Eric Dugat-Bony

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.

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.

Investigation of the Activity of the Microorganisms in a Reblochon-Style Cheese by Metatranscriptomic Analysis

The microbial communities in cheeses are composed of varying bacteria, yeasts, and molds, which contribute to the development of their typical sensory properties. In situ studies are needed to better understand their growth and activity during cheese ripening. Our objective was to investigate the activity of the microorganisms used for manufacturing a surface-ripened cheese by means of metatranscriptomic analysis. The cheeses were produced using two lactic acid bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), one ripening bacterium (Brevibacterium aurantiacum), and two yeasts (Debaryomyces hansenii and Geotrichum candidum). RNA was extracted from the cheese rinds and, after depletion of most ribosomal RNA, sequencing was performed using a short-read sequencing technology that generated ~75 million reads per sample. Except for B. aurantiacum, which failed to grow in the cheeses, a large number of CDS reads were generated for the inoculated species, making it possible to investigate their individual transcriptome over time. From day 5 to 35, G. candidum accounted for the largest proportion of CDS reads, suggesting that this species was the most active. Only minor changes occurred in the transcriptomes of the lactic acid bacteria. For the two yeasts, we compared the expression of genes involved in the catabolism of lactose, galactose, lactate, amino acids, and free fatty acids. During ripening, genes involved in ammonia assimilation and galactose catabolism were down-regulated in the two species. Genes involved in amino acid catabolism were up-regulated in G. candidum from day 14 to day 35, whereas in D. hansenii, they were up-regulated mainly at day 35, suggesting that this species catabolized the cheese amino acids later. In addition, after 35 days of ripening, there was a down-regulation of genes involved in the electron transport chain, suggesting a lower cellular activity. The present study has exemplified how metatranscriptomic analyses provide insight into the activity of cheese microbial communities for which reference genome sequences are available. In the future, such studies will be facilitated by the progress in DNA sequencing technologies and by the greater availability of the genome sequences of cheese microorganisms.

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.

Highlighting the microbial diversity of 12 French cheese varieties

Surface-ripened cheeses host complex microbial communities responsible for the transformation of milk into cheese as well as the development of important properties in terms of texture, color and sensory perception. In this study, we used high-throughput amplicon sequencing to decipher the bacterial and fungal diversity of 60 cheeses belonging to 12 popular French cheese varieties. Using this approach, 76 bacterial and 44 fungal phylotypes were identified. Major differences were observed between rind and core samples and also according to cheese varieties and manufacturing processes. Occurrence analysis revealed the presence of widespread taxa as well as operational taxonomic units (OTUs) specific to one or several cheese varieties. Finally, we observed patterns specific to the cheese production facility, supporting the importance of indigenous microorganisms for the microbial assemblage of cheese microbiota.

Distribution of Dehalococcoidia in the Anaerobic Deep Water of a Remote Meromictic Crater Lake and Detection of Dehalococcoidia-Derived Reductive Dehalogenase Homologous Genes

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin.

MetaExploArrays: A Large-Scale Oligonucleotide Probe Design Software for Explorative DNA Microarrays

The selection of oligonucleotide probes for micro arrays is still very difficult task. With the rapid growth of environmental databases (metagenomics programs coupled to next generation sequencing), the computational capacity requirements of probe design algorithms have hugely increased. The use of parallel and distributed architectures can considerably reduce the complexity and the computational time of these algorithms. In this paper we present a new efficient algorithm of oligonucleotide probe selection for an individual specific nucleic acid sequence or a group of sequences. We used a model driven engineering approach to simultaneous design of thousands of sensitive, specific, isothermal and explorative probes, on both PC, multiprocessor, cluster and grid computing with on the one hand a significant computing speedup and on the other hand an improved quality of the resulting probes when compared to equivalent software.

Changes in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine

Escherichia coli O157:H7 is a foodborne pathogen that colonizes ruminants. Cattle are considered the primary reservoir of E. coli O157:H7 with super-shedders, defined as individuals excreting > 104 E. coli O157:H7 CFU g-1 feces. The mechanisms leading to the super-shedding condition are largely unknown. Here, we used 16S rRNA gene pyrosequencing to examine the composition of the fecal bacterial community in order to investigate changes in the bacterial microbiota at several locations along the digestive tract (from the duodenum to the rectal-anal junction) in 5 steers previously identified as super-shedders and 5 non-shedders. The overall bacterial community structure did not differ by E. coli O157:H7 shedding status; but several differences in the relative abundance of taxa and OTUs were noted between the two groups. The genus Prevotella was most enriched in the non-shedders while the genus Ruminococcus and the Bacteroidetes phylum were notably enriched in the super-shedders. There was greater bacterial diversity and richness in samples collected from the lower- as compared to the upper gastrointestinal tract (GI). The spiral colon was the only GI location that differed in terms of bacterial diversity between super-shedders and non-shedders. These findings reinforced linkages between E. coli O157:H7 colonization in cattle and the nature of the microbial community inhabiting the digestive tract of super-shedders.

Comparative genomic analysis of Brevibacterium strains: insights into key genetic determinants involved in adaptation to the cheese habitat

BackgroundBrevibacterium strains are widely used for the manufacturing of surface-ripened cheeses, contributing to the breakdown of lipids and proteins and producing volatile sulfur compounds and red-orange pigments. The objective of the present study was to perform comparative genomic analyses in order to better understand the mechanisms involved in their ability to grow on the cheese surface and the differences between the strains.ResultsThe genomes of 23 Brevibacterium strains, including twelve strains isolated from cheeses, were compared for their gene repertoire involved in salt tolerance, iron acquisition, bacteriocin production and the ability to use the energy compounds present in cheeses. All or almost all the genomes encode the enzymes involved in ethanol, acetate, lactate, 4-aminobutyrate and glycerol catabolism, and in the synthesis of the osmoprotectants ectoine, glycine-betaine and trehalose. Most of the genomes contain two contiguous genes encoding extracellular proteases, one of which was previously characterized for its activity on caseins. Genes encoding a secreted triacylglycerol lipase or involved in the catabolism of galactose and D-galactonate or in the synthesis of a hydroxamate-type siderophore are present in part of the genomes. Numerous Fe3+/siderophore ABC transport components are present, part of them resulting from horizontal gene transfers. Two cheese-associated strains have also acquired catecholate-type siderophore biosynthesis gene clusters by horizontal gene transfer. Predicted bacteriocin biosynthesis genes are present in most of the strains, and one of the corresponding gene clusters is located in a probable conjugative transposon that was only found in cheese-associated strains.ConclusionsBrevibacterium strains show differences in their gene repertoire potentially involved in the ability to grow on the cheese surface. Part of these differences can be explained by different phylogenetic positions or by horizontal gene transfer events. Some of the distinguishing features concern biotic interactions with other strains such as the secretion of proteases and triacylglycerol lipases, and competition for iron or bacteriocin production. In the future, it would be interesting to take the properties deduced from genomic analyses into account in order to improve the screening and selection of Brevibacterium strains, and their association with other ripening culture components.