Didier Debroas

Genetic Diversity of Small Eukaryotes in Lakes Differing by Their Trophic Status

ABSTRACT Small eukaryotes, cells with a diameter of less than 5 μm, are fundamental components of lacustrine planktonic systems. In this study, small-eukaryote diversity was determined by sequencing cloned 18S rRNA genes in three libraries from lakes of differing trophic status in the Massif Central, France: the oligotrophic Lake Godivelle, the oligomesotrophic Lake Pavin, and the eutrophic Lake Aydat. This analysis shows that the least diversified library was in the eutrophic lake (12 operational taxonomic units [OTUs]) and the most diversified was in the oligomesotrophic lake (26 OTUs). Certain groups were present in at least two ecosystems, while the others were specific to one lake on the sampling date. Cryptophyta, Chrysophyceae, and the strictly heterotrophic eukaryotes, Ciliophora and fungi, were identified in the three libraries. Among the small eukaryotes found only in two lakes, Choanoflagellida and environmental sequences (LKM11) were not detected in the eutrophic system whereas Cercozoa were confined to the oligomesotrophic and eutrophic lakes. Three OTUs, linked to the Perkinsozoa, were detected only in the Aydat library, where they represented 60% of the clones of the library. Chlorophyta and Haptophyta lineages were represented by a single clone and were present only in Godivelle and Pavin, respectively. Of the 127 clones studied, classical pigmented organisms (autotrophs and mixotrophs) represented only a low proportion regardless of the librarys origin. This study shows that the small-eukaryote community composition may differ as a function of trophic status; certain lineages could be detected only in a single ecosystem.

Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale. To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time.

Structure of the rare archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters

Marine Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, but details regarding their community structure and long-term seasonal activity and dynamics remain largely unexplored. In this study, we monitored the interannual archaeal community composition of abundant and rare biospheres in northwestern Mediterranean Sea surface waters by pyrosequencing 16S rDNA and rRNA. A detailed analysis of the rare biosphere structure showed that the rare archaeal community was composed of three distinct fractions. One contained the rare Archaea that became abundant at different times within the same ecosystem; these cells were typically not dormant, and we hypothesize that they represent a local seed bank that is specific and essential for ecosystem functioning through cycling seasonal environmental conditions. The second fraction contained cells that were uncommon in public databases and not active, consisting of aliens to the studied ecosystem and representing a nonlocal seed bank of potential colonizers. The third fraction contained Archaea that were always rare but actively growing; their affiliation and seasonal dynamics were similar to the abundant microbes and could not be considered a seed bank. We also showed that the major archaeal groups, Thaumarchaeota marine group I and Euryarchaeota group II.B in winter and Euryarchaeota group II.A in summer, contained different ecotypes with varying activities. Our findings suggest that archaeal diversity could be associated with distinct metabolisms or life strategies, and that the rare archaeal biosphere is composed of a complex assortment of organisms with distinct histories that affect their potential for growth.

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

BackgroundMetagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome.ResultsTo face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks.ConclusionsThe new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface.

Metavir: a web server dedicated to virome analysis

SUMMARY Metavir is a web server dedicated to the analysis of viral metagenomes (viromes). In addition to classical approaches for analyzing metagenomes (general sequence characteristics, taxonomic composition), new tools developed specifically for viral sequence analysis make it possible to: (i) explore viral diversity through automatically constructed phylogenies for selected marker genes, (ii) estimate gene richness through rarefaction curves and (iii) perform cross-comparison against other viromes using sequence similarities. Metavir is thus unique as a platform that allows a comprehensive virome analysis. AVAILABILITY Metavir is freely available online at: http://metavir-meb.univ-bpclermont.fr. CONTACT simon.roux@univ-bpclermont.fr. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Anaerobic Microbial Communities in Lake Pavin, a Unique Meromictic Lake in France

ABSTRACT The Bacteria and Archaea from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of Bacteria and Archaea and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing Archaea involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and Archaea detected with probe ARCH915 represented the largest microbial group in the water column, with a mean Archaea/Eubacteria ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the Archaea community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.

Unexpected Importance of Potential Parasites in the Composition of the Freshwater Small-Eukaryote Community

ABSTRACT The diversity of small eukaryotes (0.2 to 5 μm) in a mesotrophic lake (Lake Bourget) was investigated using 18S rRNA gene library construction and fluorescent in situ hybridization coupled with tyramide signal amplification (TSA-FISH). Samples collected from the epilimnion on two dates were used to extend a data set previously obtained using similar approaches for lakes with a range of trophic types. A high level of diversity was recorded for this system with intermediate trophic status, and the main sequences from Lake Bourget were affiliated with ciliates (maximum, 19% of the operational taxonomic units [OTUs]), cryptophytes (33%), stramenopiles (13.2%), and cercozoa (9%). Although the comparison of TSA-FISH results and clone libraries suggested that the level of Chlorophyceae may have been underestimated using PCR with 18S rRNA primers, heterotrophic organisms dominated the small-eukaryote assemblage. We found that a large fraction of the sequences belonged to potential parasites of freshwater phytoplankton, including sequences affiliated with fungi and Perkinsozoa. On average, these sequences represented 30% of the OTUs (40% of the clones) obtained for each of two dates for Lake Bourget. Our results provide information on lacustrine small-eukaryote diversity and structure, adding to the phylogenetic data available for lakes with various trophic types.

Evolution and Diversity of the Microviridae Viral Family through a Collection of 81 New Complete Genomes Assembled from Virome Reads

Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.

Metagenomic approach studying the taxonomic and functional diversity of the bacterial community in a mesotrophic lake (Lac du Bourget – France)

The main goals of this work were to identify the metabolic pathways of the bacterial community in a lacustrine ecosystem and to establish links between taxonomic composition and the relative abundances of these metabolic pathways. For this purpose, we analysed a 16S rRNA gene library obtained by gene amplification together with a sequence library of both insert ends on c. 7700 fosmids. Whatever the library used, Actinobacteria was the most abundant bacterial group, followed by Proteobacteria and Bacteroidetes. Specific aquatic clades such as acI and acIV (Actinobacteria) or LD12 and GOBB-C201 (Alphaproteobacteria) were found in both libraries. From comparative analysis of metagenomic libraries, the metagenome of this lake was characterized by overrepresentation of genes involved in the degradation of xenobiotics mainly associated with Alphaproteobacteria. Actinobacteria were mainly related to metabolic pathways involved in nucleotide metabolism, cofactors, vitamins, energy, replication and repair. Betaproteobacteria appeared to be characterized by the presence of numerous genes implicated in environmental information processing (membrane transport and signal transduction) whereas glycan and carbohydrate metabolism pathways were overrepresented in Bacteroidetes. These results prompted us to propose hypotheses on the ecological role of these bacterial classes in lacustrine ecosystems.

Succession and regulation factors of small eukaryote community composition in a lacustrine ecosystem (Lake Pavin)

ABSTRACT The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.