Corinne Biderre-Petit

Production and consumption of methane in freshwater lake ecosystems

The atmospheric concentration of methane (CH(4)), a major greenhouse gas, is mainly controlled by the activities of methane-producing (methanogens) and methane-consuming (methanotrophs) microorganisms. Freshwater lakes are identified as one of the main CH(4) sources, as it was estimated that they contribute to 6-16% of natural CH(4) emissions. It is therefore critical to better understanding the biogeochemical cycling of CH(4) in these ecosystems. In this paper, the effects of environmental factors on methanogenic and methanotrophic rates are reviewed and an inventory of the methanogens and methanotrophs at the genus/species level in freshwater lakes is given. We focus on the anaerobic oxidation of methane, which is a still poorly known process but increasingly reported in freshwater lakes.

Identification of microbial communities involved in the methane cycle of a freshwater meromictic lake

Lake Pavin is a meromictic crater lake located in the French Massif Central area. In this ecosystem, most methane (CH(4)) produced in high quantity in the anoxic bottom layers, and especially in sediments, is consumed in the water column, with only a small fraction of annual production reaching the atmosphere. This study assessed the diversity of methanogenic and methanotrophic populations along the water column and in sediments using PCR and reverse transcription-PCR-based approaches targeting functional genes, i.e. pmoA (α-subunit of the particulate methane monooxygenase) for methanotrophy and mcrA (α-subunit of the methyl-coenzyme M reductase) for methanogenesis as well as the phylogenetic 16S rRNA genes. Although methanogenesis rates were much higher in sediments, our results confirm that CH(4) production also occurs in the water column where methanogens were almost exclusively composed of hydrogenotrophic methanogens, whereas both hydrogenotrophs and acetotrophs were almost equivalent in the sediments. Sequence analysis of markers, pmoA and the 16S rRNA gene, suggested that Methylobacter may be an important group actively involved in CH(4) oxidation in the water column. Two main phylotypes were characterized, one of which could consume CH(4) under conditions where the oxygen amount is undetectable.

Detecting unknown sequences with DNA microarrays: explorative probe design strategies.

Designing environmental DNA microarrays that can be used to survey the extreme diversity of microorganisms existing in nature, represents a stimulating challenge in the field of molecular ecology. Indeed, recent efforts in metagenomics have produced a substantial amount of sequence information from various ecosystems, and will continue to accumulate large amounts of sequence data given the qualitative and quantitative improvements in the next-generation sequencing methods. It is now possible to take advantage of these data to develop comprehensive microarrays by using explorative probe design strategies. Such strategies anticipate genetic variations and thus are able to detect known and unknown sequences in environmental samples. In this review, we provide a detailed overview of the probe design strategies currently available to construct both phylogenetic and functional DNA microarrays, with emphasis on those permitting the selection of such explorative probes. Furthermore, exploration of complex environments requires particular attention on probe sensitivity and specificity criteria. Finally, these innovative probe design approaches require exploiting newly available high-density microarray formats.

Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.

Annotation of microsporidian genomes using transcriptional signals

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.

Identification of Sulfur-Cycle Prokaryotes in a Low-Sulfate Lake (Lake Pavin) Using aprA and 16S rRNA Gene Markers

Geochemical researches at Lake Pavin, a low-sulfate-containing freshwater lake, suggest that the dominant biogeochemical processes are iron and sulfate reduction, and methanogenesis. Although the sulfur cycle is one of the main active element cycles in this lake, little is known about the sulfate-reducer and sulfur-oxidizing bacteria. The aim of this study was to assess the vertical distribution of these microbes and their diversities and to test the hypothesis suggesting that only few SRP populations are involved in dissimilatory sulfate reduction and that Epsilonproteobacteria are the likely key players in the oxidative phase of sulfur cycle by using a PCR aprA gene-based approach in comparison with a 16S rRNA gene-based analysis. The results support this hypothesis. Finally, this preliminary work points strongly the likelihood of novel metabolic processes upon the availability of sulfate and other electron acceptors.

HiSpOD: probe design for functional DNA microarrays.

MOTIVATION 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/.

In situ TCE degradation mediated by complex dehalorespiring communities during biostimulation processes

The bioremediation of chloroethene contaminants in groundwater polluted systems is still a serious environmental challenge. Many previous studies have shown that cooperation of several dechlorinators is crucial for complete dechlorination of trichloroethene to ethene. In the present study, we used an explorative functional DNA microarray (DechloArray) to examine the composition of specific functional genes in groundwater samples in which chloroethene bioremediation was enhanced by delivery of hydrogen‐releasing compounds. Our results demonstrate for the first time that complete biodegradation occurs through spatial and temporal variations of a wide diversity of dehalorespiring populations involving both Sulfurospirillum, Dehalobacter, Desulfitobacterium, Geobacter and Dehalococcoides genera. Sulfurospirillum appears to be the most active in the highly contaminated source zone, while Geobacter was only detected in the slightly contaminated downstream zone. The concomitant detection of both bvcA and vcrA genes suggests that at least two different Dehalococcoides species are probably responsible for the dechlorination of dichloroethenes and vinyl chloride to ethene. These species were not detected on sites where cis‐dichloroethene accumulation was observed. These results support the notion that monitoring dechlorinators by the presence of specific functional biomarkers using a powerful tool such as DechloArray will be useful for surveying the efficiency of bioremediation strategies.

Temporal dynamics of active Archaea in oxygen-depleted zones of two deep lakes

Deep lakes are of specific interest in the study of archaeal assemblages as chemical stratification in the water column allows niche differentiation and distinct community structure. Active archaeal community and potential nitrifiers were investigated monthly over 1 year by pyrosequencing 16S rRNA transcripts and genes, and by quantification of archaeal amoA genes in two deep lakes. Our results showed that the active archaeal community patterns of spatial and temporal distribution were different between these lakes. The meromictic lake characterized by a stable redox gradient but variability in nutrient concentrations exhibited large temporal rearrangements of the dominant euryarchaeal phylotypes, suggesting a variety of ecological niches and dynamic archaeal communities in the hypolimnion of this lake. Conversely, Thaumarchaeota Marine Group I (MGI) largely dominated in the second lake where deeper water layers exhibited only short periods of complete anoxia and constant low ammonia concentrations. Investigations conducted on archaeal amoA transcripts abundance suggested that not all lacustrine Thaumarchaeota conduct the process of nitrification. A high number of 16S rRNA transcripts associated to crenarchaeal group C3 or the Miscellaneous Euryarchaeotic Group indicates the potential for these uncharacterized groups to contribute to nutrient cycling in lakes.

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

BackgroundMicroorganisms 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.ResultsFirst we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis 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.ConclusionsWe 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.