Expression of a Malassezia codon optimized mCherry fluorescent protein in a bicistronic vector

The use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labelling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

The Morphological Features of Adaptation of the Yeast of the Genus Saccharomyces to Extreme Values of Glucose and Ethanol

The influence of extreme values of glucose and ethanol on the morphological properties of the yeast of the genus Saccharomyces was studied. The ability of strains S. cerevisiae Y-503, S. cerevisiae DAW-3a, and S. oviformis M-12X to adapt to stress was found. The dependence of the functional state and morphological properties of the studied strains on ploidy was shown. It was established that the cell forms of S. cerevisiae Y-503 and S. oviformis M-12X were diverse, the cells of the haploid strain S. cerevisiae DAW-3a were characterized by a round shape and smaller sizes compared to the polyploid strain Y-503 and diploid M-12X. Adaptation to different concentrations of glucose was accompanied by a change in the shape of the cells Y-503 and DAW-3a, while the sizes of cells were practically un-changed. Under conditions of simultaneous osmotic and ethanol stress in the cells of the studied strains, the presence of granular cytoplasm was revealed, which is associated with the accumulation of reserve nutrients. A change in the shape of the cells, form, surface, profile, color, size of colonies was detected. The study of the properties of the yeast of the genus Saccharomyces in the process of adaptation to extreme conditions may serve as the basis for the selection of strains that are promising for use in food biotechnology.

Whole-Genome Sequence of an Isogenic Haploid Strain, Saccharomyces cerevisiae IR-2idA30(MATa), Established from the Industrial Diploid Strain IR-2

We present the draft genome sequence of an isogenic haploid strain, IR-2idA30(MAT a), established from Saccharomyces cerevisiae IR-2. Assembly of long reads and previously obtained contigs from the genome of diploid IR-2 resulted in 50 contigs, and the variations and sequencing errors were corrected by short reads.

Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

Calcineurin Is an Antagonist to PKA Protein Phosphorylation Required for Postmating Filamentation and Virulence, While PP2A Is Required for Viability in Ustilago maydis

Ustilago maydis is a dimorphic basidiomycete and the causal agent of corn smut disease. It serves as a genetic model for understanding dimorphism, pathogenicity, and mating response in filamentous fungi. Previous studies indicated the importance of regulated cAMP-dependent protein kinase A (PKA) for filamentous growth and pathogenicity in U. maydis. The roles of two protein phosphatases that potentially act antagonistically to PKA were assessed. A reverse genetics approach to mutate the catalytic subunits of calcineurin (CN, protein phosphatase [PP]2B) and PP2A in U. maydis was employed. A mutation in the CN catalytic subunit ucn1 caused a dramatic multiple-budding phenotype and mating between two ucn1 mutants was severely reduced. The pathogenicity of ucn1 mutant strains was also severely reduced, even in a solopathogenic haploid strain. Importantly, mutations disrupting protein phosphorylation by PKA were epistatic to ucn1 mutation, indicating a major role of ucn1 as a PKA antagonistic phosphatase. Genetic and inhibitor studies indicated that the U. maydis PP2A catalytic subunit gene (upa2) was essential.