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Summary In order to evaluate the genetic diversity of the yeast flora in Northern Spain cheese, we adapted two molecular techniques devised for Saccharomyces, RAPD and RFLP of mitochondrial DNA, to type 27 Candida zeylanoides strains and 28 Debaryomyces hansenii strains isolated from Roncal and Idiazabal cheese at different stages of manufacture in different dairies. RAPD with (GTG)5 primer, although very reproducible, only defined 2 groups of strains for both species. On the other hand, mtDNA RFLP proved to be more discriminating and defined 5 groups of strains for both species. In addition, the strains from the minor RAPD groups in both species corresponded to specific groups defined by mtDNA RFLP. Overall, this analysis has revealed an important genetic diversity, considering the limited sample of different biotopes tested. Nevertheless, the mtDNA RFLP results indicate that a large majority of the strains appeared closely related, suggesting little variability between cheese and dairies. This work has demonstrated that rapid identification methods (the typing of strains can be achieved in two days) can be applied to these poorly characterized yeast species, allowing a rapid evaluation of genetic diversity of these species in different biotopes.

A yeast strain was isolated from olive brines in a fermented black olive and olive oil manufacturing plant in the town of Nyons (France). On the basis of domains 1 and 2 (D1/D2) large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region sequence analyses, the strain CLIB 1303T was found to be closely related, but clearly distinct, from the three existing species of the genus Citeromyces: Citeromyces matritensis, Citeromyces siamensis and Citeromyces haiwaiiensis. Strain CLIB 1303T exhibited 6 bp, 7 bp and 12 bp divergences in the D1/D2 LSU rRNA gene with C. siamensis, C. matritensis and C. hawaiiensis, respectively. ITS region divergence amounted to more than 8 %, 4 % and 4.5 % with C. siamensis, C. matritensis and C. hawaiiensis, respectively, in addition to several indels. Like C. matritensis and C. siamensis strains, strain CLIB 1303T was shown to be halotolerant and osmotolerant. Phenotypically, strain CLIB 1303T can be distinguished from other species of the genus Citeromyces by its inability to assimilate trehalose. The strain CLIB 1303T ( = CBS 12700T) was assigned to a novel species, Citeromyces nyonsensis sp. nov.

Y. lipolytica harbors an unusually diverse set of transposable elements among Saccharomycotina yeasts. Among them, members of both the families of transposons, retrotransposons as well as DNA transposons, are represented. Two of the LTR retrotransposons, Ylt1 and Tyl6, are members of the Ty3/gypsy group but have some uncommon features. Ylt1 is the largest hitherto detected fungal retrotransposon and is, in contrast to the other transposons, present in a high copy number of about 35 copies/haploid genome. Its proteins are encoded by a single ORF expressed under certain conditions resulting in transposition. Tyl6 is the only one retrotransposon among Saccharomycotina yeasts which displays a program −1 ribosomal frameshifting. The LINE-like element Ylli is also unique among Saccharomycotina yeasts and forms with the C. albicans counterparts Zorro 1,2,3 a new family. It belongs to the L1 clade, which also contains the human LINEs. Like these element, the large majority of the Ylli copies are 5′ truncated, the characteristics of Ylli being that its copies are very short. The detected DNA transposon Mutator of Y. lipolytica (Mutyl) shares some similarities with several MULE elements found mainly in plants and in fungi. It is the first described DNA transposon in Saccharomycotina yeasts and like many of its counterparts, it may have invaded its host through horizontal transfer.

Abbreviations: ITS, internal transcribed spacer; LSU, large subunit of the nuclear ribosomal DNA; SSU, small subunit of the nuclear ribosomal DNA; mtSSU, small subunit of the mitochondrial ribosomal DNA; With the advances in sequencing techniques, identification of ascomycetous yeasts to the species level and phylogeny reconstruction increasingly require curated and updated taxonomic information. A specific database with nucleotide sequences of the most common markers used for yeast taxonomy and phylogeny and a user-friendly interface allowing identification, taxonomy and phylogeny of yeasts species was developed. By the 1st of September 2012, the YeastIP database contained all the described Saccharomycotina species for which sequences used for taxonomy and phylogeny, such as D1/D2 rDNA and ITS, are available. The database interface was developed to provide a maximum of relevant information and data mining tools, including the following features: (1) the Blastn program for the sequences of the YeastIP database; (2) easy retrieval of selected sequences; (3) display of the available markers for each selected group of species; and (4) a tool to concatenate marker sequences, including those provided by the user. The concatenation tool allows phylogeny reconstruction through a direct link to the Phylogeny.fr platform. YeastIP is thus a unique database in that it provides taxonomic information and guides users in their taxonomic analyses. YeastIP facilitates multigenic analysis to encourage good practice in ascomycetous yeast phylogeny.

the Consortium National de Recherche en Génomique (CNRG) to Génoscope, CNRS (GDR 2354, Génolevures), The computer framework was supported by the funding of the University of Bordeaux 1, the Aquitaine Région in the program "Génotypage et Génomique Comparée," and the ACI IMPBIO "Génolevures En Ligne."; International audience; Polyploidization is an important process in the evolution of eukaryotic genomes, but ensuing molecular mechanisms remain to be clarified. Autopolyploidization or whole-genome duplication events frequently are resolved in resulting lineages by the loss of single genes from most duplicated pairs, causing transient gene dosage imbalance and accelerating speciation through meiotic infertility. Allopolyploidization or formation of interspecies hybrids raises the problem of genetic incompatibility (Bateson-Dobzhansky-Muller effect) and may be resolved by the accumulation of mutational changes in resulting lineages. In this article, we show that an osmotolerant yeast species, Pichia sorbitophila, recently isolated in a concentrated sorbitol solution in industry, illustrates this last situation. Its genome is a mosaic of homologous and homeologous chromosomes, or parts thereof, that corresponds to a recently formed hybrid in the process of evolution. The respective parental contributions to this genome were characterized using existing variations in GC content. The genomic changes that occurred during the short period since hybrid formation were identified (e.g., loss of heterozygosity, unilateral loss of rDNA, reciprocal exchange) and distinguished from those undergone by the two parental genomes after separation from their common ancestor (i.e., NUMT (NUclear sequences of MiTochondrial origin) insertions, gene acquisitions, gene location movements, reciprocal translocation). We found that the physiological characteristics of this new yeast species are determined by specific but unequal contributions of its two parents, one of which could be identified as very closely related to an extant Pichia farinosa strain.

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δμ) 2 genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates.

Among ascomycetous yeasts, the CTG clade is so-called because its constituent species translate CTG as serine instead of leucine. Though the biology of certain pathogenic species such as Candida albicans has been much studied, little is known about the life cycles of non-pathogen species of the CTG clade. Taking advantage of the recently obtained sequence of the biotechnological Millerozyma (Pichiasorbitophila) farinosa strain CBS 7064, we used MLST to better define phylogenic relationships between most of the Millerozyma farinosa strains available in public collections. This led to the constitution of four phylogenetic clades diverging from 8% to 15% at the DNA level and possibly constituting a species complex (M. farinosa) and to the proposal of two new species:Millerozyma miso sp. nov. CBS 2004(T) ( = CLIB 1230(T)) and Candida pseudofarinosa sp. nov.NCYC 386(T)( = CLIB 1231(T)). Further analysis showed that M. farinosa isolates exist as haploid and inter-clade hybrids. Despite the sequence divergence between the clades, secondary contacts after reproductive isolation were evidenced, as revealed by both introgression and mitochondria transfer between clades. We also showed that the inter-clade hybrids do sporulate to generate mainly viable vegetative diploid spores that are not the result of meiosis, and very rarely aneuploid spores possibly through the loss of heterozygosity during sporulation. Taken together, these results show that in this part of the CTG clade, non-Mendelian genetic exchanges occur at high rates through hybridization between divergent strains from distinct clades and subsequent massive loss of heterozygosity. This combination of mechanisms could constitute an alternative sexuality leading to an unsuspected biodiversity.

The evolution of the metabolism of sulfur compounds among yeast species was investigated. Differences between species were observed in the cysteine biosynthesis pathway. Most yeast species possess two pathways leading to cysteine production, the transsulfuration pathway and the O-acetyl-serine (OAS) pathway, with the exception of Saccharomyces cerevisiae and Candida glabrata, which only display the transsulfuration pathway, and Schizosaccharomyces pombe, which only have the OAS pathway. An examination of the components of the regulatory network in the different species shows that it is conserved in all the species analyzed, as its central component Met4p was shown to keep its functional domains and its partners were present. The analysis of the presence of genes involved in the catabolic pathway shows that it is evolutionarily conserved in the sulfur metabolism and leads us to propose a role for two gene families which appeared to be highly conserved. This survey has provided ways to understand the diversity of sulfur metabolism products among yeast species through the reconstruction of these pathways. This diversity could account for the difference in metabolic potentialities of the species with a biotechnological interest.

DNA sequencing has revolutionized yeast taxonomy. Although initially rDNA sequences proved to be universal and convenient for assigning phylogenetic relationships, it was eventually supplanted by multigene analysis, which provided more discriminating and robust results. This led to a new classification of the major yeast clades, which is still used as a reference today. More recently, the availability of a large number of complete genome sequences has given a new perspective on the molecular taxonomy of yeasts by providing a high number of genes to compare. It also highlighted an unexpected aspect of yeast genome evolution: the existence of interspecific hybrids outside of the industrial Saccharomyces clade. Together with the loss of heterozygosity in interspecific hybrids and a reduced sexuality leading to clonal propagation, this observation obliges us to reexamine the present concept of species. In parallel, the ongoing challenge is to find a universal molecular marker, to improve fast authentication and, if possible, phylogeny of yeasts. The future of yeast taxonomy will involve the sequencing of more genomes, thorough analysis of populations to obtain a good representation of the biodiversity and integration of these data into dedicated databases. (C) 2011 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d'Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d'Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.

C. R. Biol. ISI Document Delivery No.: 809CL Times Cited: 2 Cited Reference Count: 75 Dequin, Sylvie Casaregola, Serge INRA; European Community [FP7-228310] This work was supported by INRA. This work has received funding from the European Community's Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project). Elsevier france-editions scientifiques medicales elsevier Paris Si; Many different yeast species can take part in spontaneous fermentations, but the species of the genus Saccharomyces, including Saccharomyces cerevisiae in particular, play a leading role in the production of fermented beverages and food. In recent years, the development of whole-genome scanning techniques, such as DNA chip-based analysis and high-throughput sequencing methods, has considerably increased our knowledge of fermentative Saccharomyces genomes, shedding new light on the evolutionary history of domesticated strains and the molecular mechanisms involved in their adaptation to fermentative niches. Genetic exchange frequently occurs between fermentative Saccharomyces and is an important mechanism for generating diversity and for adaptation to specific ecological niches. We review and discuss here recent advances in the genomics of Saccharomyces species and related hybrids involved in major fermentation processes. (C) 2011 Published by Elsevier Masson SAS on behalf of Academie des sciences.

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.

The delineation of species among strains assigned to Debaryomyces hansenii was examined using a gene genealogies-based approach in order to compare spliceosomal intron sequences found in four housekeeping genes (ACT1, TUB2, RPL31 and RPL33). This revealed four distinct groups of strains containing, respectively, D. hansenii var. hansenii CBS 767(T), D. hansenii var. fabryi CBS 789(T), Candida famata var. flareri CBS 1796(T) (the anamorph of D. hansenii var. fabryi CBS 789(T)) and Debaryomyces tyrocola CBS 766(T), whose species status was no longer accepted. The sequence divergence between these groups, reaching in some cases over 20 %, unambiguously isolated the groups as separate taxa, leading to a proposal for the reinstatement of the originally described species D. hansenii CBS 767(T) and D. tyrocola CBS 766(T). The variety D. hansenii var. fabryi was further subdivided into two taxa, Debaryomyces fabryi CBS 789(T) and Candida flareri CBS 1796(T) (previously C. famata var. flareri and Blastodendrion flareri). The comparison of intron sequences therefore exposed cryptic species whose phenotypic traits are not distinguishable from known species, but which have significantly diverged from the genetic point of view. Hence, we describe the new taxon Debaryomyces macquariensis sp. nov. CBS 5571(T) is related to, but clearly distinct from, the Debaryomyces species mentioned above. The approach used in this work has also revealed the existence of populations within the newly delineated species D. hansenii and genetic exchanges between these populations, indicating an unexpected genetic diversity within this part of the genus Debaryomyces.

Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non- Saccharomyces origin. We identified Zygosaccharomyces bailii , a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non- Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.

International audience; Transfer of fragments of mtDNA to the nuclear genome is a general phenomenon that gives rise to NUMTs (NUclear sequences of MiTochondrial origin). We present here the first comparative analysis of the NUMT content of entirely sequenced species belonging to a monophyletic group, the hemiascomycetous yeasts (Candida glabrata, Kluyveromyces lactis, Kluyveromyces thermotolerans, Debaryomyces hansenii and Yarrowia lipolytica, along with the updated NUMT content of Saccharomyces cerevisiae). This study revealed a huge diversity in NUMT number and organization across the six species. Debaryomyces hansenii harbors the highest number of NUMTs (145), half of which are distributed in numerous large mosaics of up to eight NUMTs arising from multiple noncontiguous mtDNA fragments inserted at the same chromosomal locus. Most NUMTs, in all species, are found within intergenic regions including seven NUMTs in pseudogenes. However, five NUMTs overlap a gene, suggesting a positive impact of NUMTs on protein evolution. Contrary to the other species, K. lactis and K. thermotolerans harbor only a few diverged NUMTs, suggesting that mitochondrial transfer to the nuclear genome has decreased or ceased in these phylogenetic branches. The dynamics of NUMT acquisition and loss are illustrated here by their species-specific distribution.

DNA microarrays of 86 genes from the yeasts Debaryomyces hansenii , Kluyveromyces marxianus , and Yarrowia lipolytica were developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of the LAC genes of K. marxianus was observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially in D. hansenii and K. marxianus , which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved in l -methionine catabolism was also used on the array. Y. lipolytica , which efficiently assimilates l -methionine, also exhibited a high expression of the Saccharomyces cerevisiae orthologs BAT2 and ARO8 , which are involved in the l -methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures.

International audience

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from L-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher L-methionine demethiolating activity, resulting in a direct conversion of L-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis , we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae , in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis , interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of l -methionine. l -Methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, Kl ARO8.1 and Kl ARO8.2 , were found to be responsible for l -methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.

International audience; We have exploited the recently obtained complete genome sequence of Kluyveromyces lactis to compare the repertoire of transcriptional regulators between K. lactis and Saccharomyces cerevisiae. Looking for similarities with the S. cerevisiae proteins of this functional class, we observed a reduction in gene number, which is not randomly distributed among the different DNA-binding classes, the zinc binuclear cluster class (Zn(II)2Cys6), specific to ascomycetes, being one of the most affected. However, when one examines the number of proteins that, in the K. lactis genome, possess the different DNA-binding signatures, it is not reduced compared to S. cerevisiae. This indicates that transactivator proteins have strongly diverged between the two species and cannot be recognized any more, and/or that each genome has developed a specific set of regulators to adapt the cell to its specific niches. These two aspects are discussed on the basis of available data.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; In this study, rDNA ITS restriction analysis was used to identify yeasts from two naturally fermented products: French ciders and black olives. In cider musts and bottled ciders, the PCR-RFLP method generated 15 different ITS/RFLP profiles for a total of 208 isolates. The predominant yeasts corresponded to Saccharomyces bayanus, Saccharomyces cerevisiae, Lachancea cidri and Dekkera anomala. Three identified species: Candida sake, Candida tropicalis and Kluyveromyces marxianus had never been described before in ciders. For the black olive fermentation, the method allowed for identification of I I profiles for a total of 137 isolates. A sequential apparition of yeasts was observed with Pichia anomala, Candida boidinii and Debaryomyces etchellsii being the predominant species. Four isolates did not correspond to any known species based on the sequencing of the D1/D2 region of the 26S rRNA gene. By using the rDNA ITS method, valuable information on yeast population biodiversity and dynamics in the naturally fermented food products studied was obtained.

A new type of DNA transposon, Mutyl, has been identified in the sequenced genome of the yeast Yarrowia lipolytica. This transposon is 7,413 bp long and carries two open reading frames (ORFs) which potentially encode proteins of 459 and 1,178 amino acids, respectively. Whereas the first ORF shows no significant homology to previously described proteins, the second ORF shows sequence similarities with various Mutatorlike element (MULE)-encoded transposases, including the bacterial transposase signature sequence. Other MULE features shared by Mutyl include a zinc finger motif in the putative transposase, a 22-bp-long imperfect inverted repeat at each end, and a 9- to 10-bp duplication of its target site in the chromosome. Of the five copies of Mutyl present in the genome, one has a deletion of the first 8 bases, and the others are full length with a single base change in one element. The first potential gene of Mutyl, mutB, was shown to be expressed in exponentially growing cells. Its sequence contains a predicted intron with two 5 splice sites, a single branch point, and two 3 splice sites. Its mRNA is alternatively spliced, as judged by reverse transcription-PCR, and generates four mRNAs corresponding to protein-coding sequences of 128, 156, 161, and 190 amino acids. Of the three distinct lineages characterized in Y. lipolytica, strains from the German lineage and the French lineage do not carry Mutyl. A study of the distribution of Mutyl in strains of the French lineage evidenced a recent transposition event. Taken together, these results indicate that Mutyl is still active. Transposable elements (TEs) are ubiquitous, as they can be found in most genomes. Their multiplication leads to an increased number of repeated sequences in the genomes that can be involved in various processes including genome rearrangements and homologous recombination. TEs are therefore responsible for a large part of the observed genome plasticity. Two types of TEs that differ in their propagation mechanism have been described; the retrotransposons that use an RNA intermediate and a reverse transcription step make up class I, whereas the DNA transposons that can propagate as DNA with the help of a transposase constitute class II. The distribution and copy number of these TEs vary enormously according to the organism as well as the type of element considered. Among the class II elements, the Mutator (Mu) system, a

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

International audience; As part of the exploratory sequencing program G?levures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (

Background: The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant./nResults: The sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid. Conclusions: The high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications. This work was supported in part by funding from the Consortium National de Recherche en Génomique (CNRG) to Génoscope, from CNRS (GDR 2354, Génolevures), ANR (ANR-05-BLAN-0331, GENARISE). The computing framework was supported by the funding of the University of Bordeaux 1, the Aquitaine Région in the program “Génotypage et Génomique Comparée”, the ACI IMPBIO “Génolevures En Ligne” and INRIA. We thank the System and Network Administration team in LaBRI for excellent help and advice. J.A.C. is supported by the PhD Program in Computational Biology of the Instituto Gulbenkian de Ciência, Portugal (sponsored by Fundação Calouste Gulbenkian, Siemens SA, and Fundação para a Ciência e Tecnologia; SFRH/BD/33528/2008). M.C. research was supported by a grant of the Deutscher Akademischer Austauschdienst (DAAD). T.G. research was partly supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161). B.D. is a member of Institut Universitaire de France

We describe a rapid method developed to identify yeast species commonly found during cheese ripening. It is based on the isolation of species-specific sequences and their hybridization with coarse preparations of genomic DNA. Several strategies were followed for the construction of probes: PCR amplification of sequences available in databases, random cloning of genomic DNA fragments, specific RAPD fragments and PCR-amplified ribosomal DNA fragments. After validation, the probes were applied to the characterization of 400 yeast strains isolated from various French cheeses. Since the strains had been previously identified with classical diagnostic tests, we were able to compare molecular and conventional identification. In addition, the specific probes for one of the species, Debaryomyces hansenii, were used successfully in colony hybridization experiments. The probes developed here proved to be very useful for the screening of large yeast collections and for the assessment of biodiversity within the yeast flora in cheese.

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.

Autonomously replicating sequences (ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication (ORI) and centromere (CEN) DNA, both of which are necessary for extrachromosomal maintenance. To investigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-borne ORI. Our results confirm a cooperation between ORI and CEN sequences to form an ARS, since all sequences identified in this screen displayed features of centromeric DNA and included the previously characterized CEN1-1, CEN3-1 and CEN5-1 fragments. Two new centromeric DNAs were identified as they are unique, map to different chromosomes (II and IV) and induce chromosome breakage after genomic integration. A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated from chromosome I. Although these CEN sequences do not share significant sequence similarities, they display a complex pattern of short repeats, including conserved blocks of 9 to 14 bp and regions of dyad symmetry. Consistent with their A+T-richness and strong negative roll angle, Y. lipolytica CEN-derived sequences, but not ORIs, were capable of binding isolated Drosophila nuclear scaffolds. However, a Drosophila scaffold attachment region that functions as an ARS in other yeasts was unable to confer autonomous replication to an ORI-containing plasmid. Deletion analysis of CEN1-1 showed that the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance. We propose that, while Y. lipolytica CEN DNA is essential for plasmid maintenance, this function can be supplied by several sub-fragments which, together, form the active chromosomal centromere. This complex organization of Y. lipolytica centromeres is reminiscent of the regional structures described in the yeast Schizosaccharomyces pombe or in multicellular eukaryotes.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22 000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences. These sequences were first identified in a survey of D. hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested. Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767. The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese. F01pro and M18pro hybridized to the DNA of all D. hansenii var. hansenii tested, but not to DNA of other yeast species including the closely related strain of D. hansenii var. fabryii CBS 789. Hybridization patterns of F01pro and M18pro on digested genomic DNA of D. hansenii indicated that the sequences were repeated in the genome of all D. hansenii var. hansenii tested, and gave distinct polymorphic patterns. The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme. F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D. hansenii and, together with M18pro, simplifies the study of this species in complex flora.

A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2,4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n -alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t -butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.

Random sequence tags were obtained from a genomic DNA library of Saccharomyces exiguus. The mitochondrial genome appeared to be at least 25.7 kb in size, with a different organization compared to Saccharomyces cerevisiae. An unusual putative 953 bp long terminal repeated element associated to Ty3 was found. A set of 1451 genes was identified homologous to S. cerevisiae open reading frames. Only five genes were identified outside the S. cerevisiae taxon, confirming that S. exiguus is phylogenetically closely related to S. cerevisiae. Unexpectedly, numerous duplicated genes were found whereas they are unique in S. cerevisiae. The sequences are deposited at EMBL under the accession numbers: AL407377–AL409955.

The genome of Saccharomyces servazzii was analyzed with 2570 random sequence tags totalling 2.3 Mb. BLASTX comparisons revealed a minimum of 1420 putative open reading frames with significant homology to Saccharomyces cerevisiae (58% aa identity on average), two with Schizosaccharomyces pombe and one with a human protein, confirming that S. servazzii is closely related to S. cerevisiae. About 25% of the S. servazzii genes were identified, assuming that the gene complement is identical in both yeasts. S. servazzii carries very few transposable elements related to Ty elements in S. cerevisiae. Most of the mitochondrial genes were identified in eight contigs altogether spanning 25 kb for a predicted size of 29 kb. A significant match with the Kluyveromyces lactis linear DNA plasmid pGKL-1 encoded RF4 killer protein suggests that a related plasmid exists in S. servazzii. The sequences have been deposited with EMBL under the accession numbers AL402279–AL404848.

By analyzing 2830 random sequence tags (RSTs), totalling 2.7 Mb, we explored the genome of the marine, osmo- and halotolerant yeast, Debaryomyces hansenii. A contig 29 kb in length harbors the entire mitochondrial genome. The genes encoding Cox1, Cox2, Cox3, Cob, Atp6, Atp8, Atp9, several subunits of the NADH dehydrogenase complex 1 and 11 tRNAs were unambiguously identified. An equivalent number of putative transposable elements compared to Saccharomyces cerevisiae were detected, the majority of which are more related to higher eukaryote copia elements. BLASTX comparisons of RSTs with databases revealed at least 1119 putative open reading frames with homology to S. cerevisiae and 49 to other genomes. Specific functions, including transport of metabolites, are clearly over-represented in D. hansenii compared to S. cerevisiae, consistent with the observed difference in physiology of the two species. The sequences have been deposited with EMBL under the accession numbers AL436045–AL438874.

International audience

A total of 4940 random sequence tags of the dimorphic yeast Yarrowia lipolytica , totalling 4.9 Mb, were analyzed. BLASTX comparisons revealed at least 1229 novel Y. lipolytica genes 1083 genes having homology with Saccharomyces cerevisiae genes and 146 with genes from various other genomes. This confirms the rapid sequence evolution assumed for Y. lipolytica . Functional analysis of newly discovered genes revealed that several enzymatic activities were increased compared to S. cerevisiae , in particular, transport activities, ion homeostasis, and various metabolism pathways. Most of the mitochondrial genes were identified in contigs spanning more than 47 kb. Matches to retrotransposons were observed, including a S. cerevisiae Ty3 and a LINE element. The sequences have been deposited with EMBL under the accession numbers AL409956 – AL414895 .

In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series.

The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.

We have studied the role of DNA replication in turn-on and turn-off of the SOS response in Escherichia coli using a recA::lac fusion to measure levels of recA expression. An active replication fork does not seem to be necessary for mitomycin C induced recA expression: a dnaA43 initiation defective mutant, which does not induce the SOS response at non-permissive temperature, remains mitomycin C inducible after the period of residual DNA synthesis. This induction seems to be dnaC dependent since in a dnaC325 mutant recA expression not only is not induced at 42 degrees C but becomes mitomycin C non-inducible after the period of residual synthesis. Unscheduled halts in DNA replication, generally considered the primary inducing event, are not sufficient to induce the SOS response: no increase in recA expression was observed in dnaG(Ts) mutants cultivated at non-permissive temperature. The replication fork is nonetheless involved in induction, as seen by the increased spontaneous level of recA expression in these strains at permissive temperature. Turn-off of SOS functions can be extremely rapid: induction of recA expression by thymine starvation is reversed within 10 min after restoration of normal DNA replication. We conclude that the factors involved in induction--activated RecA (protease) and the activating molecular (effector)--do not persist in the presence of normal DNA replication.

The genome of Saccharomyces kluyveri was explored through 2528 random sequence tags with an average length of 981 bp. The complete nuclear ribosomal DNA unit was found to be 8656 bp in length. Sequences homologous to retroelements of the gypsy and copia types were identified as well as numerous solo long terminal repeats. We identified at least 1406 genes homologous to Saccharomyces cerevisiae open reading frames, with on average 58.1% and 72.4% amino acid identity and similarity, respectively. In addition, by comparison with completely sequenced genomes and the SwissProt database, we found 27 novel S. kluyveri genes. Most of these genes belong to pathways or have functions absent from S. cerevisiae , such as the catabolic pathway of purines or pyrimidines, melibiose fermentation, sorbitol utilization, or degradation of pollutants. The sequences are deposited in EMBL under the accession numbers AL404849 – AL407376 .

The publisher and the author regret that in the recent publication of the above article the supplementary material accompanying the article contained formatting errors hiding some of the text describing the food usage for the following species: Galactomyces candidum, Geotrichum candidum, Pichia kudriavzevii, and Pichia fermentans. The corrected supplementary data is now available online. Erratum, volume 156, issue 3, page 301, doi: 10.1016/j.ijfoodmicro.2012.04.003; Microbial food cultures have directly or indirectly come under various regulatory frameworks in the course of the last decades. Several of those regulatory frameworks put emphasis on "the history of use", "traditional food", or "general recognition of safety". Authoritative lists of microorganisms with a documented use in food have therefore come into high demand. One such list was published in 2002 as a result of a joint project between the International Dairy Federation (IDF) and the European Food and Feed Cultures Association (EFFCA). The "2002 IDF inventory" has become a de facto reference for food cultures in practical use. However, as the focus mainly was on commercially available dairy cultures, there was an unmet need for a list with a wider scope. We present an updated inventory of microorganisms used in food fermentations covering a wide range of food matrices (dairy, meat, fish, vegetables, legumes, cereals, beverages, and vinegar). We have also reviewed and updated the taxonomy of the microorganisms used in food fermentations in order to bring the taxonomy in agreement with the current standing in nomenclature.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from ‘maverick’ genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the ‘maverick’ genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear ‘Ascomycetes-specific’. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the ‘Ascomycetes-specific’ genes tend to diverge more rapidly in evolution than that of other genes. Half of the ‘Ascomycetes-specific’ genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.