HiSpOD: probe design for functional DNA microarrays.

International audienceMOTIVATION: The use of DNA microarrays allows the monitoring of the extreme microbial diversity encountered in complex samples like environmental ones as well as that of their functional capacities. However, no probe design software currently available is adapted to easily design efficient and explorative probes for functional gene arrays. RESULTS: We present a new efficient functional microarray probe design algorithm called HiSpOD (High Specific Oligo Design). This uses individual nucleic sequences or consensus sequences produced by multiple alignments to design highly specific probes. Indeed, to bypass crucial problem of cross-hybridizations, probe specificity is assessed by similarity search against a large formatted database dedicated to microbial communities containing about 10 million coding sequences (CDS). For experimental validation, a microarray targeting genes encoding enzymes involved in chlorinated solvent biodegradation was built. The results obtained from a contaminated environmental sample proved the specificity and the sensitivity of probes designed with the HiSpOD program. AVAILABILITY: http://fc.isima.fr/~g2im/hispod/

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

PhylArray: phylogenetic probe design algorithm for microarray

International audienceMOTIVATION: Microbial diversity is still largely unknown in most environments, such as soils. In order to get access to this microbial 'black-box', the development of powerful tools such as microarrays are necessary. However, the reliability of this approach relies on probe efficiency, in particular sensitivity, specificity and explorative power, in order to obtain an image of the microbial communities that is close to reality. RESULTS: We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria

Oryza Tag Line, a phenotypic mutant database for the Génoplante rice insertion line library

To organize data resulting from the phenotypic characterization of a library of 30 000 T-DNA enhancer trap (ET) insertion lines of rice (Oryza sativa L cv. Nipponbare), we developed the Oryza Tag Line (OTL) database (http://urgi.versailles.inra.fr/OryzaTagLine/). OTL structure facilitates forward genetic search for specific phenotypes, putatively resulting from gene disruption, and/or for GUSA or GFP reporter gene expression patterns, reflecting ET-mediated endogenous gene detection. In the latest version, OTL gathers the detailed morpho-physiological alterations observed during field evaluation and specific screens in a first set of 13 928 lines. Detection of GUS or GFP activity in specific organ/tissues in a subset of the library is also provided. Search in OTL can be achieved through trait ontology category, organ and/or developmental stage, keywords, expression of reporter gene in specific organ/tissue as well as line identification number. OTL now contains the description of 9721 mutant phenotypic traits observed in 2636 lines and 1234 GUS or GFP expression patterns. Each insertion line is documented through a generic passport data including production records, seed stocks and FST information. 8004 and 6101 of the 13 928 lines are characterized by at least one T-DNA and one Tos17 FST, respectively that OTL links to the rice genome browser OryGenesDB

New tools for isolation and identification of micro-organisms.

The microbial world contains a highly heterogeneous group of organisms sharing only one common characteristic, their small sizes. These organisms make up two (out of three) entire Domains of life on earth, the prokaryotic Bacteria and Archaea (Woese, 1987). Within the third Domain Eukarya, the majority of the phylogenetic diversity is contained within eukaryotic micro-organisms such as protozoa, algae, and fungi. Micro-organisms are estimated to make up more than a third of Earth's biomass (Whitman, et al., 1998). Because culturing provided the platform for building the depth and detail of modern microbiological knowledge, for a long time microbiologists ignored the challenge to identify and characterize uncultured organisms. One of the indicators that cultured micro-organisms did not represent much of the microbial world was the oft-observed "great plate count anomaly" (Staley and Konopka, 1985). The discrepancy between the sizes of populations estimated by dilution plating and by microscopy is particularly dramatic in some aquatic environments, in which plate counts and viable cells estimated by acridine orange staining can differ by four to six orders of magnitude, and in soil, in which 0.1 to 1% of bacteria are readily culturable on common media under standard conditions (Torsvik and Ovreas, 2002). Understanding the extend and importance of the genetic and biochemical diversity among strains of the same or very closely related species is a cornerstone issue for many microbiological disciplines such as biotechnology, taxonomy, diagnosis, diversity surveys, etc. We are only at the beginning of the history of the discovery of the amazing microbial diversity

Caractérisation des capacités métaboliques des populations microbiennes impliquées dans les processus de bioremédiation des chloroéthènes par des approches moléculaires haut débit (les biopuces ADN fonctionnelles)

Les chloroéthènes sont les polluants majeurs des eaux souterraines et des nappes phréatiques. De par leur toxicité et leur effet cancérigène, ils représentent une préoccupation majeure pour les autorités publiques et sanitaires. La restauration des sites contaminés est possible par des techniques de dépollution biologique impliquant les microorganismes (bioremédiation microbienne). Cependant, la réussite des traitements dépend à la fois des conditions physicochimiques du site pollué et des capacités de dégradation de la microflore indigène. Ainsi, pour optimiser les processus de décontamination, l identification et le suivi des différentes populations microbiennes sont indispensables avant et pendant le traitement. Les biopuces ADN fonctionnelles (FGA, Functional Gene Array), outils moléculaires haut débit, sont particulièrement bien adaptées pour des applications en bioremédiation. Leur élaboration nécessite de disposer de logiciels performants pour le design de sondes qui combinent à la fois une forte sensibilité, une très bonne spécificité et un caractère exploratoire, ce dernier étant indispensable pour la détection des séquences connues mais surtout de celles encore jamais décrites au sein d échantillons environnementaux. Un nouveau logiciel, autorisant la sélection de sondes combinant tous ces critères, a été développé et nommé HiSpOD. Son utilisation pour la construction d une FGA dédiée aux voies de biodégradation des chloroéthènes a permis d évaluer l effet de traitements de biostimulation sur la microflore indigène pour plusieurs sites industriels contaminés. Les données révèlent différentes associations entre microorganismes déhalorespirants qui sont fonction des paramètres environnementaux.Chlorinated solvents are among the most frequent contaminants found in groundwater and subsurface ecosystems. Because of their high toxicity and carcinogenicity, they represent a serious risk for human health and the environment. Thus, such polluted sites need a rehabilitation treatment. Among remediation solutions, microbial bioremediation represents a less invasive and expensive alternative than physico-chemical treatments. However, the process efficiency greatly depends on the environmental conditions and the microbial populations biodegradation capacities. Therefore, bioremediation treatment optimization requires the identification and monitoring of such capacities before and during the treatment. Functional Gene Arrays (FGA), by profiling environmental communities in a flexible and easy-to-use manner, are well adapted for an application in bioremediation. But, constructing efficient microarrays dedicated to microbial ecology requires a probe design step allowing the selection of highly sensitive, specific and explorative oligonucleotides. After a detailed state of the art on probe design strategies suitable for microbial ecology studies, we present new software, called HiSpOD, generating efficient explorative probes for FGA dedicated to environmental applications. Finally, this bioinformatics tool was used to construct a FGA targeting most genes involved in chloroethenes biodegradation pathways which allowed the evaluation of biostimulation treatments conducted on indigenous bacterial populations for several industrial contaminated sites.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

New insights into the microbial contribution to the chlorine cycle in aquatic ecosystems

Chapter 17Microorganisms hold key positions in ecosystem functioning, and thus in biogeochemical cycles. Among these cycles, some, such as chlorine (Cl), are still poorly understood. Recent works have revealed that natural chlorination and dechlorination of organic matter (OM) in most of the ecosystems were much more extensive and ubiquitous than previously suggested. Currently, there are clear evidences that natural chlorination is tightly linked to different defence mechanisms and antagonistic reactions among microorganisms. Likewise, it has been clearly demonstrated that organochlorine (Clorg) formation is also linked to OM degradation, possibly affecting carbon cycle. The chlorination rate of OM depends on several parameters including OM content and quality, microbial activity, chloride (Cl−) input and pH. Once produced, Clorg undergoes oxidative or reductive degradation in the environment depending on the surrounding physico-chemical conditions. Among all enzyme-mediated processes described, the organohalide respiration (an anaerobic bacterial respiratory process) is the only known mechanism leading to the removal of halogens from highly chlorinated compounds, transforming them into biodegradable metabolites. However, despite a significant growth in the literature since the early 1990s, the biogeochemistry of Cl in natural environment is still poorly documented. For instance, the Cl cycling in aquatic environments including Cl− and Clorg pools in sediment and water, are largely missing. The present chapter seeks to review the literature on the natural Cl cycling in environment, with a focus on a freshwater ecosystem, the Lake Pavin

A drift‐barrier model drives the genomic landscape of a structured bacterial population

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New insights into the pelagic microorganisms involved in the methane cycle in the meromictic Lake Pavin through metagenomics

International audienceAdvances in metagenomics have given rise to the possibility of obtaining genome sequences from uncultured microorganisms, even for those poorly represented in the microbial community, thereby providing an important means to study their ecology and evolution. In this study, metagenomic sequencing was carried out at four sampling depths having different oxygen concentrations or environmental conditions in the water column of Lake Pavin. By analyzing the sequenced reads and matching the contigs to the proxy genomes of the closest cultivated relatives, we evaluated the metabolic potential of the dominant planktonic species involved in the methane cycle. We demonstrated that methane-producing communities were dominated by the genus Methanoregula while methane-consuming communities were dominated by the genus Methylobacter, thus confirming prior observations. Our work allowed the reconstruction of a draft of their core metabolic pathways. Hydrogenotrophs, the genes required for acetate activation in the methanogen genome, were also detected. Regarding methanotrophy, Methylobacter was present in the same areas as the non-methanotrophic, methylotrophic Methylotenera, which could suggest a relationship between these two groups. Furthermore, the presence of a large gene inventory for nitrogen metabolism (nitrate transport, denitrification, nitrite assimilation and nitrogen fixation, for instance) was detected in the Methylobacter genome