C. bracarensis, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. inconspicua, C. kefyr, C. krusei, C. lipolytica, C. lusitaniae, C. nivariensis, C. norvegensis, C. parapsilosis, C. tropicalis and C. sojae, and Saccharomyces cerevisiae and one species pair, i.e. C. metaspsilosis/C. orthopsilosis. Starting from a cultured isolate, ITS2-MCA led to differentiation of these species within 6 h. According to our findings, ITS2-MCA offers a simple, rapid and cost-effective method for identification of cultured isolates of the clinically most relevant and prevalent Candida species. Further studies will be necessary to evaluate how it performs on mixed samples and clinical samples.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Candida albicans, Ascomycota, Candidiasis, Yeasts

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Cryptococcus gattii is responsible for an expanding epidemic of serious infections in Western Canada and the Northwestern United States (NW), some patients with these infections respond poorly to azole antifungals, and high azole MICs have been reported in NW C. gattii. In this study, 25 NW C. gattii had higher MICs for multiple azoles (but not amphotericin B) than did 34 non-NW C. gattii or 20 Cryptococcus neoformans strains. We therefore examined the roles in azole resistance of overexpression of or mutations in the gene (ERG11) encoding the azole target enzyme. ERG11/ACT1 mRNA ratios were higher in C. gattii than in C. neoformans, but these ratios did not differ in NW and non-NW C. gattii strains, nor did they correlate with fluconazole MICs within any group. Three NW C. gattii strains with low azole MICs and 2 with high azole MICs had deduced Erg11p sequences that differed at one or more position from that of the fully-sequenced NW C. gattii strain R265. However, the azole MICs of conditional S. cerevisiae erg11 mutants expressing the 5 variant ERG11s were within 2-fold of the azole MICs of S. cerevisiae expressing the ERG11s from C. gattii R265, non-NW C. gattii strain WM276, or C. neoformans strains H99 or JEC21. We conclude that neither ERG11 overexpression nor variations in ERG11 coding sequences was responsible for the high azole MICs observed in the NW C. gattii strains we studied.

Concepts: Fungus, Yeast, Cryptococcus neoformans, Flucytosine, Basidiomycota, Cryptococcus, Yeasts, Cryptococcosis

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The opportunistic fungal pathogen Candida albicans is one of the leading agents of life threatening infections affecting immunocompromised individuals. Many factors make C. albicans a successful pathogen. These include the ability to switch between yeast and invasive hyphal morphologies in addition to an arsenal of cell wall virulence factors such as lipases, proteases, dismutases and adhesins that promote the attachment to host, a prerequisite for invasive growth. We have previously characterized Hwp2 a C. albicans cell wall protein which we found necessary for proper oxidative stress, biofilm formation and adhesion to host cells. Baker’s yeast Saccharomyces cerevisiae also possesses adhesins that promote aggregation and flocculence. Flo11 is one such adhesin that has sequence similarity to Hwp2. Here we determined that transforming an HWP2 cassette can complement the lack of filamentation of an S. cerevisiae flo11 null strain, and impart on S. cerevisiae adhesive properties similar to a pathogen.

Concepts: Bacteria, Microbiology, Fungus, Yeast, Cell cycle, Saccharomyces cerevisiae, Ascomycota, Yeasts

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Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Ascomycetes in either the Saccharomycotina or the Taphrinomycotina have one to three GGTs, whereas members of the Pezizomycotina have two to four GGTs. A Bayesian analysis indicates there are three well-supported main clades of GGTs in the Ascomycota. 1) A Saccharomycotina and a Taphrinomycotina-specific GGT sub-clade form a yeast main clade. This clade has the three relatively well-characterized fungal GGTs: (Saccharomyces cerevisiae CIS2 and Schizosaccharomyces pombe Ggt1 and Ggt2) and most of its members have all 14 of the highly conserved and critical amino acids that are found in GGTs in the other kingdoms. 2) In contrast, a main clade (GGT3) differs in 11 of the 14 highly conserved amino acids that are found in GGTs in the other kingdoms. All of the 44 Pezizomycotina analyzed have either one or two GGT3s. 3) There is a Pezizomycotina-only GGT clade that has two well-supported sub-clades (GGT1 and GGT2); this clade differs in only two of the 14 highly conserved amino acids found in GGTs in the other kingdoms. Because the Pezizomycotina GGTs differ in apparently critical amino acids from the cross-kingdom consensus, a putative GGT from Colletotrichum graminicola, a member of the Pezizomycotina, was cloned and the protein product was expressed as a secreted protein in Pichia pastoris. A GGT enzyme assay of the P. pastoris supernatant showed that the recombinant protein was active, thereby demonstrating that CgGGT1 is a bona fide GGT.

Concepts: Amino acid, Fungus, Yeast, Saccharomyces cerevisiae, Ascomycota, Yeasts, Saccharomycetes, Saccharomycotina

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The ability of Candida shehatae, Saccharomyces cerevisiae, or the combination of these two yeasts in converting the mixed sugar composition of rice hull hydrolysate (RHH) as substrate for ethanol production is presented. In shake flask experiments, co-cultures showed ethanol yields (Y) of 0.42 and 0.51 in synthetic medium simulating the sugar composition of RHH and in RHH, respectively, with both glucose and xylose being completely depleted, while pure cultures of C. shehatae produced slightly lower ethanol yields (0.40). Experiments were scaled-up to bioreactors, in which anaerobiosis and oxygen limitation conditions were tested. Bioreactor co-cultures produced similar ethanol yields in both conditions (0.50-0.51) in synthetic medium, while in RHH, yields of 0.48 and 0.44 were obtained, respectively. The results showed near-theoretical yields of ethanol. Results suggest the feasibility of co-cultures of C. shehatae, a newly isolated strain, and S. cerevisiae in RHH as substrate for second-generation ethanol production.

Concepts: Fungus, Yeast, Model organism, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Yeasts, Saccharomycetes

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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is emerging in laboratories as a new diagnostic tool for microorganism identification. We prospectively compared the performances of the Biflex III-Biotyper (Bruker Daltonics) and of the Axima (Shimadzu)-SARAMIS (Anagnostec) systems for the identification of 312 yeasts isolated from clinical specimens (249 Candida spp. including 19 C. albicans and 230 non-albicans species, 63 isolates belonging to different species of the genera Saccharomyces (20), Rhodotorula (8), Cryptococcus (8) Trichosporon (7), Pichia (7), Geotrichum (12) and 1 Sporopachydermia cereana). Species were identified by using routine conventional phenotypical methods and ITS sequencing in case of discrepancy. We used expanded thresholds for species identification (log score ≥ 1.7 with 3 identical consecutive propositions and no discrepancy between the duplicates for the Bruker Daltonics system and similitude ≥ 40% with 5 successive identical propositions and no discrepancy between the duplicates for the Shimadzu system). Of the 312 isolates, 272 (87.2%) and 258 (82.7%) were successfully identified on Bruker Daltonics and Shimadzu systems, respectively. All isolates were successfully identified within the most frequent and clinically-relevant Candida species on the two systems. Non valid results corresponded mainly to species not or poorly represented in the databases. A major misidentification was observed for 2 isolates (0.6%) on Bruker Daltonics and 4 isolates (1.3 %) on the Shimadzu system. In conclusion, the performances of the Bruker Daltonics and the Shimazu systems for yeast identification were good and comparable under clinical routine conditions, despite their differences in sample preparation, database content and spectra analysis.

Concepts: Mass spectrometry, Species, Yeast, Candida albicans, Matrix-assisted laser desorption/ionization, Candidiasis, Yeasts, Bruker

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P-bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping-enzymes where mRNA decay can occur. Here we described the characterization of P-bodies in the fission yeast Schizosaccharomyces pombe. Most informations on the property and function of P-bodies stem from studies in the distant related budding yeast Saccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike in S. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping-enzyme, with a limited-similarity to plant Edc4/Varicose that required for the assembly of P-body was identified (TAP-MALDI/MS/MS). Pdc1 interacts with Dcp2 through its C-terminal and contains a colied-coli region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridging different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although not required for the interaction between Dcp1 and Dcp2, our data suggests that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping-enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein composition between different species and might help to provide insight into their evolutional path.

Concepts: Protein, Molecular biology, Fungus, Yeast, Model organism, Saccharomyces cerevisiae, Ascomycota, Yeasts

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Due to increased occurrence of infections and food spoilage caused by yeast, there is an unmet need for new antifungal agents. The arginine-β-(2,5,7-tri-tert-butylindol-3-yl)alanine-arginine (R-Tbt-R) motif was previously proved useful in the design of an antifungal tripeptide. Here, an array of peptidomimetics based on this motif was investigated for antifungal and hemolytic activity. The five most promising modified tetrapeptide analogues ( 6: and 9-12: contain an additional C-terminal hydrophobic residue, and these were found to exhibit antifungal activity against Saccharomyces cerevisiae (MIC 6 and 12 μg mL(-1)) and Zygosaccharomyces bailii (MIC 6-25 μg mL(-1)). Four compounds ( 6: and 9-11: , had limited hemolytic activity (<10% hemolysis at 8 × MIC). Determination of their killing kinetics revealed that compound 9: displayed fungicidal effect. Testing against cells from an S. cerevisiae deletion mutant library indicated that interaction with yeast-specific fungal sphingolipids, most likely constitutes a crucial step in the mode of action of these. Interestingly, a lack of activity of peptidomimetics 6: and 9-11: towards Candida spp. was shown to be due to degradation or sequestering by the yeast. Due to their ultrashort nature, antifungal activity and low toxicity the four compounds may have potential as leads for novel preservatives.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Yeasts, Saccharomycetes, Saccharomycetaceae