A multiphase multiobjective dynamic genome-scale model shows different redox balancing among yeast species of the saccharomyces genus in fermentation

Yeasts constitute over 1,500 species with great potential for biotechnology. Still, the yeast Saccharomyces cerevisiae dominates industrial applications, and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here, we propose a multiphase multiobjective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kinetic …

Characterisation of four species of the genus Kluyveromyces by mitochondrial DNA restriction analysis

Summary In the present work, we determine the relationships at the within-species level among strains of Kluyveromyces dobzhanskii, K. lactis, K. marxianus, and K. thermotolerans, through the restriction analysis of their mtDNAs. The three first species showed a high level of intraspecific mtDNA divergence, this polymorphism is correlated to the varieties or species defined according to the original taxonomy of the genus, which is in concordance with that shown by other phenotypic or genotypic markers codified for by the nuclear genome. In these species, the analysis of the relationships among strains based on mtDNA restriction data agrees with previous classifications based on morphologica…

Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking

Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains. In this work we evaluated the usefulness of three dif…

Natural hybrids fromSaccharomyces cerevisiae,Saccharomyces bayanusandSaccharomyces kudriavzeviiin wine fermentations

Several wine isolates of Saccharomyces were analysed for six molecular markers, five nuclear and one mitochondrial, and new natural interspecific hybrids were identified. The molecular characterization of these Saccharomyces hybrids was performed based on the restriction analysis of five nuclear genes ( CAT8 , CYR1 , GSY1 , MET6 and OPY1 , located in different chromosomes), the ribosomal region encompassing the 5.8S rRNA gene and the two internal transcribed spacers, and sequence analysis of the mitochondrial gene COX2 . This method allowed us to identify and characterize new hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii , between S. cerevisiae and Saccharomyces ba…

The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity

New double- and triple-hybrid Saccharomyces yeasts were characterized using PCR-restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double-hybrid S. cerevisiae×S. kudriavzevii in non-fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in …

GroEL and the maintenance of bacterial endosymbiosis

Many eukaryotic organisms have symbiotic associations with obligate intracellular bacteria. The clonal transmission of endosymbionts between host generations should lead to the irreversible fixation of slightly deleterious mutations in their non-recombinant genome by genetic drift. However, the stability of endosymbiosis indicates that some mechanism is involved in the amelioration of the effects of these mutations. We propose that the chaperone GroEL was involved in the acquisition of an endosymbiotic lifestyle not only by means of its over-production, as proposed by Moran, but also by its adaptive evolution mediated by positive selection to improve the interaction with the unstable endosy…

Genome structure reveals the diversity of mating mechanisms in Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrids, and the genomic instability that promotes phenotypic diversity

Interspecific hybridization has played an important role in the evolution of eukaryotic organisms by favouring genetic interchange between divergent lineages to generate new phenotypic diversity involved in the adaptation to new environments. This way, hybridization between Saccharomyces species, involving the fusion between their metabolic capabilities, is a recurrent adaptive strategy in industrial environments. In the present study, whole-genome sequences of natural hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii were obtained to unveil the mechanisms involved in the origin and evolution of hybrids, as well as the ecological and geographic contexts in which sponta…

A multi-phase multi-objective dynamic genome-scale model shows different redox balancing among yeast species in fermentation

ABSTRACTYeasts constitute over 1500 species with great potential for biotechnology. Still, the yeastSaccharomyces cerevisiaedominates industrial applications and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here we propose a multi-phase multi-objective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kin…

Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome-c oxidase II gene.

A phylogenetic analysis of 17 species belonging to the genus Kluyveromyces and 12 reference and outgroup species was performed using mitochondrial cytochrome-c oxidase II gene sequences. The genus Kluyveromyces appears as a polyphyletic taxon formed by species included within the following four main groups. The Kluyveromyces phaffii group encompasses the species Kluyveromyces blattae, K. phaffii and Kluyveromyces yarrowii. The Kluyveromyces marxianus group is a monophyletic group consisting of the species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, K. marxianus and Kluyveromyces wickerhamii. The monophyletic Kluyveromyces thermotolerans group is formed by K. th…

Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA

We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (affinis and obscura) does not correspond to the phylogeny of the group, which shows two well-defined clusters (the Nearctic affinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency to evolve toward high A+T values. This leads to a "saturation" effect that (1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDN…

Mitochondrial inheritance and fermentative : oxidative balance in hybrids between Saccharomyces cerevisiae and Saccharomyces uvarum.

Breeding between Saccharomyces species is a useful tool for obtaining improved wine yeast strains, combining fermentative features of parental species. In this work, 25 artificial Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed by spore conjugation. A multi-locus PCR‐restriction fragment length polymorphism (PCR‐RFLP) analysis, targeting six nuclear gene markers and the ribosomal region including the 5.8S rRNA gene and the two internal transcribed spacers, showed that the hybrid genome is the result of two chromosome sets, one coming from S. cerevisiae and the other from S. uvarum. Mitochondrial DNA (mtDNA) typing showed uniparental inheritance in all hybrids. Furth…

The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection

The heat-shock protein GroEL is a double-ring-structured chaperonin that assists the folding of many newly synthesized proteins in Escherichia coli and the refolding in vitro, with the cochaperonin GroES, of conformationally damaged proteins. This protein is constitutively overexpressed in the primary symbiotic bacteria of many insects, constituting approximately 10% of the total protein in Buchnera, the primary endosymbiont of aphids. In the present study, we perform a maximum likelihood (ML) analysis to unveil the selective constraints in GroEL. In addition, we apply a new statistical approach to determine the patterns of evolution in this highly interesting protein. The main conclusion d…

Centennial olive trees as a reservoir of genetic diversity

†Background and Aims Genetic characterization and phylogenetic analysis of the oldest trees could be a powerful tool both for germplasm collection and for understanding the earliest origins of clonally propagated fruit crops. The olive tree (Olea europaea L.) is a suitable model to study the origin of cultivars due to its long lifespan, resulting in the existence of both centennial and millennial trees across the Mediterranean Basin. †Methods The genetic identity and diversity as well as the phylogenetic relationships among the oldest wild and cultivated olives of southern Spain were evaluated by analysing simple sequence repeat markers. Samples from both the canopy and the roots of each tr…

Temperature Adaptation Markedly Determines Evolution within the Genus Saccharomyces

12 pages, 7 figures, 3 tables

Susceptibility and resistance to ethanol in Saccharomyces strains isolated from wild and fermentative environments

11 pages, 3 figures, 3 tables.-- Article first published online: 8 SEP 2010

Identification of Colletotrichum species responsible for anthracnose of strawberry based on the internal transcribed spacers of the ribosomal region.

In recent years, different molecular techniques have led to an important progress in the characterisation of Colletotrichum species, but there are no available methods which permit the easy identification of Colletotrichum strains and their assignation to classical species. In the present work, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene, were used to identify a total of 80 strains of Colletotrichum, the majority of them isolated from strawberry. One of the most interesting results derived from this study was the easy and reliable distinction, using the endonuclease MvnI, between Colletotrichum fragariae…

Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity.

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae × S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1…

An analysis of inter- and intraspecific genetic variabilities in theKluyveromyces marxianusgroup of yeast species for the reconsideration of theK. lactistaxon

In the present work, we analyse the sequences of the 5.8S rRNA gene and the two internal transcribed spacers 1 and 2 (5.8S-ITS region), obtained from 39 strains belonging to the species Kluyveromyces aestuarii, K. dobzhanskii, K. lactis and K. marxianus, K. nonfermentans and K. wickerhamii, to solve the phylogenetic relationships among these species and also to determine the possible genetic basis for the delimitation of the two currently accepted K. lactis varieties: lactis, including lactose-positive strains isolated from dairy products, and drosophilarum, comprising lactose-negative strains isolated from insects and plant exudates. The determination of the phylogenetic relationships with…

Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied t…

Phylogenetic reconstruction of the yeast genus Kluyveromyces: restriction map analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

Summary We have constructed restriction site maps of the 5.8S rRNA gene and the two ITS regions in 60 strains of Kluyveromyces genus. We test the value of this region as a phylogenetic indicator, and its possible use as a fast and easy method to identify species of this genus. Despite some minor incongruences, our results are in good agreement with previous phylogenetic reconstructions based on the 18S rRNA gene sequencing (Cai et al., 1996; James et al., 1997). A highly significant monophyletic group was formed by K. lactis, K. marxianus, K. aestuarii, K. dobzhanskii and K. wickerhamii, which should be considered the true Kluyveromyces genus. The other species of the genus were grouped wit…

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

8 pages, 1 figure, 4 tables.-- Online version published: May 2009.-- The definitive version is available at www3.interscience.wiley.com

Chimeric Genomes of Natural Hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii

11 pages, 6 figures.-- PMID: 19251887 [PubMed].-- Printed version published Apr 2009.

Aroma production and fermentation performance of S. cerevisiae × S. kudriavzevii natural hybrids under cold oenological conditions

This work aims to describe the wine fermentation characteristics of 23 natural S. cerevisiae × S. kudriavzevii hybrid yeasts related to fermentative environments isolated from different regions and their significance for the aroma spectra of the produced wines. Fermentations were performed at 12 °C in artificial must, and S. cerevisiae and S. kudriavzevii pure species strains were used for comparison purposes. We determined the relevant kinetic parameters of fermentation, the concentration of the main metabolites and the main aroma-related compounds produced after fermentation. The results revealed that some strains that show well-rounded characteristics could be profitable yeast starters f…

Rapid characterization of four species of the Saccharomyces sensu stricto complex according to mitochondrial DNA patterns

Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were anal…

Molecular evolution in yeast of biotechnological interest

The importance of yeast in the food and beverage industries was only realized about 1860, when the role of these organisms in food manufacture became evident. Since they grow on a wide range of substrates and can tolerate extreme physicochemical conditions, yeasts, especially the genera Saccharomyces and Kluyveromyces, have been applied to many industrial processes, Industrial strains of these genera are highly specialized organisms that have evolved to utilize a range of environments and ecological niches to their full potential. This adaptation is called "domestication". This review describes the phylogenetic relationships among Saccharomyces and Kluyveromyces species and the different me…

Exclusion of Saccharomyces kudriavzevii from a wine model system mediated by Saccharomyces cerevisiae.

This study investigated the competition and potential hybrid generation between the species Saccharomyces cerevisiae and S. kudriavzevii in a wine-model environment. Our main goal was to understand why S. kudriavzevii has not been found in wine fermentations whilst their hybrids are present. Auxotrophic mutants (Ura(-) and Lys(-)) were used to favour the selection of hybrids and to specifically differentiate the two species in mixed fermentations carried out at different temperatures (17 °C, 24 °C and 31 °C). Both yeasts showed a reduction in their maximum specific growth rates in mixed fermentations, indicating a clear antagonistic effect between the two microorganisms. Temperature played …

Quantifying the individual effects of ethanol and temperature on the fitness advantage of Saccharomyces cerevisiae

The presence of Saccharomyces cerevisiae in grape berries and fresh musts is usually very low. However, as fermentation progresses, the population levels of this species considerably increase. In this study, we use the concept of fitness advantage to measure how increasing ethanol concentrations (0-25%) and temperature values (4-46 °C) in wine fermentations affects competition between S. cerevisiae and several non-Saccharomyces yeasts (Hanseniaspora uvarum, Torulaspora delbrueckii, Candida zemplinina, Pichia fermentans and Kluyveromyces marxianus). We used a mathematical approach to model the hypothetical time needed for S. cerevisiae to impose itself on a mixed population of the non-Saccha…

RNAseq-based transcriptome comparison of Saccharomyces cerevisiae strains isolated from diverse fermentative environments.

Transcriptome analyses play a central role in unraveling the complexity of gene expression regulation in Saccharomyces cerevisiae. This species, one of the most important microorganisms for humans given its industrial applications, shows an astonishing degree of genetic and phenotypic variability among different strains adapted to specific environments. In order to gain novel insights into the Saccharomyces cerevisiae biology of strains adapted to different fermentative environments, we analyzed the whole transcriptome of three strains isolated from wine, flor wine or mezcal fermentations. An RNA-seq transcriptome comparison of the different yeasts in the samples obtained during synthetic m…

Mitochondrial introgression suggests extensive ancestral hybridization events among Saccharomyces species.

Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several wor…

Mitochondrial DNA variability of striped dolphins (Stenella coeruleoalba) in the Spanish Mediterranean waters

Frozen muscle samples from 44 striped dolphins stranded on the Spanish Mediterranean coasts from 1990 to 1993 have been studied by means of mitochondrial DNA (mtDNA) restriction site analysis. Thirty-five of these dolphins were affected by a die-off occurring during this time in the western Mediterranean Sea. The mtDNA from each dolphin was digested with 15 restriction endonucleases that recognized 61 different restriction sites. The specific location of these sites on the mitochondrial gene map allowed us to determine the distribution of variability along this molecule. From the restriction analysis, a total of 15 different composite patterns or haplotypes was obtained and their phylogenet…

Dominance of wine Saccharomyces cerevisiae strains over S. kudriavzevii in industrial fermentation competitions is related to an acceleration of nutrient uptake and utilization

Grape must is a sugar‐rich habitat for a complex microbiota which is replaced by Saccharomyces cerevisiae strains during the first fermentation stages. Interest on yeast competitive interactions has recently been propelled due to the use of alternative yeasts in the wine industry to respond to new market demands. The main issue resides in the persistence of these yeasts due to the specific competitive activity of S. cerevisiae. To gather deeper knowledge of the molecular mechanisms involved, we performed a comparative transcriptomic analysis during fermentation carried out by a wine S. cerevisiae strain and a strain representative of the cryophilic S. kudriavzevii, which exhibits high genet…

A DNA region ofTorulaspora delbrueckii containing theHIS3 gene: sequence, gene order and evolution

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae his3 mutant strain. DNA sequence analysis revealed that the fragment contained two complete ORFs, which share a high similarity with S. cerevisiae His3p and Mrp51p, respectively. The cloned TdHIS3 gene fully complemented the his3 mutation of S. cerevisiae, confirming that it encodes for the imidazoleglycerol-phosphate dehydrate of T. delbrueckii. Two additional ORFs, with a high homology to S. cerevisiae PET56 and DED1 genes, were mapped upstream and downstream from TdHIS3 and TdMRP51, respectively. This genetic organization is analogous to that previously found in Saccharo…

Yeasts Isolated from the Alcoholic Fermentation ofAgave duranguensisDuring Mezcal Production

Mezcal is a spirit produced in some regions of Mexico. In the state of Durango, mezcal is produced via traditional fermentation of the Agave duranguensis plant. To better understand traditional fermentation processes, it is necessary to know which yeast species are present in fermentations in different producer regions. The aim of this research was to study yeasts involved in traditional mezcal fermentation in Durango, Mexico, and investigate the phylogeny of the native Saccharomyces cerevisiae strains involved in this process. The 5.8S-ITS genomic region was analyzed to identify strains present in the fermentation process samples in this study. To differentiate strains belonging to the gen…

Genetic Polymorphism in Wine Yeasts: Mechanisms and Methods for Its Detection

The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the…

Characterization of wine yeast strains of the Saccharomyces genus on the basis of molecular markers: Relationships between genetic distance and geographic or ecological origin

Summary We identify and characterize 31 Saccharomyces strains from different wine regions, deposited at the Spanish Type Culture Collection, according to mtDNA restriction patterns and chromosomal profiles. By using this kind of information we analyze the correlation between genetic distances and ecological or geographical factors by means of a cluster analysis, assessed by an analysis of the molecular variance (AMOVA). From these analyses, red wine strains are significantly grouped according to their geographic origin, independently of the wine type and the grapevine cultivar, and white wine strians according to ecological factors (wine type of grapevine cultivars). This study also confirm…

A Comparative Study of Different Methods of Yeast Strain Characterization

Summary An extensive survey of different methods of yeast strain identification (classical microbiological tests, whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction analysis show differences between strains. Both techniques are very complex to be used in bio technological industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on mtDNA restriction analysis.

Saccharomyces uvarum is responsible for the traditional fermentation of apple chicha in Patagonia

Apple chicha is a fresh low alcoholic beverage elaborated by aboriginal communities of Andean Patagonia (Argentina and Chile). In the present work, we identified the yeast microbiota associated with this fermentation, and characterized genetically those belonging to the genus Saccharomyces. Both Saccharomyces cerevisiae and S. uvarum were found in the analyzed fermentations. Phylogenetic and population structure analyses based on genes sequence analysis were carried out for both S. cerevisiae and S. uvarum strains obtained in this study and a set of additional strains from diverse origins. The results demonstrate that S. cerevisiae strains from apple chicha belong to the big group of wine/E…

Enological characterization of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii.

The effect of yeasts on wine flavor response is of primary importance. The genus Saccharomyces, and mainly the species Saccharomyces cerevisiae, is responsible for alcoholic fermentation. Recently, several novel yeast isolates from wines have been described as hybrid yeasts between S. cerevisiae x S. kudriavzevii. We have analyzed their influence on two grape musts (Macabeo and Tempranillo) in fermentations conducted at four different temperatures (14, 18, 22 and 32 degrees C) by studying volatile compound production, sugar assimilation and other characteristics influencing the enological properties of wine caused by the impact of yeast. Hybrid yeasts behave particularly well at 14, 18 and …

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 x 10(-5) to 3 x 10(-5) per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker an…

Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars th…

Saccharomyces eubayanusandSaccharomyces uvarumassociated with the fermentation ofAraucaria araucanaseeds in Patagonia

Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this …

Characterization of the length polymorphism in the A + T-rich region of the Drosophila obscura group species

In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A + T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin. The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex. By comparing the nucleotide sequence of the conserved region in D. ambigu…

Phylogeny of the Drosophila obscura species group deduced from mitochondrial DNA sequences

Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., affinis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two…

Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain)

Aims:  The present study was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied from 14 samples taken during fermentations carried out in the 2002 vintage, from 11 wineries belonging to five wine regions on Tenerife Island. Methods and Results:  Yeasts’ strains were identified and characterized through restriction analysis of the 5·8S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species were found, where 14 species were present in significant frequencies in only one sa…

Four new Candida cretensis strains isolated from Spanish fermented sausages (chorizo): Taxonomic and phylogenetic implications

Four yeast strains were isolated from Spanish traditional fermented sausages (chorizo) spoiled by gas production. Using the classical identification procedures, they were identified as Debaryomyces hansenii. However, they fermented galactose and did not produce positive results in Debaryomyces differential medium (DDM), a growth medium highly specific for this species. Phylogenetic analysis showed identical sequences for the D1/D2 domain of the 26S rRNA gene and almost identical sequences for the 5.8S-ITS region with those of the recently described yeast species Candida cretensis. This result was confirmed by sequencing the gene encoding actin of the type and the new strains. Candida creten…

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during sponta…

Molecular and enological characterization of a natural Saccharomyces uvarum and Saccharomyces cerevisiae hybrid

Available online 17 March 2015

GWideCodeML: A python package for testing evolutionary hypotheses at the genome-wide level

One of the most widely used programs for detecting positive selection, at the molecular level, is the program codeml, which is implemented in the Phylogenetic Analysis by Maximum Likelihood (PAML) package. However, it has a limitation when it comes to genome-wide studies, as it runs on a gene-by-gene basis. Furthermore, the size of such studies will depend on the number of orthologous genes the genomes have income and these are often restricted to only account for instances where a one-to-one relationship is observed between the genomes. In this work, we present GWideCodeML, a Python package, which runs a genome-wide codeml with the option of parallelization. To maximize the number of analy…

Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex.

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most impor…

Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity.

The genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two approximately 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both Taq and Pfu DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by Taq amplification (20 clones from each region), whereas amplification of the same samples with Pfu revealed between 0 and 5 mutations. PCR fidelity assays, performed under the same PCR conditions as those used in the population analysis, showed that the Taq error-rate estimate of 0.27 x 10(-4) misinco…

Convergent adaptation of Saccharomyces uvarum to sulfite, an antimicrobial preservative widely used in human-driven fermentations

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that …

Adaptive evolution in GroEL from distantly related endosymbiotic bacteria of insects

Many symbioses between bacteria and insects resulted from ancient infections followed by strict vertical transmission within host lineages. The strong bottlenecks under which this transmission occurs promote the neutral fixation of slightly deleterious mutations by genetic drift. As predicted by Muller's ratchet, this fixation will drive endosymbiotic bacteria through an irreversible dynamics of fitness loss. The chaperonin GroEL has been proposed as a compensatory mechanism whereby endosymbiotic bacteria of aphids persist. Here, we show that endosymbiotic bacteria of insects from two phylogenetically very distant bacterial phyla have fixed amino acid substitutions by positive selection in …

A Sliding Window-Based Method to Detect Selective Constraints in Protein-Coding Genes and Its Application to RNA Viruses

Here we present a new sliding window-based method specially designed to detect selective constraints in specific regions of a multiple protein-coding sequence alignment. In contrast to previous window-based procedures, our method is based on a nonarbitrary statistical approach to find the appropriate codon-window size to test deviations of synonymous (d(S)) and nonsynonymous (d(N)) nucleotide substitutions from the expectation. The probabilities of d(N) and d(S) are obtained from simulated data and used to detect significant deviations of d(N) and d(S) in a specific window region of the real sequence alignment. The nonsynonymous-to-synonymous rate ratio (w = d(N)/d(S)) was used to highlight…

Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802(T), and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscrib…

Comparative Genomics Between Saccharomyces kudriavzevii and S. cerevisiae Applied to Identify Mechanisms Involved in Adaptation

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis…

Differences in the glucose and fructose consumption profiles in diverse Saccharomyces wine species and their hybrids during grape juice fermentation

7 pages, 4 figures, 5 tables.

GroEL buffers against deleterious mutations

GroEL, a heat-shock protein that acts as a molecular chaperone1, is overproduced in endosymbiotic but not in free-living bacteria2,3,4, presumably to assist in the folding of conformationally damaged proteins. Here we show that the overproduction of GroEL in Escherichia coli masks the effects of harmful mutations that have accumulated during a simulated process of vertical transmission. This molecular mechanism, which may be an adaptation to the bacterium's intracellular lifestyle, is able to rescue lineages from a progressive fitness decline resulting from the fixation of deleterious mutations under strong genetic drift5,6.

Differences in activation of MAP kinases and variability in the polyglutamine tract of Slt2 in clinical and non-clinical isolates of Saccharomyces cerevisiae

The concept of Saccharomyces cerevisiae as an emerging opportunistic pathogen is relatively new and it is due to an increasing number of human infections during the past 20 years. There are still few studies addressing the mechanisms of infection of this yeast species. Moreover, little is known about how S. cerevisiae cells sense and respond to the harsh conditions imposed by the host, and whether this response is different between clinical isolates and non-pathogenic strains. In this regard, mitogen-activated protein kinase (MAPK) pathways constitute one of the major mechanisms for controlling transcriptional responses and, in some cases, virulence in fungi. Here we show differences among …

New Trends in the Uses of Yeasts in Oenology

Abstract The most important factor in winemaking is the quality of the final product and the new trends in oenology are dictated by wine consumers and producers. Traditionally the red wine is the most consumed and more popular; however, in the last times, the wine companies try to attract other groups of populations, especially young people and women that prefer sweet, whites or rose wines, very fruity and with low alcohol content. Besides the new trends in consumer preferences, there are also increased concerns on the effects of alcohol consumption on health and the effects of global climate change on grape ripening and wine composition producing wines with high alcohol content. Although S…

Human-associated migration of Holarctic Saccharomyces uvarum strains to Patagonia

Our results show that the greatest S. uvarum population diversity worldwide is observed in Patagonia, where strains of this species can be isolated from industrial and traditional fermentations as well as from natural environments. This greater Patagonian diversity is due to the presence of strains belonging to two genetically differentiated populations, South America B (SA-B), and Holarctic/South America A (H/SA-A). The H/SA-A population of Patagonia is directly related to apple fermentation environments, mainly from cider fermentations but also, to a lesser extent, from traditional apple chicha. Our data suggest that strains from the Holarctic population colonized Patagonia. This is possi…

Genomic instability in an interspecific hybrid of the genus Saccharomyces: a matter of adaptability

Ancient events of polyploidy have been linked to huge evolutionary leaps in the tree of life, while increasing evidence shows that newly established polyploids have adaptive advantages in certain stress conditions compared to their relatives with a lower ploidy. The genus Saccharomyces is a good model for studying such events, as it contains an ancient whole-genome duplication event and many sequenced Saccharomyces cerevisiae are, evolutionary speaking, newly formed polyploids. Many polyploids have unstable genomes and go through large genome erosions; however, it is still unknown what mechanisms govern this reduction. Here, we sequenced and studied the natural S. cerevisiae × Saccharomyces…

Identification of a yeast strain as a potential stuck wine fermentation restarter: a kinetic characterization

Restarter yeast strains are used to consume residual sugar (mainly fructose) in stuck wine fermentations. Forty-three yeast strains were evaluated initially. Strain ITD-00068 showed the highest values for the maximum growth rate, maximum fructose consumption rate, and maximum ethanol production rate (0.143, 0.268, and 0.231 h−1, respectively). Therefore, it was selected for further molecular and kinetic analyses. RFLP analysis identified this isolate as Saccharomyces cerevisiae. Strain ITD-00068 consumed 100% and 36% of the fructose present (at 20°C and 30°C, respectively), when cultured in medium with 12% (v/v) ethanol. The fructose consumption rate was reduced by 97% at 30°C, when ethanol…

The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity.

11 pages, 2 tables, 4 figures. --Article first published online: 6 JAN 2012. --This is the pre-peer reviewed version of the following article: Peris, D., Belloch, C., Lopandić, K., Álvarez-Pérez, J. M., Querol, A. and Barrio, E. (2012), The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity. Yeast, 29: 81–91. which has been published in final form at http://dx.doi.org/10.1002/yea.2891

Alternative yeasts for winemaking: Saccharomyces non-cerevisiae and its hybrids

Wine fermentation has not significantly changed since ancient times and the most traditional aspects are seen by the market as elements that uplift wine nuances and quality. In recent years, new trends have emerged from the sector in line with consumer preferences, and due to the effects of global climate change on grape ripening. In the first cases, the consumers are looking for wines with less ethanol and fruitier aromas and in the second cases the wineries want to reduce the wine alcohol levels and/or astringency. New yeast starters of alternative Saccharomyces species and their hybrids can help to solve some problems that wineries face. In this article we review several physiological an…

The Use of Mixed Populations of Saccharomyces cerevisiae and S. kudriavzevii to Reduce Ethanol Content in Wine: Limited Aeration, Inoculum Proportions, and Sequential Inoculation

Saccharomyces cerevisiae is the most widespread microorganism responsible for wine alcoholic fermentation. Nevertheless, the wine industry is currently facing new challenges, some of them associate with climate change, which have a negative effect on ethanol content and wine quality. Numerous and varied strategies have been carried out to overcome these concerns. From a biotechnological point of view, the use of alternative non-Saccharomyces yeasts, yielding lower ethanol concentrations and sometimes giving rise to new and interesting aroma, is one of the trendiest approaches. However, S. cerevisiae usually outcompetes other Saccharomyces species due to its better adaptation to the fermenta…

Inter- and intraspecific chromosome pattern variation in the yeast genusKluyveromyces

The analysis of the electrophoretic chromosome patterns of the species of the genus Kluyveromyces, reveals a high polymorphism in size, number and intensity of bands. DiVerent sets of electrophoresis running conditions were used to establish species-specific patterns and also to detect intraspecific variation. According to their karyotypes, the species of this genus can be divided into two major groups. The first group includes the species K. africanus, K. bacillisporus, K. delphensis, K. lodderae, K. phaY, K. polysporus and K. yarrowii, composing the so-called ‘Saccharomyces cerevisiae-like’ group, because their karyotypes resemble that of the species S. cerevisiae. The second group compri…

On the complexity of the Saccharomyces bayanus taxon: Hybridization and potential hybrid speciation

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequenc…

Kluyveromyces osmophilus is not a synonym of Zygosaccharomyces mellis; reinstatement as Zygosaccharomyces osmophilus comb. nov.

Kluyveromyces osmophilus, a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis. Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis. During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata, as well as ripe honey of Tetragonisca angustula. The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they rep…

Molecular Mechanisms Involved in the Adaptive Evolution of Industrial Yeasts

Analysis of the genetic variability in the species of theSaccharomyces sensu strictocomplex

Random amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR) analysis was applied to differentiate the sibling species Saccharomyces bayanus, S. cerevisiae, S. paradoxus and S. pastorianus, which constitute the most common strains of the Saccharomyces sensu stricto complex. Six decamer primers of arbitrary sequences were used to amplify the DNA of 58 strains. Species-specific (diagnostic) bands were obtained for each species. Two phylogenetic trees constructed by the neighbour-joining and maximum parsimony methods clearly showed that the delimitation of these related yeast species is possible by using RAPD analysis. Four groups of strains, corresponding to the species S. bayanus, S…

On the origins and industrial applications ofSaccharomyces cerevisiae×Saccharomyces kudriavzeviihybrids

Companies based on alcoholic fermentation products, such as wine, beer and biofuels, use yeasts to make their products. Each industrial process utilizes different media conditions, which differ in sugar content, the presence of inhibitors and fermentation temperature. Saccharomyces cerevisiae has traditionally been the main yeast responsible for most fermentation processes. However, the market is changing due to consumer demand and external factors such as climate change. Some processes, such as biofuel production or winemaking, require new yeasts to solve specific challenges, especially those associated with sustainability, novel flavours and altered alcohol content. One of the proposed so…

A new PCR-based method for monitoring inoculated wine fermentations.

A new PCR-based method has been developed to monitor inoculated wine fermentations. The method is based on the variation in the number and position of introns in the mitochondrial gene COX1. Oligonucleotide primers homologous to the regions flanking the Saccharomyces cerevisiae COX1 introns have been designed and tested for S. cerevisiae wine yeast strain differentiation. Four primers were selected for their subsequent use in a multiplex PCR reaction and have proved to be very effective in uncovering polymorphism in natural and commercial yeast strains. An important point is that the speed and simplicity of the technique, which does not require the isolation of DNA, allows early detection o…

Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid

The effects of temperature, pH and sugar concentration (50% glucose + 50% fructose) on the growth parameters of Saccharomyces cerevisiae T73, S. kudriavzevii IFO 1802T and the hybrid strain S. cerevisiae × S. kudriavzevii W27 were studied by means of response surface methodology based in a central composite circumscribed design. Lag phase could not be properly modelled in the wine model system, where yeasts started the fermentation in few hours after inoculation. In the case of the maximum specific growth rate (μ max), the temperature was the most important variable for three yeasts, although the effects of sugar concentration (in T73 and W27) and pH (W27 and 1802) were also significan…

Metabolic differences between a wild and a wine strain of Saccharomyces cerevisiae during fermentation unveiled by multi‐omic analysis

Saccharomyces cerevisiae, a widespread yeast present both in the wild and in fermentative processes, like winemaking. During the colonization of these human‐associated fermentative environments, certain strains of S. cerevisiae acquired differential adaptive traits that enhanced their physiological properties to cope with the challenges imposed by these new ecological niches. The advent of omics technologies allowed unveiling some details of the molecular bases responsible for the peculiar traits of S. cerevisiae wine strains. However, the metabolic diversity within yeasts remained poorly explored, in particular that existing between wine and wild strains of S. cerevisiae. For this purpose,…

Effect of temperature on the prevalence of Saccharomyces non-cerevisiae species against a S. cerevisiae wine strain in wine fermentation: competition, physiological fitness, and influence in final wine composition

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations,…

Evolutionary advantage conferred by an eukaryote-to-eukaryote gene transfer event in wine yeasts

Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a s…

A comparison of the performance of natural hybrids Saccharomyces cerevisiae × Saccharomyces kudriavzevii at low temperatures reveals the crucial role of their S. kudriavzevii genomic contribution.

Fermentation performance at low temperature is a common approach to obtain wines with better aroma, and is critical in industrial applications. Natural hybrids S. cerevisiae × S. kudriavzevii, isolated from fermentations in cold-climate European countries, have provided an understanding of the mechanisms of adaptation to grow at low temperature. In this work, we studied the performance of 23 S. cerevisiae × S. kudriavzevii hybrids at low temperature (8, 12 and 24 °C) to characterize their phenotypes. Kinetic parameters and spot tests revealed a different ability to grow at low temperature. Interestingly, the genome content of the S. kudriavzevii in hybrids was moderately correlated with a s…

Aneuploidy and Ethanol Tolerance in Saccharomyces cerevisiae

Response to environmental stresses is a key factor for microbial organism growth. One of the major stresses for yeasts in fermentative environments is ethanol. Saccharomyces cerevisiae is the most tolerant species in its genus, but intraspecific ethanol-tolerance variation exists. Although, much effort has been done in the last years to discover evolutionary paths to improve ethanol tolerance, this phenotype is still hardly understood. Here, we selected five strains with different ethanol tolerances, and used comparative genomics to determine the main factors that can explain these phenotypic differences. Surprisingly, the main genomic feature, shared only by the highest ethanol-tolerant st…

Molecular characterization and cytonuclear disequilibria of two Drosophila subobscura mitochondrial haplotypes.

According to restriction site analyses of mitochondrial DNA, Drosophila subobscura shows a polymorphism that consists of two frequent haplotypes that are evenly distributed all over the Old World and several rare haplotypes never present in more than one locality. To ascertain the causes responsible for such distribution, three different mtDNA fragments from haplotypes I and II sampled in a population from Zürich have been partially sequenced. Only three silent nucleotide changes have been detected in the ND5 gene. One of them implies the loss of the HaeIII restriction site, which differentiates haplotype I from haplotype II. On the basis of these results as well as on others involving the…

A rapid and simple method for the preparation of yeast mitochondrial DNA

Evidence for Positive Selection in the Capsid Protein-Coding Region of the Foot-and-Mouth Disease Virus (FMDV) Subjected to Experimental Passage Regimens

We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (dN/dS) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our …

Lipid Composition Analysis Reveals Mechanisms of Ethanol Tolerance in the Model YeastSaccharomyces cerevisiae

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and the food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyze five representatives with various ethanol tolerances. The most tolerant strain, AJ4, was dominant in coculture at 0 and 10% ethanol. Unexpectedly, although it does not have the highest noninhibitory concentration or MIC, MY29 was the dominant strain in coculture at 6% ethanol, which may be linked to differen…

Adaptive evolution of wine yeast.

Abstract Alcoholic fermentation is one of the main phases in wine production. It is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. Industrial S. cerevisiae strains are highly specialized organisms, which have evolved to utilize to their full potential the different environments or ecological niches. So, during the alcoholic fermentation, the yeast has been adapted to different kinds of stress conditions; this adaptation is call “domestication”. In this review, we describe the different mechanisms involved in the adaptive evolution of wine yeast strains.

The evolution of RNA viruses: A population genetics view.

RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested th…

A multi-phase multi-objective genome-scale model shows diverse redox balance strategies in yeasts

Yeasts constitute over 1500 species with great potential for biotechnology. Still, the yeastSaccharomyces cerevisiaedominates industrial applications and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here we propose a multi-phase multi-objective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kinetic mod…

Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethan…

Mitochondrial introgression suggests extensive ancestral hybridization events amongSaccharomycesspecies

1.AbstractHorizontal Gene Transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested betweenS. cerevisiaeandS. paradoxus. However, few strains have been explored due to the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur and their role in cytonuclear incompatibilities and fitness. In fact, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. In this study, we have explored the mitochondrial inher…

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. G…

Molecular Characterization of a Chromosomal Rearrangement Involved in the Adaptive Evolution of Yeast Strains

Wine yeast strains show a high level of chromosome length polymorphism. This polymorphism is mainly generated by illegitimate recombination mediated by Ty transposons or subtelomeric repeated sequences. We have found, however, that the SSU1-R allele, which confers sulfite resistance to yeast cells, is the product of a reciprocal translocation between chromosomes VIII and XVI due to unequal crossing-over mediated by microhomology between very short sequences on the 5' upstream regions of the SSU1 and ECM34 genes. We also show that this translocation is only present in wine yeast strains, suggesting that the use for millennia of sulfite as a preservative in wine production could have favored …

Molecular Identification and Characterization of Wine Yeasts

The transformation of grape must into wine is a complex microbiological process involving the sequential growth of bacteria and yeasts, although only the yeasts are responsible for alcoholic fermentation. In the past, winemaking was purely empirical, but it is now a well-understood, controlled process that has been gradually improved over time. Advances have largely been made possible by the development of molecular techniques to identify and characterize wine yeasts based on analysis of their DNA. These methods are rapid, reproducible, and sensitive, and continue to be used for a variety of purposes, such as analyzing variation in naturally occurring and inoculated yeast populations, monit…

Natural hybrids of S. cerevisiae×S. kudriavzevii share alleles with European wild populations of Saccharomyces kudriavzevii

Saccharomyces kudriavzevii, a yeast species described from a pair of strains isolated from decayed leaves in Japan, has recently been isolated from oak barks in Portugal. Some data suggest that these European S. kudriavzevii populations could be closely related to the S. kudriavzevii genetic background present in natural hybrids isolated from wines and beers in different regions of Europe. However, a more exhaustive study of European S. kudriavzevii natural populations is necessary to confirm this observation. In this study, new S. kudriavzevii isolates were recovered from oak trees in different areas in Spain, and identified and characterized according to their molecular and physiological …

A new chromosomal rearrangement improves the adaptation of wine yeasts to sulfite

Sulfite‐generating compounds are widely used during winemaking as preservatives because of its antimicrobial and antioxidant properties. Thus, wine yeast strains have developed different genetic strategies to increase its sulfite resistance. The most efficient sulfite detoxification mechanism in Saccharomyces cerevisiae uses a plasma membrane protein called Ssu1 to efflux sulfite. In wine yeast strains, two chromosomal translocations (VIIItXVI and XVtXVI) involving the SSU1 promoter region have been shown to upregulate SSU1 expression and, as a result, increase sulfite tolerance. In this study, we have identified a novel chromosomal rearrangement that triggers wine yeast sulfite adaptation.…

Molecular Characterization of New Natural Hybrids of Saccharomyces cerevisiae and S. kudriavzevii in Brewing▿ †

ABSTRACT We analyzed 24 beer strains from different origins by using PCR-restriction fragment length polymorphism analysis of different gene regions, and six new Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrid strains were found. This is the first time that the presence in brewing of this new type of hybrid has been demonstrated. From the comparative molecular analysis of these natural hybrids with respect to those described in wines, it can be concluded that these originated from at least two hybridization events and that some brewing hybrids share a common origin with wine hybrids. Finally, a reduction of the S. kudriavzevii fraction of the hybrid genomes was observed, but th…

Comparative genomics among Saccharomyces cerevisiae x Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins

Abstract Background Interspecific hybrids between S. cerevisiae × S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae × S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids. Results Comparative genomic hybridization (CGH) and ploidy analyses carried out in thi…

Reconstruction of the evolutionary history of Saccharomyces cerevisiae x S. kudriavzevii hybrids based on multilocus sequence analysis.

In recent years, interspecific hybridization and introgression are increasingly recognized as significant events in the evolution of Saccharomyces yeasts. These mechanisms have probably been involved in the origin of novel yeast genotypes and phenotypes, which in due course were to colonize and predominate in the new fermentative environments created by human manipulation. The particular conditions in which hybrids arose are still unknown, as well as the number of possible hybridization events that generated the whole set of natural hybrids described in the literature during recent years. In this study, we could infer at least six different hybridization events that originated a set of 26 S…