A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast.

Microbe domestication has a major applied relevance but is still poorly understood from an evolutionary perspective. The yeast Torulaspora delbrueckii is gaining importance for biotechnology but little is known about its population structure, variation in gene content or possible domestication routes. Here, we show that T. delbrueckii is composed of five major clades. Among the three European clades, a lineage associated with the wild arboreal niche is sister to the two other lineages that are linked to anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix-Anthropic clade). Using 64 genomes we assembled the pangenome and the variable genome of T. delbrueckii. A comparison with Saccharomyces cerevisiae indicated that the weight of the variable genome in the pangenome of T. delbrueckii is considerably smaller. An association of gene content and ecology supported the hypothesis that the Mix-Anthropic clade has the most specialized genome and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. In contrast to S. cerevisiae, domestication in T. delbrueckii is not primarily driven by alcoholic fermentation but rather by adaptation to dairy and bread-production niches. This study expands our views on the processes of microbe domestication and on the trajectories leading to adaptation to anthropic niches.

Giant GAL gene clusters for the melibiose-galactose pathway in Torulaspora.

In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5-gene cluster containing GAL 7-1-10-4-2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.

Genome Features of a New Double-Stranded RNA Helper Virus (LBCbarr) from Wine Torulaspora delbrueckii Killer Strains.

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.

Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter.

Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km = 6.5 ± 2.0 mM) and a high-affinity (Km = 0.10 ± 0.01 mM) component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5 ± 1.5 mM). This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry.

Yeast communities of primary and secondary peat swamp forests in southern Thailand.

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

Early adaptation strategies of Saccharomyces cerevisiae and Torulaspora delbrueckii to co-inoculation in high sugar grape must-like media.

Torulaspora delbrueckii and Saccharomyces cerevisiae are yeast species found concurrently in wine. In order to commence fermentation, they adapt to the initial harsh environment, maintaining cellular homeostasis and promoting metabolism. These actions involve an intricate regulation of stress tolerance, growth and metabolic genes. Their phenotypes are influenced by the fermentation environment and physiological state of the cell, but such gene-environment interactions are poorly understood. This study aimed to compare the cell physiology of the two species, through genome-wide analysis of gene expression, coupling Oxford Nanopore MinION and Illumina Hiseq sequencing platforms. The early transcriptional responses to stress, nutrients and cell-to-cell communication were analysed. Particular attention was given to the fundamental gene modulations, leading to an understanding of the physiological changes needed to maintain cellular homeostasis, exit the quiescent state and establish dominance in the fermentation. Our findings suggest the existence of species-specific adaptation strategies in response to growth in a high sugar synthetic grape juice medium.

A Metagenomic-Based Approach for the Characterization of Bacterial Diversity Associated with Spontaneous Malolactic Fermentations in Wine.

This study reports the first application of a next generation sequencing (NGS) analysis. The analysis was designed to monitor the effect of the management of microbial resources associated with alcoholic fermentation on spontaneous malolactic consortium. Together with the analysis of 16S rRNA genes from the metagenome, we monitored the principal parameters linked to MLF (e.g., malic and lactic acid concentration, pH). We encompass seven dissimilar concrete practices to manage microorganisms associated with alcoholic fermentation: Un-inoculated must (UM), pied-de-cuve (PdC), Saccharomyces cerevisiae (SC), S. cerevisiae and Torulaspora delbrueckii co-inoculated and sequentially inoculated, as well as S. cerevisiae and Metschnikowia pulcherrima co-inoculated and sequentially inoculated. Surprisingly, each experimental modes led to different taxonomic composition of the bacterial communities of the malolactic consortia, in terms of prokaryotic phyla and genera. Our findings indicated that, uncontrolled AF (UM, PdC) led to heterogeneous consortia associated with MLF (with a relevant presence of the genera Acetobacter and Gluconobacter), when compared with controlled AF (SC) (showing a clear dominance of the genus Oenococcus). Effectively, the SC trial malic acid was completely degraded in about two weeks after the end of AF, while, on the contrary, malic acid decarboxylation remained uncomplete after 7 weeks in the case of UM and PdC. In addition, for the first time, we demonstrated that both (i) the inoculation of different non-Saccharomyces (T. delbrueckii and M. pulcherrima) and, (ii) the inoculation time of the non-Saccharomyces with respect to S. cerevisiae resources (co-inoculated and sequentially inoculated) influence the composition of the connected MLF consortia, modulating MLF performance. Finally, we demonstrated the first findings of delayed and inhibited MLF when M. pulcherrima, and T. delbrueckii were inoculated, respectively. In addition, as a further control test, we also assessed the effect of the inoculation with Oenococcus oeni and Lactobacillus plantarum at the end of alcoholic fermentation, as MLF starter cultures. Our study suggests the potential interest in the application of NGS analysis, to monitor the effect of alcoholic fermentation on the spontaneous malolactic consortium, in relation to wine.

Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model.

This study describes a screening system for future brewing yeasts focusing on non-Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off-flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by-products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre-fermentation as a bio-flavouring agent for beers that have been post-fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour-forming properties.

Clade- and species-specific features of genome evolution in the Saccharomycetaceae.

Many aspects of the genomes of yeast species in the family Saccharomycetaceae have been well conserved during evolution. They have similar genome sizes, genome contents, and extensive collinearity of gene order along chromosomes. Gene functions can often be inferred reliably by using information from Saccharomyces cerevisiae. Beyond this conservative picture however, there are many instances where a species or a clade diverges substantially from the S. cerevisiae paradigm-for example, by the amplification of a gene family, or by the absence of a biochemical pathway or a protein complex. Here, we review clade-specific features, focusing on genomes sequenced in our laboratory from the post-WGD genera Naumovozyma, Kazachstania and Tetrapisispora, and from the non-WGD species Torulaspora delbrueckii. Examples include the loss of the pathway for histidine synthesis in the cockroach-associated species Tetrapisispora blattae; the presence of a large telomeric GAL gene cluster in To. delbrueckii; losses of the dynein and dynactin complexes in several independent yeast lineages; fragmentation of the MAT locus and loss of the HO gene in Kazachstania africana; and the patchy phylogenetic distribution of RNAi pathway components.

Torulaspora indica a novel yeast species isolated from coal mine soils.

Four yeast strains (APSS 805, APSS 806, APSS 815 and AP-18) belonging to a novel Torulaspora species were isolated from coal mine soils of Singareni in Andhra Pradesh state, India. Another strain (PBA-22) was isolated from agricultural field soil from Gujarat state, India. The vegetative cells of all these strains were round, haploid and produced asci by conjugation between independent cells or mother cell and bud, with rough ascospores, suggesting their possible relation to ascomycetous yeast genus Torulaspora. Phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene and Internal Transcribed Spacer (ITS) regions revealed that, among the five strains, three viz. APSS 805, APSS 806 and APSS 815 have identical sequences. The other two strains (AP-18 and PBA-22) differed from the other three strains in less than 1% nucleotide substitutions in the combined D1/D2 domain and ITS sequences, indicating that all of them (five strains) may belong to the same species. These five strains were closely related to Torulaspora globosa, but showed more than 3-7% sequence divergence from T. globosa and all other species in the genus Torulaspora in the combined sequence analysis of D1/D2 domain and ITS region of rRNA gene. In addition, these strains also showed distinct microsatellite finger-printing pattern from related species and differed in several physiological responses suggesting that these strains belong to a novel species of Torulaspora. We propose to name these strains as Torulaspora indica sp. nov., and designate APSS 805(T) = MTCC 9772 (T) = CBS 12408 (T) as the type strain of this novel species. The Mycobank number of the novel species is MB 563738.

The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein.

To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a nonduplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1 at the silencer. Here, we examined whether the emergence of Sir1 coincided with Orc1 acting as a silencer-binding protein. In the nonduplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC-binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae.

Isolation and characterization of the carbon catabolite-derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii.

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41-293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP-binding region (positions 48-71) and a consensus Ser/Thr protein kinase active site (positions 160-172).

Phenotypic and transcriptional analysis of Saccharomyces cerevisiae during wine fermentation in response to nitrogen nutrition and co-inoculation with Torulaspora delbrueckii.

Nitrogen content of grape musts strongly impacts on fermentation performance and wine metabolite production. As nitrogen is a limiting nutrient in most grape musts, nitrogen supplementation is a common practice that ensures yeast growth during fermentation. However, preferred nitrogen sources -as ammonium- repress the genes related to alternative nitrogen sources consumption, usually involved in aromatic compounds production. Here, we describe the effect of high ammonium doses in Saccharomyces cerevisiae fermentation performance and wine properties, and how it is affected by yeast co-inoculation in mixed (S. cerevisiae + Torulaspora delbrueckii) fermentations. In addition, an RNA-seq analysis allowed us to study the S. cerevisiae transcriptional response to ammonium nutrition and yeast interaction, demonstrating that T. delbrueckii presence affects the global S. cerevisiae transcriptional response, reducing ammonium effects at both phenotypic -fermentation kinetics and metabolite production- and transcriptional levels, under experimental conditions.

Fermentation in fine cocoa type Scavina: Change in standard quality as the effect of use of starters yeast in fermentation.

In the present work we aimed to demonstrate the influence of inoculum starter in support high quality fermentation. Cocoa fermentations were performed in wooden boxes and eight yeasts strains were used in separated fermentations of fine cocoa, type Scavina, as starter inoculum. Temperature, pH, titirable acidity, reducing sugar and free amino acids were evaluated during or after fermentation. The influence of starters yeasts on the decrease of acidity, sugar concentration and free amino acids was significant. The strains Candida parapsilosis, Torulaspora delbrueckii and Pichia kluyveri showed greater changes in the reducing sugar and free amino acids in fermented cocoa beans. These results indicate the ability of yeast used as inoculum starter to modify the end condition and further enhance the quality of fine cocoa beans.

Cloning and functional characterization of xylitol dehydrogenase genes from Issatchenkia orientalis and Torulaspora delbrueckii.

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.

Improved gene disruption method for Torulaspora delbrueckii.

PCR-based disruption cassettes are one of the most commonly used strategies for gene targeting in Saccharomyces cerevisiae. The efficiencies of gene disruption using this conventional method are highly variable among species, and often quite low with nonconventional yeasts. Here we describe an improved strategy to obtain deletion mutants in baker's yeast Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species, present in home-made corn and rye bread dough.

Torulaspora quercuum sp. nov. and Candida pseudohumilis sp. nov., novel yeasts from human and forest habitats.

Strains XZ-46A, XZ-105, XZ-129 and XZ-281(T) isolated from the oral cavities of healthy Tibetan volunteers were revealed to represent two novel ascomycetous yeast species by molecular taxonomic characterizations. Strain XZ-281(T) was most closely related to Candida humilis, but differed from the type strain of the species by eight (1.2%) substitutions in the 26S rRNA gene D1/D2 domain and by >100 (>20%) mismatches in the internal transcribed spacer (ITS) region. Strains XZ-46A, XZ-105 and XZ-129 had identical or similar D1/D2 and ITS sequences with each other and with strain 17YF(T) isolated from a leaf of an oak tree (Quercus sp.). The closest relative of this group was Torulaspora microellipsoides. They differed from the type strain of the species by five (0.9%) substitutions in the D1/D2 domain and >70 (>15%) mismatches in the ITS region. A sexual state was observed in strain 17YF(T), but not in the other four oral strains. An anamorphic name Candida pseudohumilis sp. nov. is proposed for strain XZ-281(T) (=AS 2.3956(T)=CBS 11404(T)) and a teleomorphic name Torulaspora quercuum sp. nov. is proposed for strain 17YF(T) (=AS 2.3768(T)=CBS 11403(T)) and the other three oral strains.

Linking gene expression and oenological traits: Comparison between Torulaspora delbrueckii and Saccharomyces cerevisiae strains.

Wine fermentations typically involve the yeast Saccharomyces cerevisiae. However, many other yeast species participate to the fermentation process, some with interesting oenological traits. In this study the species Torulaspora delbrueckii, used occasionally in mixed or sequential fermentation with S. cerevisiae to improve wine sensory profile, was investigated to understand the physiological differences between the two. Next generation sequencing was used to characterize the transcriptome of T. delbrueckii and highlight the different genomic response of these yeasts during growth under wine-like conditions. Of particular interest were the basic differences in the glucose fermentation pathway and the formation of aromatic and flavour compounds such as glycerol, esters and acetic acid. Paralog genes were missing in glycolysis and glycerol biosynthesis in T. delbrueckii. Results indicate the tendency of T. delbrueckii to produce less acetic acid relied on a higher expression of alcoholic fermentation related genes, whereas acetate esters were influenced by the absence of esterases, ATF1-2. Additionally, in the Δbap2 S. cerevisiae strain, the final concentration of short branched chain ethyl esters (SBCEEs) was related to branched chain amino acid (BCAA) uptake. In conclusion, different adaption strategies are apparent for T. delbrueckii and S. cerevisiae yeasts, an understanding of which will allow winemakers to make better use of such microbial tools to achieve a desired wine sensory outcome.

Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested.

New Insights into the Genome Organization of Yeast Killer Viruses Based on "Atypical" Killer Strains Characterized by High-Throughput Sequencing.

Viral M-dsRNAs encoding yeast killer toxins share similar genomic organization, but no overall sequence identity. The dsRNA full-length sequences of several known M-viruses either have yet to be completed, or they were shorter than estimated by agarose gel electrophoresis. High-throughput sequencing was used to analyze some M-dsRNAs previously sequenced by traditional techniques, and new dsRNAs from atypical killer strains of Saccharomyces cerevisiae and Torulaspora delbrueckii. All dsRNAs expected to be present in a given yeast strain were reliably detected and sequenced, and the previously-known sequences were confirmed. The few discrepancies between viral variants were mostly located around the central poly(A) region. A continuous sequence of the ScV-M2 genome was obtained for the first time. M1 virus was found for the first time in wine yeasts, coexisting with Mbarr-1 virus in T. delbrueckii. Extra 5'- and 3'-sequences were found in all M-genomes. The presence of repeated short sequences in the non-coding 3'-region of most M-genomes indicates that they have a common phylogenetic origin. High identity between amino acid sequences of killer toxins and some unclassified proteins of yeast, bacteria, and wine grapes suggests that killer viruses recruited some sequences from the genome of these organisms, or vice versa, during evolution.