Yeast最新文献

With the steady rise in antifungal resistance amongst clinically important yeasts, antifungal drug discovery remains of the utmost importance. To determine the potential of some honeys as alternative antifungal agents, we quantified the antifungal activity of 12 Western Australian honey samples, two Manuka honey samples and an artificial honey against 10 yeast isolates including clinical and reference strains. Results showed that the tested honeys varied in activity, and yeasts species also differed in susceptibility, with minimum inhibitory concentrations (MICs) determined by broth microdilution ranging from 8% to >44% w/v honey. Honeys with the highest overall activity were derived from Blackbutt (Eucalyptus patens), Jarrah (E. marginata), and Karri (E. diversicolor). The optical density of each MIC microtitre plate was determined after incubation and showed that at relatively low concentrations of honey the growth of all yeasts was enhanced compared to the untreated control, whereas at and above approximately 12% w/v, honeys exerted a dose-dependent growth inhibitory effect, the extent of which varied by honey type. Time-kill studies with 64% w/v honey showed that all eight of the natural honeys tested had greater fungicidal activity than the comparator artificial honey. Our findings suggest that the specific nectar-derived phytochemicals present within each honey play an important role in antifungal activity, and support the notion that activity is due to a combination of factors including osmotic activity, hydrogen peroxide and phytochemical compounds. These data indicate that honey is worthy of further investigation as a potential therapeutic agent for superficial yeast infections.

The evolution of protein sequence is driven not only by factors directly related to protein function and shape but also by nonfunctional factors. Such factors in protein evolution might be categorized as those connected to energetic costs, synthesis efficiency, and avoidance of misfolding and toxicity. A common approach to studying them is correlational analysis contrasting them with some characteristics of the protein, like amino acid composition, but these features are interdependent. To avoid possible bias, empirical studies are needed, and not enough work has been done to date. In this review, we describe the role of nonfunctional factors in protein evolution and present an experimental approach using yeast as a suitable model organism. The focus of the proposed approach is on the potential negative impact on the fitness of mutations that change protein properties not related to function and the frequency of mutations that change these properties. Experimental results of testing the misfolding avoidance hypothesis as an explanation for why highly expressed proteins evolve slowly are inconsistent with correlational research results. Therefore, more efforts should be made to empirically test the effects of nonfunctional factors in protein evolution and to contrast these results with the results of the correlational analysis approach.

The CRISPR-Cas9 system is extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae, and other yeast species. We have previously reported the use of an inducible CRISPR-Cas9 system in Candida glabrata, which allows genome editing but also the study of double-strand break (DSB) repair. We report, in this study, a comparable system for C. glabrata, relying on a new plasmid, which is more stable than the previous one. We also report the use of this plasmid to induce DSBs in two additional human pathogens, Candida bracarensis and Candida nivariensis. We examine lethality induced by an in vivo DSB in the three species and describe the different types of nonhomologous end-joining (NHEJ) events detected in these three pathogens.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying a variety of critical cellular processes. Traditional methods to knock in or -out at specific yeast loci utilize polymerase chain reaction-based techniques, in which marker cassettes with gene-specific homologies are integrated into the genome via homologous recombination. While simple and cost-effective, these methods are limited by marker availability when multiple edits are desired. More recently, CRISPR-Cas9 technology has introduced methods to edit the yeast genome without the need for selectable markers. Although efficient, this method is hindered by additional reagents and lengthy protocols to design and test unique guide RNAs and donor templates for each desired edit. In this study, we have combined these two approaches and have developed a highly efficient economical method to edit the yeast genome marker-free. We have designed two universal donor templates that efficiently repair commonly used selectable markers when targeted by a novel guideRNA-Cas9 designed to promoter regions in Ashbya gossypii found in most integration modules. Furthermore, we find our newly designed guideRNA-Cas9 successfully multiplexes when multiple markers are present. Using these new tools, we have significantly improved the cost and efficiency to generate single or multiple marker-free genetic modifications. In this study, we demonstrate the effectiveness of these new tools by marker-free ablating PRC1, PEP4, and PRB1 vacuolar proteases typically inactivated before many biochemical and membrane-trafficking studies using budding yeast.

The toxicity of non-proteinogenic amino acids has been known for decades. Numerous reports describe their antimicrobial/anticancer potential. However, these molecules are often toxic to the host as well; thus, a synthetic lethality approach that reduces the dose of these toxins while maintaining toxicity can be beneficial. Here we investigate synthetic lethality between toxic amino acids, the retrograde pathway, and molecular chaperones. In Saccharomyces cerevisiae, mitochondrial retrograde (RTG) pathway activation induces transcription of RTG-target genes to replenish alpha-ketoglutarate and its downstream product glutamate; both metabolites are required for arginine and lysine biosynthesis. We previously reported that tolerance of canavanine, a toxic arginine derivative, requires an intact RTG pathway, and low-dose canavanine exposure reduces the expression of RTG-target genes. Here we show that only a few of the examined chaperone mutants are sensitive to sublethal doses of canavanine. To predict synthetic lethality potential between RTG-target genes and chaperones, we measured the expression of RTG-target genes in canavanine-sensitive and canavanine-tolerant chaperone mutants. Most RTG-target genes were induced in all chaperone mutants starved for arginine; the same trend was not observed under lysine starvation. Canavanine exposure under arginine starvation attenuated and even reversed RTG-target-gene expression in the tested chaperone mutants. Importantly, under nearly all tested genetic and pharmacological conditions, the expression of IDH1 and/or IDH2 was induced. In agreement, idh1 and idh2 mutants are sensitive to canavanine and thialysine and show synthetic growth inhibition with chaperone mutants. Overall, we show that inhibiting molecular chaperones, RTG-target genes, or both can sensitize cells to low doses of toxic amino acids.

Cellobiose lipids are surface-active compounds or biological detergents produced by distinct Basidiomycetes yeasts, of which the most and best-described ones belong to the Ustilaginomycetes class. The molecules display slight variation in congener type, which is linked to the hydroxylation position of the long fatty acid, acetylation profile of the cellobiose unit, and presence or absence of the short fatty acid. In general, this variation is strain specific. Although cellobiose lipid biosynthesis has been described for about 11 yeast species, hitherto only two types of biosynthetic gene clusters are identified, and this for only three species. This work adds six more biosynthetic gene clusters and describes for the first time a novel type of cellobiose lipid biosynthetic cluster with a simplified architecture related to specific cellobiose lipids synthesized by Trichosporonaceae family members.

Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.

To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest.

Smk1 is a MAPK homolog in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore wall assembly. During this program, haploid cells are surrounded by a layer of mannan and then a layer of glucan. These inner layers of the spore wall resemble the vegetative cell wall. Next, the outer layers consisting of chitin/chitosan and then dityrosine are assembled. The outer layers are spore-specific and provide protection against environmental stressors. Smk1 is required for the proper assembly of spore walls. However, the protective properties of the outer layers have limited our understanding of how Smk1 controls this morphogenetic program. Mutants lacking the chitin deacetylases, Cda1 and Cda2, form spores that lack the outer layers of the spore wall. In this study, cda1,2∆ cells were used to demonstrate that Smk1 promotes deposition of the glucan layer of the spore wall through the partially redundant glucan synthases Gsc2 and Fks3. Although Gsc2 is localized to sites of spore wall assembly in the wild type, it is mislocalized in the mother cell cytoplasm in the smk1∆ mutant. These findings suggest that Smk1 controls assembly of the spore wall by regulating the localization of Gsc2 during sporogenesis.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' → 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' → 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.