Olivier Lespinet

The genome sequence of the model ascomycete fungus Podospora anserina

BackgroundThe dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.ResultsWe present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown.ConclusionThe features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.

Comparative genomics of emerging pathogens in the Candida glabrata clade

BackgroundCandida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts.ResultsOur results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata.ConclusionsRemarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces.Sequence Accession NumbersNakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186

Genome quality control: RIP (repeat‐induced point mutation) comes to Podospora

RIP (repeat‐induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C‐G to T‐A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP‐like event was observed after several rounds of sexual reproduction in a strain with a 40u2003kb tandem duplication resulting from homologous integration of a cosmid in the mating‐type region. The 9u2003kb sequenced show 106 C‐G to T‐A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP‐like event extended to single‐copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C‐methylation. Interestingly, the N. crassa zeta–eta sequence, which acts as a potent de novo C‐methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.

Survival trade-offs in plant roots during colonization by closely related beneficial and pathogenic fungi

The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.

Assessment of phylogenetic diversity of bacterial microflora in drinking water using serial analysis of ribosomal sequence tags.

We examined chlorinated drinking water samples from three different surface water treatment plants for bacterial 16S rDNA diversity using the serial analysis of V6 ribosomal sequence tag (SARST-V6) method. A considerable degree of diversity was observed in each sample, with an estimated richness ranging from 173 to 333 phylotypes. The community structure shows that there are differences in bacterial evenness between sampled sites. The taxonomic composition of the microbial communities was found to be dominated by members of the Proteobacteria (57.2-77.4%), broadly distributed among the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria. Additionally, a large proportion of sequences (6.3-36.5%) were found to be distantly related to database sequences of unknown phylogenetic affiliation. Given the apparent significance of this bacterial group in drinking water, a 16S rDNA analysis was performed and confirmed their presence and phylogeny. Notwithstanding the potential under-representation of certain bacterial phyla using the SARST-V6 primer pairs, as revealed by a refined computer algorithm, our results suggest that 16S rDNA corresponding to a variety of eubacterial groups can be detected in finished drinking water, suggesting that this water may contain a higher level of bacterial diversity than previously observed.

Assessing the evolutionary rate of positional orthologous genes in prokaryotes using synteny data.

BackgroundComparison of completely sequenced microbial genomes has revealed how fluid these genomes are. Detecting synteny blocks requires reliable methods to determining the orthologs among the whole set of homologs detected by exhaustive comparisons between each pair of completely sequenced genomes. This is a complex and difficult problem in the field of comparative genomics but will help to better understand the way prokaryotic genomes are evolving.ResultsWe have developed a suite of programs that automate three essential steps to study conservation of gene order, and validated them with a set of 107 bacteria and archaea that cover the majority of the prokaryotic taxonomic space. We identified the whole set of shared homologs between two or more species and computed the evolutionary distance separating each pair of homologs. We applied two strategies to extract from the set of homologs a collection of valid orthologs shared by at least two genomes. The first computes the Reciprocal Smallest Distance (RSD) using the PAM distances separating pairs of homologs. The second method groups homologs in families and reconstructs each familys evolutionary tree, distinguishing bona fide orthologs as well as paralogs created after the last speciation event. Although the phylogenetic tree method often succeeds where RSD fails, the reverse could occasionally be true. Accordingly, we used the data obtained with either methods or their intersection to number the orthologs that are adjacent in for each pair of genomes, the Positional Orthologous Genes (POGs), and to further study their properties. Once all these synteny blocks have been detected, we showed that POGs are subject to more evolutionary constraints than orthologs outside synteny groups, whichever the taxonomic distance separating the compared organisms.ConclusionThe suite of programs described in this paper allows a reliable detection of orthologs and is useful for evaluating gene order conservation in prokaryotes whichever their taxonomic distance. Thus, our approach will make easy the rapid identification of POGS in the next few years as we are expecting to be inundated with thousands of completely sequenced microbial genomes.

FUNGIpath: a tool to assess fungal metabolic pathways predicted by orthology

BackgroundMore and more completely sequenced fungal genomes are becoming available and many more sequencing projects are in progress. This deluge of data should improve our knowledge of the various primary and secondary metabolisms of Fungi, including their synthesis of useful compounds such as antibiotics or toxic molecules such as mycotoxins. Functional annotation of many fungal genomes is imperfect, especially of genes encoding enzymes, so we need dedicated tools to analyze their metabolic pathways in depth.DescriptionFUNGIpath is a new tool built using a two-stage approach. Groups of orthologous proteins predicted using complementary methods of detection were collected in a relational database. Each group was further mapped on to steps in the metabolic pathways published in the public databases KEGG and MetaCyc. As a result, FUNGIpath allows the primary and secondary metabolisms of the different fungal species represented in the database to be compared easily, making it possible to assess the level of specificity of various pathways at different taxonomic distances. It is freely accessible at http://www.fungipath.u-psud.fr.ConclusionsAs more and more fungal genomes are expected to be sequenced during the coming years, FUNGIpath should help progressively to reconstruct the ancestral primary and secondary metabolisms of the main branches of the fungal tree of life and to elucidate the evolution of these ancestral fungal metabolisms to various specific derived metabolisms.

ORENZA: a web resource for studying ORphan ENZyme activities

BackgroundDespite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap.DescriptionWe designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases.ConclusionORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene sequences to the relevant enzymes.

A lophotrochozoan twist gene is expressed in the ectomesoderm of the gastropod mollusk Patella vulgata.

SUMMARY The twist gene is known to be involved in mesoderm formation in two of the three clades of bilaterally symmetrical animals: viz. deuterostomes (such as vertebrates) and ecdysozoans (such as arthropods and nematodes). There are currently no data on the spatiotemporal expression of this gene in the third clade, the lophotrochozoans (such as mollusks and annelids). To approach the question of mesoderm homology across bilaterians, we decided to analyze orthologs of this gene in the gastropod mollusk Patella vulgata that belongs to the lophotrochozoans. We present here the cloning, characterization, and phylogenetic analysis of a Patella twist ortholog, Pv‐twi, and determine the early spatiotemporal expression pattern of this gene. Pv‐twi expression was found in the trochophore larva in a subset of the ectomesoderm, one of the two sources of mesoderm in Patella. These data support the idea that twist genes were ancestrally involved in mesoderm differentiation. The absence of Pv‐twi in the second mesodermal source, the endomesoderm, suggests that also other genes must be involved in lophotrochozoan mesoderm differentiation. It therefore remains a question if the mesoderm of all bilaterians is homologous.

Characterisation of two snail genes in the gastropod mollusc Patella vulgata. Implications for understanding the ancestral function of the snail-related genes in Bilateria

Abstract.Snail genes have been found to play a role in mesoderm formation in two of the three clades of bilaterians, deuterostomes (comprising the chordates) and ecdysozoans (comprising the arthropods). No clear data are available on the role these genes play in development of the mesoderm in the third clade, that of lophotrochozoans (comprising annelids and molluscs). We identified two new members of the snail gene family in the gastropod mollusc Patellavulgata. Phylogenetic analysis showed that the two genes clearly belong to the snail sub-family. Their expression patterns do not indicate a role during early mesoderm formation. In fact, contrary to expectations, the snail genes of Patella were mostly expressed in the ectoderm. In view of the location of their expression sites, we suggest that these genes could be involved in regulating epithelial-mesenchymal transitions (EMT) and cell motility, as has recently been demonstrated for snail genes in vertebrates. This may well correspond to the ancestral function of these genes. The results are discussed in the light of the evolutionary origin of the mesoderm. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00427-002-0228-1.