Whole-Genome Transformation of Yeast Promotes Rare Host Mutations with a Single Causative SNP Enhancing Acetic Acid Tolerance.

Whole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes-most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805G→T], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.

Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance.

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 "novel" hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1 The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research.IMPORTANCECandida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of multidrug resistance (MDR) in C. auris is monitored in vitro Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

Expression of stress-related genes in diapause of European corn borer (Ostrinia nubilalis Hbn.).

Diapause is a state of arrested development during which insects cope with many external and internal stressful factors. European corn borer, Ostrinia nubilalis, overwinters as a fifth instar freeze-tolerant diapausing larva. In order to explore diapause-linked stress tolerance processes, the expression of selected genes coding for stress-related proteins-glutathione S-transferase (Gst), thioredoxin (Trx), glutaredoxin (Grx), ferritin (Fer), metallothionein (Mtn), and heat shock proteins Hsp90, Hsc70, Hsp20.4, and Hsp20.1-was assessed in the fat body of diapause-destined, warm (22 °C) and cold (5 °C) acclimated diapausing larvae using the quantitative real-time PCR. Gene expression was normalised to mRNA transcripts for Actin and Rps03, and relative expression was calculated using non-diapausing larvae as a control group. During the initiation phase of diapause, the abundance of mRNA transcripts of Grx, Hsp90, Hsc70, and Hsp20.1 was significantly upregulated, Trx, Fer, Mtn, and Hsp20.1 were unchanged, while only Gst was clearly downregulated in comparison to non-diapause control. Later, in the early phase of diapause, the expression of most genes (except Trx and Hsp20.1) was upregulated in warm-acclimated larvae, while only Trx and Hsp90 were upregulated in cold-acclimated larvae. Furthermore, the relative expression of all genes (except Trx) increased gradually throughout the diapause in cold-acclimated larvae. This result indicates that the half-life of mRNAs is prolonged during diapause at low temperature, which may lead to a gradual accumulation of mRNA transcripts. Our results show that both diapause programming and temperatures affect the expression of stress-related genes in Ostrinia nubilalis.