The halotolerant Debaryomyces hansenii, the Cinderella of non-conventional yeasts.

Debaryomyces hansenii is a halotolerant yeast with a high biotechnological potential, particularly in the food industry. However, research in this yeast is limited by its molecular peculiarities. In this review we summarize the state of the art of research in this microorganisms, describing both pros and cons. We discuss (i) its halotolerance, (ii) the molecular factors involved in saline and osmotic stress, (iii) its high gene density and ambiguous CUG decoding, and (iv) its biotechnological and medical interests. We trust that all the bottlenecks in its study will soon be overcome and D. hansenii will become a fundamental organism for food biotechnological processes. Copyright © 2016 John Wiley & Sons, Ltd.

Filling annotation gaps in yeast genomes using genome-wide contact maps.

MOTIVATIONS: De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. RESULTS: Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. AVAILABILITY AND IMPLEMENTATION: The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. CONTACT: romain.koszul@pasteur.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

Population polymorphism of nuclear mitochondrial DNA insertions reveals widespread diploidy associated with loss of heterozygosity in Debaryomyces hansenii.

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767(T), and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.

Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis.

BACKGROUND: To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available. RESULTS: A dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset. Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not confidently resolve whether Candida glabrata or Saccharomyces castellii lies at the base of the WGD clade. CONCLUSION: We have constructed robust phylogenies for fungi based on whole genome analysis. Overall, our phylogenies provide strong support for the classification of phyla, sub-phyla, classes and orders. We have resolved the relationship of the classes Leotiomyctes and Sordariomycetes, and have identified two classes within the CTG clade of the Saccharomycotina that may correlate with sexual status.