Search

Next generation sequencing offers several ways to study microbial communities. For agri-food sciences, identifying species in diverse food ecosystems is key for both food sustainability and food security. The aim of this study was to compare metabarcoding pipelines and markers to determine fungal diversity in food ecosystems, from Illumina short reads. We built mock communities combining the most representative fungal species in fermented meat, cheese, wine and bread. Four barcodes (ITS1, ITS2, D1/D2 and RPB2) were tested for each mock and on real fermented products. We created a database, including all mock species sequences for each barcode to compensate for the lack of curated data in available databases. Four bioinformatics tools (DADA2, QIIME, FROGS and a combination of DADA2 and FROGS) were compared. Our results clearly showed that the combined DADA2 and FROGS tool gave the most accurate results. Most mock community species were not identified by the RPB2 barcode due to unsuccessful barcode amplification. When comparing the three rDNA markers, ITS markers performed better than D1D2, as they are better represented in public databases and have better specificity to distinguish species. Between ITS1 and ITS2, differences in the best marker were observed according to the studied ecosystem. While ITS2 is best suited to characterize cheese, wine and fermented meat communities, ITS1 performs better for sourdough bread communities. Our results also emphasized the need for a dedicated database and enriched fungal-specific public databases with novel barcode sequences for 118 major species in food ecosystems.

International audience; Aims The pea seeds (Pisum sativum) hosts a complex microbial community that is exposed to environmental constraints modulated by biotic, abiotic and anthropogenic factors. The aim of this study was to explore the effect of geographical origins on the composition of the microbial communities associated with pea seeds from storage locations in different countries. These seeds are used by Roquette Freres (R) as source proteins for human food after industrial extraction process of their proteins. Methods The high-throughput sequencing of two phylogenetic markers (V3-V4 regions of the 16S rRNA gene and ITS2 region) was used to evaluate the structure of the microbial community associated with pea seeds. The pea seeds were harvested by different suppliers from three countries (France, Sweden and Canada) with different climates and stored in six cooperatives. Representative microbial strains of dominant species were isolated and characterized using phenotypic methods. Results Although the pea seed microbiota shared a dominant core microbiota at the genus level, seeds showed a species-specific, highly unique microbial signature with variable abundances according to origin. Origin was identified as the main driver of the composition of the pea seed microbiota. Conclusions These results provide an initial insight into the broad factors such as country and cooperative that may determine the assembly of the pea microbiota before processing.

International audience; Bacteriophage ecology has raised an increasing attention over the last few years, thanks to the improvement and contributions of microscopy, comparative genomics and viral metagenomics methods. Fermented foods host dense and diverse microbial communities and, therefore, represent an ideal biotope for bacteriophages. If their occurrence in such environments has been demonstrated decades ago, data highlighting their impact on mixed communities and their ecological roles are scarce when compared to other microbial ecosystems. This review summarizes most recent knowledge into the bacteriophage diversity of fermented foods and stress evidences suggesting the impact of these entities on the dynamics of food microbial communities. The main ecological roles played by bacteriophages in microbial ecosystems are also addressed. Understanding the impact of bacteriophages in fermented foods will further help in designing adapted microbial consortia and thus providing a better control of the food fermentations.

International audience; The temperature of the feed is known to be an important factor affecting the formation and the growth rates of bacterial communities on dairy filtration membranes. However, decades after the integration of filtration processes in the dairy industry, there is still questioning whether filtration should be performed at cold (48 degrees C) temperature. A biofilm reactor designed to mimic a filtration system was used to provide answers to this question. Bacteria adhering and growing on ultrafiltration membranes in contact with pasteurised skim milk were characterised at 15 degrees C and 50 degrees C through a metabarcoding approach. Our results suggested that the processing time should be limited to 10 h at 50 degrees C to avoid the exponential growth of thermophilic spore-former bacteria, while the use of 15 degrees C combined with daily cleaning procedures appeared the best way to retard the formation of biofilms on membranes.

International audience; In order to encourage Western populations to increase their consumption of vegetables, we suggest turning legumes into novel, healthy foods by applying an old, previously widespread method of food preservation: fermentation. In the present study, a total of 55 strains from different microbial species (isolated from cheese or plants) were investigated for their ability to: (i) grow on a emulsion containing 100% pea proteins and no carbohydrates or on a 50:50 pea:milk protein emulsion containing lactose, (ii) increase aroma quality and reduce sensory off-flavors; and (iii) compete against endogenous micro organisms. The presence of carbohydrates in the mixed pea:milk emulsion markedly influenced the fermentation by strongly reducing the pH through lactic fermentation, whereas the absence of carbohydrates in the pea emulsion promoted alkaline or neutral fermentation. Lactic acid bacteria assigned to Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis and Lactobacillus casei species grew well in both the pea and pea:milk emulsions. Most of the fungal strains tested (particularly those belonging to the Mucor and Geotrichum genera) were also able to grow on both emulsions. Although most Actinobacteria and Proteobacteria did not compete with endogenous microbiota (Bacillus), some species such as Hafnia alvei, Acinetobacter johnsonii and Glutamicibacter arilaitensis grew strongly and appeared to restrict the development of the endogenous microbiota when the pea emulsion was inoculated with a combination of three to nine strains. In the mixed emulsions, lactic fermentation inhibited Actinobacteria and Proteobacteria (e.g. Brevibacterium casei, Corynebacterium casei, Staphylococcus lentos) to the greatest extent but also inhibited Bacillus (e.g. Bacillus subtilis and Bacillus licheniformis). Overall, this procedure enabled us to select two microbial consortia able to colonize pea-based products and positively influence the release of volatile compounds by generating a roasted/grilled aroma for the 100% pea emulsion, and a fruity, lactic aroma for the 50:50 pea:milk emulsion. Moreover, the fermentation in the pea-based emulsions reduced the level of hexanal, which otherwise leads to an undesired green pea aroma. Our present results show how the assembly of multiple microbial cultures can help to develop an innovative food product.

Basaltic rocks play a significant role in CO2 sequestration from the atmosphere during their weathering. Moreover, the primary microorganisms that colonize them, by providing mineral elements and nutrients, are shown to promote growth of diverse heterotrophic communities and plants, therefore positively impacting Earth's long-term climate balance. However, the first steps of microbial colonization and subsequent rock weathering remain poorly understood, especially regarding microbial communities over a chronological sequence. Here, we analyzed the microbial communities inhabiting the soil developed in crevices on lava flows derived from different eruptions on Fogo Island. Investigated soils show typically low carbon and nitrogen content and are relatively similar to one another regarding their phylogenetic composition, and similar to what was recorded in large soil surveys with dominance of Actinobacteria and Proteobacteria. Moreover, our results suggest a stronger effect of the organic carbon than the lava flow age in shaping microbial communities as well as the possibility of exogenous sources of bacteria as important colonizers. Furthermore, archaea reach up to 8.4% of the total microbial community, dominated by the Soil Crenarchaeotic Group, including the ammonium-oxidizer Candidatus Nitrososphaera sp. Therefore, this group might be largely responsible for ammonia oxidation under the environmental conditions found on Fogo.

International audience; The structure and functioning of microbial communities from fermented foods, including cheese, have beenextensively studied during the past decade. However, there is still a lack of information about both the occur-rence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems.Viral metagenomics was recently applied to a wide variety of environmental samples and standardized proce-dures for recovering viral particles from different type of materials has emerged. In this study, we adapted aprocedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysisof cheese surface. We tested and validated the positive impact of both addition of afiltration step prior to virusconcentration and substitution of purification by density gradient ultracentrifugation by a simple chloroformtreatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesiclesample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembledfrom the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even frombacterial chromosomal DNA. The best procedure was then chosen, and used to describe thefirst cheese surfacevirome, using Epoisses cheese as example. This study provides the basis of future investigations regarding theecological importance of viruses in cheese microbial ecosystems.

Sodium reduction in the human diet is currently one of the main concerns for public health agencies and, consequently, has become a challenge for the food industries. In this study, the impact of reduced sodium chloride content (20%) or its partial substitution with potassium chloride in soft (“Camembert”-type) and semi-hard (“Reblochon”-type) cheeses was evaluated. Analyses included physicochemical and biochemical composition, microbial counts, 16S rRNA gene metabarcoding and metatranscriptomic analysis, volatile aroma compounds and sensory analysis. Regarding soft cheeses, the salt content of cheeses affected proteolysis at 21 days of ripening. RNA sequencing revealed that the relative activity of G. candidum increased, whereas that of P. camemberti decreased in reduced salt cheeses in comparison to the controls. Higher global intensity of odor and taste was observed in cheeses with reduced salt content, consistent with higher levels of alcohol and ester components. Regarding semi-hard cheeses, modifications of salt content did not significantly affect either their biochemical parameters and sensory characteristics or their technological microbial composition at day 21 of ripening. Finally, no impact of salt content was observed on the growth of the spoiler Yarrowia lipolytica in soft cheeses. In contrast, reducing salt content increased spoiler growth in semi-hard cheeses, as highlighted by a greater development of Pseudomonas that led to an increase in cheese proteolysis and lipolysis. In conclusion, the effect of reducing salt content is highly dependent on the cheese type. This factor should thus be taken into account by the dairy industry when the reduction of salt content is being considered. Moreover, the quality of raw products, in particular, the level of spoiler microorganisms, must be controlled before use during dairy processes.

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering virus-like particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the Epoisses cheese virome. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.

The production system of freeze-dried lactic acid bacteria involves several processes, but its impact on bacteria resistance is still not well understood. This system can be defined as a complex one since it depends on multiple scales: the Genomic, the Cellular and the Population scale. The scarcity of data available for building models leads us to propose an approach that makes use of expert knowledge. In this paper we present a semiautomatic modelling tool, LIDEOGRAM and discuss how it contributes to insight formulation and rapid hypothesis testing. New results show that LIDEOGRAM is able to produce more robust modelling hypotheses when experts can interact and revisit the genomic data preprocessing.

This study evaluated the ability of dairy matrices, different in composition (with and without fat) and structure (liquid and gel), to enhance microorganisms survival through digestion. The viability of three dairy microorganisms Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei was measured during in vitro and in vivo digestion. S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. LIVE/DEAD confocal microscopy's images, probed the effect of low pH on microorganisms survival. However, in vivo analyses (16S rRNA gene metabarcoding) failed to confirm in vitro observations since tested microorganisms were not detected. Despite of the different evolutions during digestion on buffer capacity, lipolysis, and rheological characteristics, we did not observe any protective effect of the dairy matrices on microorganisms survival.

Cheese rinds host a specific microbiota composed of both prokaryotes (such as Actinobacteria, Firmicutes and Proteobacteria) and eukaryotes (primarily yeasts and moulds). By combining modern molecular biology tools with conventional, culture-based techniques, it has now become possible to create a catalogue of the biodiversity that inhabits this special environment. Here, we review the microbial genera detected on the cheese surface and highlight the previously unsuspected importance of non-inoculated microflora--raising the question of the latter's environmental sources and their role in shaping microbial communities. There is now a clear need to revise the current view of the cheese rind ecosystem (i.e. that of a well-defined, perfectly controlled ecosystem). Inclusion of these new findings should enable us to better understand the cheese-making process.

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.

Background Brevibacterium 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. Results The 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. Conclusions Brevibacterium 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. Electronic supplementary material The online version of this article (10.1186/s12864-017-4322-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD–aadA–rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.

Chapter 6; Oligonucleotide microarrays have been widely used for gene detection and/or quantification of gene expression in various samples ranging from a single organism to a complex microbial assemblage. The success of a microarray experiment, however, strongly relies on the quality of designed probes. Consequently, probe design is of critical importance and therefore multiple parameters should be considered for each probe in order to ensure high specificity, sensitivity, and uniformity as well as potentially quantitative power. Moreover, to assess the complete gene repertoire of complex biological samples such as those studied in the field of microbial ecology, exploratory probe design strategies must be also implemented to target not-yet-described sequences. To design such probes, two algorithms, KASpOD and HiSpOD, have been developed and they are available via two user-friendly web services. Here, we describe the use of this software necessary for the design of highly effective probes especially in the context of microbial oligonucleotide microarrays by taking into account all the crucial parameters.

Lactobacillus delbrueckii subsp. bulgaricus ( L. bulgaricus ) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes.

Chapter 17; Microorganisms 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.

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.

Many health authorities have targeted salt reduction in food products as a means to reduce dietary sodium intake due to the harmful effects associated with its excessive consumption. In the present work, we evaluated the effect of reducing sodium chloride (NaCl) content on the microbiological and biochemical characteristics of an experimental surface-ripened cheese. A control cheese (1.8% NaCl) and a cheese with a reduced NaCl content (1.3% NaCl) were sampled weekly over a period of 27d. Reducing NaCl content induced microbial perturbations such as the lesser development of the yeast Debaryomyces hansenii and the greater development of the gram-negative bacterium Hafnia alvei. This was accompanied by changes in proteolytic kinetics and in profiles of volatile aroma compounds and biogenic amine production. Finally, the development of the spoilage microorganism Pseudomonas fragi was significantly higher in the cheese with a reduced salt content.

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.

Here, we report the draft genome sequence of Corynebacterium variabile Mu292, which was originally isolated from the surface of Munster, a French smear-ripened cheese. This genome investigation will improve our knowledge on the molecular determinants potentially involved in the adaptation of this strain during the Munster-type cheese manufacturing process.

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 approximately 75 million reads per sample. Except for Brevibacterium 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 day 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.

Oligonucleotide microarrays have been widely used for gene detection and/or quantification of gene expression in various samples ranging from a single organism to a complex microbial assemblage. The success of a microarray experiment, however, strongly relies on the quality of designed probes. Consequently, probe design is of critical importance and therefore multiple parameters should be considered for each probe in order to ensure high specificity, sensitivity, and uniformity as well as potentially quantitative power. Moreover, to assess the complete gene repertoire of complex biological samples such as those studied in the field of microbial ecology, exploratory probe design strategies must be also implemented to target not-yet-described sequences. To design such probes, two algorithms, KASpOD and HiSpOD, have been developed and they are available via two user-friendly web services. Here, we describe the use of this software necessary for the design of highly effective probes especially in the context of microbial oligonucleotide microarrays by taking into account all the crucial parameters.

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157: H7 at >10(4) CFU/g feces have been termed "super-shedders". A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a "normal" microbiota prevents E. coli O157: H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157: H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.

International audience; 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.

Chantier qualité GA; International audience; Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over-represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over-represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate-reducing bacteria and sulphur-oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico-chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage

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.

EA GenoSol CT3; International audience; 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.

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/. Contact: pierre.peyret@univ-bpclermont.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Background 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. Results First 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. Conclusions 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.