Hemiascomycetous Yeasts Research Articles

Family relationships: should consensus reign?—consensus clustering for protein families

Reliable identification of protein families is key to phylogenetic analysis, functional annotation and the exploration of protein function diversity in a given phylogenetic branch. As more and more complete genomes are sequenced, there is a need for powerful and reliable algorithms facilitating protein families construction. We have formulated the problem of protein families construction as an instance of consensus clustering, for which we designed a novel algorithm that is computationally efficient in practice and produces high quality results. Our algorithm uses an election method to construct consensus families from competing clustering computations. Our consensus clustering algorithm is tailored to serve the specific needs of comparative genomics projects. First, it provides a robust means to incorporate results from different and complementary clustering methods, thus avoiding the need for an a priori choice that may introduce computational bias in the results. Second, it is suited to large-scale projects due to the practical efficiency. And third, it produces high quality results where families tend to represent groupings by biological function. This method has been used for Génolevures project to compute protein families of Hemiascomycetous yeasts. The data are available online at http://cbi.labri.fr/Genolevures/fam/

A Phylogenetic Analysis of the Sugar Porters in Hemiascomycetous Yeasts

A total of 214 members of the sugar porter (SP) family (TC 2.A.1.1) from eight hemiascomycetous yeasts: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Ashbya (Eremothecium) gossypii, Debaryomyces hansenii, Yarrowia lipolytica, Candida albicans and Pichia stipitis, were identified. The yeast SPs were classified in 13 different phylogenetic clusters. Specific sugar substrates could be allocated to nine phylogenetic clusters, including two novel TC clusters that are specific to fungi, i.e. the glycerol:H⁺ symporter (2.A.1.1.38) and the high-affinity glucose transporter (2.A.1.1.39). Four phylogenetic clusters are identified by the preliminary fifth number Z23, Z24, Z25 and Z26 and the substrates of their members remain undetermined. The amplification of the SP clusters across the Hemiascomycetes reflects adaptation to specific carbon and energy sources available in the habitat of each yeast species.

Yarrowia lipolytica vesicle-mediated protein transport pathways

BackgroundProtein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.ResultsWe identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae.ConclusionThese results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae.

Phylogenetic Analysis of Fungal Centromere H3 Proteins

Centromere H3 proteins (CenH3's) are variants of histone H3 specialized for packaging centromere DNA. Unlike canonical H3, which is among the most conserved of eukaryotic proteins, CenH3's are rapidly evolving, raising questions about orthology and conservation of function across species. To gain insight on CenH3 evolution and function, a phylogenetic analysis was undertaken on CenH3 proteins drawn from a single, ancient lineage, the Fungi. Using maximum-likelihood methods, a credible phylogeny was derived for the conserved histone fold domain (HFD) of 25 fungal CenH3's. The collection consisted mostly of hemiascomycetous yeasts, but also included basidiomycetes, euascomycetes, and an archaeascomycete. The HFD phylogeny closely recapitulated known evolutionary relationships between the species, supporting CenH3 orthology. The fungal CenH3's lacked significant homology in their N termini except for those of the Saccharomyces/Kluyveromyces clade that all contained a region homologous to the essential N-terminal domain found in Saccharomyces cerevisiae Cse4. The ability of several heterologous CenH3's to function in S. cerevisiae was tested and found to correlate with evolutionary distance. Domain swapping between S. cerevisiae Cse4 and the noncomplementing Pichia angusta ortholog showed that species specificity could not be explained by the presence or absence of any recognized secondary structural element of the HFD.

Evolution of gene families: the multidrug resistance transporter genes in five related yeast species

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.

Highly Variable Rates of Genome Rearrangements between Hemiascomycetous Yeast Lineages

Hemiascomycete yeasts cover an evolutionary span comparable to that of the entire phylum of chordates. Since this group currently contains the largest number of complete genome sequences it presents unique opportunities to understand the evolution of genome organization in eukaryotes. We inferred rates of genome instability on all branches of a phylogenetic tree for 11 species and calculated species-specific rates of genome rearrangements. We characterized all inversion events that occurred within synteny blocks between six representatives of the different lineages. We show that the rates of macro- and microrearrangements of gene order are correlated within individual lineages but are highly variable across different lineages. The most unstable genomes correspond to the pathogenic yeasts Candida albicans and Candida glabrata. Chromosomal maps have been intensively shuffled by numerous interchromosomal rearrangements, even between species that have retained a very high physical fraction of their genomes within small synteny blocks. Despite this intensive reshuffling of gene positions, essential genes, which cluster in low recombination regions in the genome of Saccharomyces cerevisiae, tend to remain syntenic during evolution. This work reveals that the high plasticity of eukaryotic genomes results from rearrangement rates that vary between lineages but also at different evolutionary times of a given lineage.

Visualizing syntenic relationships among the hemiascomycetes with the Yeast Gene Order Browser.

The Yeast Gene Order Browser (YGOB) is an online tool designed to facilitate the comparative genomic visualization and appraisal of synteny within and between the genomes of seven hemiascomycete yeast species. Three of these genomes are polyploid, and hence contain intra-genomic syntenic regions, the correct assembly of which is a particular success of YGOB. Designed to accurately assemble, display and score gene order relationships, YGOB is both an interactive tool for browsing genomic data, and a software engine now being used for evolutionary analyses on a whole-genome scale. Underlying the online interface is the YGOB database, which consists of homology assignments across the species, extensively curated based on sequence similarity and novelly, an appraisal of genomic context (synteny) in multiple genomes. Currently the YGOB database incorporates genome data from Saccharomyces cerevisiae, Candida glabrata, Saccharomyces castellii, Ashbya gossypii, Kluyveromyces lactis, Kluyveromyces waltii and Saccharomyces kluyveri, but the system is scaleable to accommodate additional genomes. This paper discusses the usage and utility of version 1.0 of YGOB, which is publicly available at .

Genolevures complete genomes provide data and tools for comparative genomics of hemiascomycetous yeasts

The Génolevures online database () provides tools and data relative to 4 complete and 10 partial genome sequences determined and manually annotated by the Génolevures Consortium, to facilitate comparative genomic studies of hemiascomycetous yeasts. With their relatively small and compact genomes, yeasts offer a unique opportunity for exploring eukaryotic genome evolution. The new version of the Génolevures database provides truly complete (subtelomere to subtelomere) chromosome sequences, 25 000 protein-coding and tRNA genes, and in silico analyses for each gene element. A new feature of the database is a novel collection of conserved multi-species protein families and their mapping to metabolic pathways, coupled with an advanced search feature. Data are presented with a focus on relations between genes and genomes: conservation of genes and gene families, speciation, chromosomal reorganization and synteny. The Génolevures site includes an area for specific studies by members of its international community.

ESCRT-I components of the endocytic machinery are required for Rim101-dependent ambient pH regulation in the yeast Yarrowia lipolytica

Ambient pH signalling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Insertional mutagenesis in the yeast Yarrowia lipolytica identified two components of the endosome-associated ESCRT-I complex involved in multivesicular body (MVB) vesicle formation, YlVps28p and YlVps23p. They were shown to be required at alkaline pH, like Rim factors, for transcriptional activation of alkaline-induced genes and repression of acid-induced genes. The constitutively active YlRIM101-1119 allele, which suppresses the pH-signalling defects of Ylrim mutations, also suppresses Ylvps defects in pH response, but not in endocytosis. The contribution of the ESCRT-III component Snf7p could not be assessed due to the essential nature of this component in Y. lipolytica. Unlike Rim factors, YlVps4p, a component of the MVB pathway acting downstream from ESCRT complexes, seems not to be required for the alkaline response. In Y. lipolytica, all vps mutations including those affecting YlVPS4, affected growth at acidic pH, a feature not exhibited by Ylrim mutations. These results suggest that Rim and Vps pathways cooperate in ambient pH signalling and that this relation is conserved across the full range of hemiascomycetous yeasts.

Molecular Evolution of Minisatellites in Hemiascomycetous Yeasts

Minisatellites are DNA tandem repeats exhibiting size polymorphism among individuals of a population. This polymorphism is generated by two different mechanisms, both in human and yeast cells, "replication slippage" during S-phase DNA synthesis and "repair slippage" associated to meiotic gene conversion. The Saccharomyces cerevisiae genome contains numerous natural minisatellites. They are located on all chromosomes without any obvious distribution bias. Minisatellites found in protein-coding genes have longer repeat units and on the average more repeat units than minisatellites in noncoding regions. They show an excess of cytosines on the coding strand, as compared to guanines (negative GC skew). They are always multiples of three, encode serine- and threonine-rich amino acid repeats, and are found preferably within genes encoding cell wall proteins, suggesting that they are positively selected in this particular class of genes. Genome-wide, there is no statistically significant association between minisatellites and meiotic recombination hot spots. In addition, minisatellites that are located in the vicinity of a meiotic hot spot are not more polymorphic than minisatellites located far from any hot spot. This suggests that minisatellites, in S. cerevisiae, evolve probably by strand slippage during replication or mitotic recombination. Finally, evolution of minisatellites among hemiascomycetous yeasts shows that even though many minisatellite-containing genes are conserved, most of the time the minisatellite itself is not conserved. The diversity of minisatellite sequences found in orthologous genes of different species suggests that minisatellites are differentially acquired and lost during evolution of hemiascomycetous yeasts at a pace faster than the genes containing them.

A surprisingly large RNase P RNA in Candida glabrata

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast Yarrowia lipolytica are regulated differentially in the absence of oxygen

Yarrowia lipolytica is a strictly aerobic fungus, which differs from the extensively studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its physiology, genetics and dimorphic growth habit. We isolated and sequenced cDNA and genomic clones (YlAAC1) from Y. lipolytica that encode a mitochondrial ADP/ATP carrier. The YlAAC1 gene can complement the S. cerevisiae Deltaaac2 deletion mutant. Southern hybridization, analysis of Yarrowia clones obtained in the course of the Genolevures project, and further sequencing revealed the existence of two paralogs of the YlAAC1 gene, which were named YlAAC2 and YlAAC3, respectively. Phylogenetic analysis showed that YlAAC1 and YlAAC2 were more closely related to each other than to YlAAC3, and are likely to represent the products of a recent gene duplication. All three Y. lipolytica YlAAC genes group together on the phylogenetic tree, suggesting that YlAAC3 is derived from a more ancient duplication within the Y. lipolytica lineage. A similar branching pattern for the three ScAAC paralogs in the facultative anaerobe S. cerevisiae demonstrates that two rounds of duplication of AAC genes occurred independently at least twice in the evolution of hemiascomycetous yeasts. Surprisingly, in both the aerobic Y. lipolytica and the facultative anaerobe S. cerevisiae, the three paralogs are differentially regulated in the absence of oxygen. Apparently, Y. lipolytica can sense hypoxia and down-regulate target genes in response.

Comparative Genomics of Hemiascomycete Yeasts: Genes Involved in DNA Replication, Repair, and Recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.

Highly variable rates of genome rearrangements between Hemiascomycetous yeast lineages

Highly variable rates of genome rearrangements between Hemiascomycetous yeast lineages

Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts

In a quantitative comparative study, we elucidated the glucose metabolism in fourteen hemiascomycetous yeasts from the Genolevures project. The metabolic networks of these different species were first established by (13)C-labeling data and the inventory of the genomes. This information was subsequently used for metabolic-flux ratio analysis to quantify the intracellular carbon flux distributions in these yeast species. Firstly, we found that compartmentation of amino acid biosynthesis in most species was identical to that in Saccharomyces cerevisiae. Exceptions were the mitochondrial origin of aspartate biosynthesis in Yarrowia lipolytica and the cytosolic origin of alanine biosynthesis in S. kluyveri. Secondly, the control of flux through the TCA cycle was inversely correlated with the ethanol production rate, with S. cerevisiae being the yeast with the highest ethanol production capacity. The classification between respiratory and respiro-fermentative metabolism, however, was not qualitatively exclusive but quantitatively gradual. Thirdly, the flux through the pentose phosphate (PP) pathway was correlated to the yield of biomass, suggesting a balanced production and consumption of NADPH. Generally, this implies the lack of active transhydrogenase-like activities in hemiascomycetous yeasts under the tested growth condition, with Pichia angusta as the sole exception. In the latter case, about 40% of the NADPH was produced in the PP pathway in excess of the requirements for biomass production, which strongly suggests the operation of a yet unidentified mechanism for NADPH reoxidation in this species. In most yeasts, the PP pathway activity appears to be driven exclusively by the demand for NADPH.

Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis

Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.

Yeast orthologues associated with glycerol transport and metabolism.

Glycerol is a key compound in the regulation of several metabolic pathways in Saccharomyces cerevisiae. From this yeast most of the genes involved in glycerol consumption, production and transport are now available. Some of the mechanisms involving glycerol metabolism and transport are common to other yeasts. This work presents a search for GPD1/2, GUT1, GUP1/2 and FPS1 orthologues in a series of hemiascomycetous yeasts. All the genes cloned were able to complement S. cerevisiae mutant phenotypes and presented a high degree of similarity to the corresponding genes in this yeast. A phylogenetic analysis is presented. The allocation of GUP genes in the membrane bound O-acyl transferases (MBOAT) family is suggested as more consistent than their inclusion in the TC-DB/glycerol uptake family.

Genome evolution in yeasts.

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.

Génolevures: comparative genomics and molecular evolution of hemiascomycetous yeasts.

The Génolevures online database (http://cbi.labri.fr/Genolevures/) provides data and tools to facilitate comparative genomic studies on hemiascomycetous yeasts. Now, four complete genome sequences recently determined (Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica) have been added to the partial sequences of 13 species previously analysed by a random approach. The database also includes the reference genome Saccharomyces cerevisiae. Data are presented with a focus on relations between genes and genomes: conservation of genes and gene families, speciation, chromosomal reorganization and synteny. The Génolevures site includes a community area for specific studies by members of the international community.

Unique distribution of GAL genes on chromosome XI in the yeast Saccharomyces naganishii.

A region of DNA extending from GAL7 to GAL1 was cloned in the yeast Saccharomyces naganishii. Sequence analysis revealed that GAL7 and GAL1 are separated by approximately 2 kbp and share a common promoter region. Although GAL7, GAL10, and GAL1 are clustered in this order in previously studied hemiascomycetous yeasts, GAL10 was not found between GAL7 and GAL1 in S. naganishii. Southern blotting of S. naganishii chromosomal DNA showed that both the GAL7-GAL1 region and GAL10 are located on chromosome XI, but that GAL10 is located more than 10 kbp away from GAL7-GAL1. Thus, S. naganishii and S. cerevisiae, while related phylogenetically, do not share the same orientation with respect to GAL genes. These data are highly relevant to studies of chromosomal evolution in yeast.