Apport des nouvelles générations de séquençage pour accéder à la diversité des communautés microbiennes du sol : nécessité d’un ‘pipeline’ bio-informatique pour les biologistes

Communication orale, résuméLa diversité microbienne d’un sol est difficile à caractériser. Ceci s’explique par une accessibilité plus ou moins importante des populations au sein d’une matrice hétérogène et structurée, mais aussi par l’incapacité à résoudre une information constituée de 100 000 à 1 000 000 d’espèces différentes par gramme de sol. Toutefois, récemment, d’importantes avancées en biologie moléculaire ont permis de mieux caractériser la diversité des communautés microbiennes du sol in situ et ce sans a priori. Ainsi, la puissance des nouvelles générations de séquençage comme le pyroséquençage permettent de travailler en haut-débit afin d’obtenir plusieurs dizaines, voire plusieurs centaines de milliers de séquences à partir d’un ADN méta-génomique. De premières études ont déjà été réalisées avec cette technique afin d’aborder la diversité bactérienne des sols. Ces études ont, pour la première fois, permis de quantifier de façon exhaustive la diversité microbienne de sols en termes de richesse spécifique et de démontrer la pertinence, la faisabilité et la robustesse de cette approche. Cette approche est maintenant unanimement reconnue pour sa pertinence et ses potentialités très importantes, et ce afin de déterminer la diversité des microorganismes telluriques. Notre approche consiste en la caractérisation de la diversité taxonomique (bactérienne et fongique) de sols sur des échantillonnages de grande ampleur dans le temps et dans l’espace, avec comme objectifs : (i) de faire un inventaire exhaustif de la diversité microbienne tellurique, (ii) d’évaluer sa distribution spatiale, (iii) de mieux comprendre sa régulation et, (iv) in fine, de pouvoir relier cette diversité en fonctionnement biologique du sol et en services écosystémiques [1-3]. Cependant, l’étude d’un aussi grand nombre d’échantillons va entraîner la production massive de séquences. Ce caractère massif, ainsi que les caractéristiques inhérentes aux séquences obtenues par cette technique requièrent le développement d’outils bioinformatiques adaptés, optimisés et évalués, afin d’analyser rapidement et efficacement ce type de données. Ce nouveau pipeline d’analyse doit tout d’abord être facile d’utilisation et répondre aux attentes des différents utilisateurs, qu’ils soient compétents en bio-informatique, ou novices dans l’analyse de tels jeux de données. Il doit également permettre de gérer un grand nombre de séquences et d’automatiser les grandes étapes d’analyse (prétraitement, filtration, clustérisation, assignation taxonomique, calculs d’indices d’abondance et de diversité, taux de couverture,…). L’ensemble du système devra enfin être transféré sur un serveur de calcul et accessible au travers d’un serveur Web pour être accessible à la collectivité des écologistes microbiens. L’objectif étant de coupler, sur un grand nombre d’échantillons, cette approche avec des mesures d’activités et de faire le lien entre la diversité microbienne et l’aptitude des sols à rendre des services

Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard

Les machines : architecture des ordinateurs - d'une introduction historique à la définition d'une machine virtuelle universelle -

Cet article est une présentation de ce qu'on appelle communément l'architecture des ordinateurs en Informatique. Il est destiné aux étu-diants de niveau Licence ou Master en Informatique, notamment à ceux préparant un CAPES d'informatique, comme aux enseignants du secondaire qui souhaitent accompagner l'apparition de la discipline Informatique au lycée. Il suit un plan en 5 parties :-La genèse des ordinateurs où l'on introduit progressivement et en suivant la voie historique les étapes qui ont conduit aux fon-dements de l'architecture que l'on connaît actuellement-L'architecture de base des ordinateurs en exposant les grands principes communs à toutes les réalisations-La présentation d'un Ordinateur Réduit Facile Évolutif Universel (que nous appelons ORFEU) illustrant ce type d'architecture et son langage d'assemblage (LAMOR). Cet ordinateur et ce lan-gage pouvant également servir de base à l'élaboration de séances d'enseignement.-Quelques extensions facilitant la programmation d'un ordinateur de base-Les architectures évoluées du processeur et des mémoires Il est suivi d'une brève conclusion et de quelques annexes Table des matières 1 La genèse 4

Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones

The transcriptome of the venom duct of the Atlantic piscivorous cone species Chelyconus ermineus (Born, 1778) was determined. The venom repertoire of this species includes at least 378 conotoxin precursors, which could be ascribed to 33 known and 22 new (unassigned) protein superfamilies, respectively.Most abundant superfamilies were T,W, O1, M, O2, and Z, accounting for 57% of all detected diversity. A total of three individuals were sequenced showing considerable intraspecific variation: each individual had many exclusive conotoxin precursors, and only 20% of all inferred mature peptides were common to all individuals. Three different regions (distal, medium, and proximal with respect to the venom bulb) of the venom duct were analyzed independently. Diversity (in terms of number of distinct members) of conotoxin precursor superfamilies increased toward the distal region whereas transcripts detected toward the proximal region showed higher expression levels. Only the superfamilies A and I3 showed statistically significant differential expression across regions of the venom duct. Sequences belonging to the alpha (motor cabal) and kappa (lightning-strike cabal) subfamilies of the superfamily A were mainly detected in the proximal region of the venom duct. The mature peptides of the alpha subfamily had the a4/4 cysteine spacing pattern, which has been shown to selectively target muscle nicotinic-acetylcholine receptors, ultimately producing paralysis. This function is performed by mature peptides having a a3/5 cysteine spacing pattern in piscivorous cone species from the Indo-Pacific region, thereby supporting a convergent evolution of piscivory in cones

A Polychaete’s Powerful Punch: Venom Gland Transcriptomics of Glycera Reveals a Complex Cocktail of Toxin Homologs

© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. The article attached is the publisher's pdf

Identification of transcriptional signals in Encephalitozoon cuniculi widespread among Microsporidia phylum: support for accurate structural genome annotation

<p>Abstract</p> <p>Background</p> <p>Microsporidia are obligate intracellular eukaryotic parasites with genomes ranging in size from 2.3 Mbp to more than 20 Mbp. The extremely small (2.9 Mbp) and highly compact (~1 gene/kb) genome of the human parasite <it>Encephalitozoon cuniculi </it>has been fully sequenced. The aim of this study was to characterize noncoding motifs that could be involved in regulation of gene expression in <it>E. cuniculi </it>and to show whether these motifs are conserved among the phylum Microsporidia.</p> <p>Results</p> <p>To identify such signals, 5' and 3'RACE-PCR experiments were performed on different E. cuniculi mRNAs. This analysis confirmed that transcription overrun occurs in E. cuniculi and may result from stochastic recognition of the AAUAAA polyadenylation signal. Such experiments also showed highly reduced 5'UTR's (<7 nts). Most of the <it>E. cuniculi </it>genes presented a CCC-like motif immediately upstream from the coding start. To characterize other signals involved in differential transcriptional regulation, we then focused our attention on the gene family coding for ribosomal proteins. An AAATTT-like signal was identified upstream from the CCC-like motif. In rare cases the cytosine triplet was shown to be substituted by a GGG-like motif. Comparative genomic studies confirmed that these different signals are also located upstream from genes encoding ribosomal proteins in other microsporidian species including <it>Antonospora locustae</it>, <it>Enterocytozoon bieneusi</it>, <it>Anncaliia algerae </it>(syn. <it>Brachiola algerae</it>) and <it>Nosema ceranae</it>. Based on these results a systematic analysis of the ~2000 E. cuniculi coding DNA sequences was then performed and brings to highlight that 364 translation initiation codons (18.29% of total CDSs) had been badly predicted.</p> <p>Conclusion</p> <p>We identified various signals involved in the maturation of E. cuniculi mRNAs. Presence of such signals, in phylogenetically distant microsporidian species, suggests that a common regulatory mechanism exists among the microsporidia. Furthermore, 5'UTRs being strongly reduced, these signals can be used to ensure the accurate prediction of translation initiation codons for microsporidian genes and to improve microsporidian genome annotation.</p

Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

<p>Abstract</p> <p>Background</p> <p>Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes.</p> <p>Results</p> <p>First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain <it>Sphingomonas paucimobilis </it>sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently.</p> <p>Conclusions</p> <p>We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to study any group of genes. The Metabolic Design software is freely available from the authors and can be downloaded and modified under general public license.</p

Shifts in microbial diversity through land use intensity as drivers of carbon mineralization in soil

Land use practices alter the biomass and structure of soil microbial communities. However, the impact of land management intensity on soil microbial diversity (i.e. richness and evenness) and consequences for functioning is still poorly understood. Here, we addressed this question by coupling molecular characterization of microbial diversity with measurements of carbon (C) mineralization in soils obtained from three locations across Europe, each representing a gradient of land management intensity under different soil and environmental conditions. Bacterial and fungal diversity were characterized by high throughput sequencing of ribosomal genes. Carbon cycling activities (i.e., mineralization of autochthonous soil organic matter, mineralization of allochthonous plant residues) were measured by quantifying 12C- and 13C-CO2 release after soils had been amended, or not, with 13C-labelled wheat residues. Variation partitioning analysis was used to rank biological and physicochemical soil parameters according to their relative contribution to these activities. Across all three locations, microbial diversity was greatest at intermediate levels of land use intensity, indicating that optimal management of soil microbial diversity might not be achieved under the least intensive agriculture. Microbial richness was the best predictor of the C-cycling activities, with bacterial and fungal richness explaining 32.2 and 17% of the intensity of autochthonous soil organic matter mineralization; and fungal richness explaining 77% of the intensity of wheat residues mineralization. Altogether, our results provide evidence that there is scope for improvement in soil management to enhance microbial biodiversity and optimize C transformations mediated by microbial communities in soil

Recruitment of Glycosyl Hydrolase Proteins in a Cone Snail Venomous Arsenal: Further Insights into Biomolecular Features of Conus Venoms

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms (“injectable venom” stands for the venom variety obtained by milking of the snails. This is in contrast to the “dissected venom”, which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms

Can cyanobacterial diversity in the source predict the diversity in sludge and the risk of toxin release in a drinking water treatment plant?

ABSTRACT: Conventional processes (coagulation, flocculation, sedimentation, and filtration) are widely used in drinking water treatment plants and are considered a good treatment strategy to eliminate cyanobacterial cells and cell-bound cyanotoxins. The diversity of cyanobacteria was investigated using taxonomic cell counts and shotgun metagenomics over two seasons in a drinking water treat- ment plant before, during, and after the bloom. Changes in the community structure over time at the phylum, genus, and species levels were monitored in samples retrieved from raw water (RW), sludge in the holding tank (ST), and sludge supernatant (SST). Aphanothece clathrata brevis, Microcystis aeruginosa, Dolichospermum spiroides, and Chroococcus minimus were predominant species detected in RW by taxonomic cell counts. Shotgun metagenomics revealed that Proteobacteria was the pre- dominant phylum in RW before and after the cyanobacterial bloom. Taxonomic cell counts and shotgun metagenomic showed that the Dolichospermum bloom occurred inside the plant. Cyanobac- teria and Bacteroidetes were the major bacterial phyla during the bloom. Shotgun metagenomics also showed that Synechococcus, Microcystis, and Dolichospermum were the predominant detected cyanobacterial genera in the samples. Conventional treatment removed more than 92% of cyanobac- terial cells but led to cell accumulation in the sludge up to 31 times more than in the RW influx. Coagulation/sedimentation selectively removed more than 96% of Microcystis and Dolichospermum. Cyanobacterial community in the sludge varied from raw water to sludge during sludge storage (1–13 days). This variation was due to the selective removal of coagulation/sedimentation as well as the accumulation of captured cells over the period of storage time. However, the prediction of the cyanobacterial community composition in the SST remained a challenge. Among nutrient parameters, orthophosphate availability was related to community profile in RW samples, whereas communities in ST were influenced by total nitrogen, Kjeldahl nitrogen (N- Kjeldahl), total and particulate phos- phorous, and total organic carbon (TOC). No trend was observed on the impact of nutrients on SST communities. This study profiled new health-related, environmental, and technical challenges for the production of drinking water due to the complex fate of cyanobacteria in cyanobacteria-laden sludge and supernatant