High-throughput functional metagenomics for the discovery of glycan metabolizing pathways

Glycans are widely distributed in nature. Produced by almost all organisms, they are involved in numerous cellular processes, such as energy supply and storage, cell structuration, protein maturation and signalling, and cell recognition. Glycans are thus key elements mediating the interactions between mammals, plants, bacteria, fungi and even viruses. They also represent a reliable source of carbon for microbes, which have developed complex strategies to face their structural diversity and to harvest them. However between 70 and 99% of these microorganisms are still uncultured, while they represent a goldmine for the discovery of new enzymes. In order to boost their identification and characterization, a functional metagenomic approach was developed, based on the design of various high-throughput, robust and sensitive screening strategies. The functional potential of Gbp of metagenomic DNA from various origins was explored, revealing dozens of novel enzyme families and functions. Integration of biochemical, structural, meta-omic and omic data allowed us to decipher, from the molecular to the ecosystemic scale, novel mechanisms of plant, microbial and mammal glycan metabolization. These new metabolic pathways involve batteries of glycoside-hydrolases, glycoside-phosphorylases and sugar transporters. These fascinating proteins appear as new targets to control host-microbe interactions. They also constitute very efficient biotechnological tools for biorefineries and synthetic biology

Development and application of metagenomic tools for characterization of toluene degradation in hydrocarbon polluted sediments

A metagenomic procedure was developed to assess microbial potential for toluene degradation within the sediment of a polluted aquifer at the tar-oil-contaminated site of Flingern, Germany. We targeted genes encoding the α-subunit of the toluene-4-monooxygenase (tmoA genes) to find new toluene degradation genes and pathways. Good quality and high molecular weight environmental DNA was extracted and cloned in BAC and fosmid vectors in Escherichia coli. The BAC library was transferred to other hosts, Cupriavidus metallidurans (Proteobacteria) and Edaphobacter aggregans (Acidobacteria). The use of an acidobacterial strain for the first time as an alternative host implied further characterization of its interaction with broad host range (BHR) plasmids (pMOL98, pKT230, and RP4). New ecological insight regarding host range of these plasmids was gained, which opened up new perspectives of developing BHR vectors for metagenomic applications. Exploitation of the acidobacterial-hosted BAC library led to the isolation of an as-yet unsequenced fragment from an Acidobacterium source that contains genes involved in aerobic and anaerobic toluene degradation. The functional screening of the BAC library in C. metallidurans CH34 led to the capture of 3 novel toluene monoxygenase-encoding operons located on IncP1 plasmids. The genetic screening using an innovative high throughput hybridization procedure allowed recovery of 16 significantly divergent tmoA-like genes. Their source fragments belonged exclusively to Proteobacteria and more specifically to Acinetobacter species. This unique metagenomic study applied to the Flingern groundwater sediment has opened future horizons for more applications.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201

Metagenomics for bioremediation

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Metagenomics: Probing pollutant fate in natural and engineered ecosystems

Polluted environments are a reservoir of microbial species able to degrade or to convert pollutants to harmless compounds. The proper management of microbial resources requires a comprehensive characterization of their genetic pool to assess the fate of contaminants and increase the efficiency of bioremediation processes. Metagenomics offers appropriate tools to describe microbial communities in their whole complexity without lab-based cultivation of individual strains. After a decade of use of metagenomics to study microbiomes, the scientific community has made significant progress in this field. In this review, we survey the main steps of metagenomics applied to environments contaminated with organic compounds or heavy metals. We emphasize technical solutions proposed to overcome encountered obstacles. We then compare two metagenomic approaches, i.e. library-based targeted metagenomics and direct sequencing of metagenomes. In the former, environmental DNA is cloned inside a host, and then clones of interest are selected based on (i) their expression of biodegradative functions or (ii) sequence homology with probes and primers designed from relevant, already known sequences. The highest score for the discovery of novel genes and degradation pathways has been achieved so far by functional screening of large clone libraries. On the other hand, direct sequencing of metagenomes without a cloning step has been more often applied to polluted environments for characterization of the taxonomic and functional composition of microbial communities and their dynamics. In this case, the analysis has focused on 16S rRNA genes and marker genes of biodegradation. Advances in next generation sequencing and in bioinformatic analysis of sequencing data have opened up new opportunities for assessing the potential of biodegradation by microbes, but annotation of collected genes is still hampered by a limited number of available reference sequences in databases. Although metagenomics is still facing technical and computational challenges, our review of the recent literature highlights its value as an aid to efficiently monitor the clean-up of contaminated environments and develop successful strategies to mitigate the impact of pollutants on ecosystems.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Metagenomics: Probing pollutant fate in natural and engineered ecosystems

Polluted environments are a reservoir of microbial species able to degrade or to convert pollutants to harmless compounds. The proper management of microbial resources requires a comprehensive characterization of their genetic pool to assess the fate of contaminants and increase the efficiency of bioremediation processes. Metagenomics offers appropriate tools to describe microbial communities in their whole complexity without lab-based cultivation of individual strains. After a decade of use of metagenomics to study microbiomes, the scientific community has made significant progress in this field. In this review, we survey the main steps of metagenomics applied to environments contaminated with organic compounds or heavy metals. We emphasize technical solutions proposed to overcome encountered obstacles. We then compare two metagenomic approaches, i.e. library-based targeted metagenomics and direct sequencing of metagenomes. In the former, environmental DNA is cloned inside a host, and then clones of interest are selected based on (i) their expression of biodegradative functions or (ii) sequence homology with probes and primers designed from relevant, already known sequences. The highest score for the discovery of novel genes and degradation pathways has been achieved so far by functional screening of large clone libraries. On the other hand, direct sequencing of metagenomes without a cloning step has been more often applied to polluted environments for characterization of the taxonomic and functional composition of microbial communities and their dynamics. In this case, the analysis has focused on 16S rRNA genes and marker genes of biodegradation. Advances in next generation sequencing and in bioinformatic analysis of sequencing data have opened up new opportunities for assessing the potential of biodegradation by microbes, but annotation of collected genes is still hampered by a limited number of available reference sequences in databases. Although metagenomics is still facing technical and computational challenges, our review of the recent literature highlights its value as an aid to efficiently monitor the clean-up of contaminated environments and develop successful strategies to mitigate the impact of pollutants on ecosystems.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Metagenomics for Bioremediation

Large amounts of toxic contaminants are released in the environment by mining, industrial, agricultural, and urban activities. These are natural compounds (fossil fuels or heavy metals discharged at high concentration) or, more commonly, chemically synthesized compounds (the so-called “xenobiotics”, such as chlorinated, halogenated, or nitroaromatic compounds). Of great concern is also the continuous discharge of various bioactive molecules in the environment (mostly endocrine disruptors and pharmaceutically active compounds), for which there is accumulating evidence of adverse effects on humans and animals. To clean up contaminated sites, one option is to use organisms (mainly bacteria, and also fungi and plants) that can oxidize/ reduce, bind, immobilize, volatize, or transform contaminants

Investigation of a metagenomic library for aerobic toluene degradation genes

Book of Abstracts P.5, poster 497Binfo:eu-repo/semantics/publishe

Development of a protocol for High Molecular Weight DNA extraction from BTEX- and PAH-contaminated sites

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Development and application of metagenomic tools for studying ecosystem potentials.

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INVESTIGATION OF A METAGENOMIC LIBRARY FOR AEROBIC TOLUENE DEGRADATION GENES

Introduction. Metagenomic analysis of polluted sites holds great promise in bioremediation. It has the potential to give access to all genes involved in pollutant degradation from cultured and not-yet-cultured bacteria. Therefore, discovering gene sequences from the little characterized or totally unknown microorganisms by this approach may help the design of novel specialized strains and enzymes useful for bioremediation. Our study was conducted on sediment from a former gasworks site located in Düsseldorf-Flingern, Germany. Our aims were: (i) to develop and to apply metagenomic tools for assessment of microbial adaptation to contaminant stress in sediments polluted with benzene, toluene, ethylene and xylene (BTEX) and (ii) to detect aerobic toluene degradation patterns based on genetic screening of the metagenomic library for the toluene 4-monooxygenase gene (tmoA) which encodes the alpha-subunit (subfamily 2) of the hydroxylase component of the multicomponent toluene monooxygenase. Methods. High quality and high molecular weight DNA was extracted from a low cell density in the sediment. The extraction method used direct lysis of bacterial cells within the sediment followed by a phenol-chloroform extraction. The DNA was then purified using a centrifugal concentrator coupled with Q-Sepharose treatment. This protocol resulted in pure DNA of appropriate size range to construct a metagenomic library in the pCC1FOS vector with the CopyControl fosmid library system (Epicentre,USA). Macroarrays containing 53,760 fosmid clones with an average insert size of 40 kb representing 537 E. coli genome equivalents were produced at Libragen, France. The library was screened by colony blot hybridization for toluene monooxygenase using a digoxigenin-labeled tmoA probe. Positive clones detected by hybridization were tested for amplicon production by PCR using primers described by Hendrickx et al (2005). Results. 1/ The developed HMW DNA extraction and purification protocol yielded 1.5 µg DNA/100 g sediment of pure DNA. 2/ DNA cloning produced a 55,296-clones library. Restriction fragment length polymorphism with BamHI showed a high variability of inserts with an average size of 40 kb. The library corresponds to 537 E. coli genome equivalents. 3/ Assuming an equal abundance of genomes within the library, about 40 % of the screened genomes contained a tmoA gene. The 79 clones detected by hybridization and showing a negative amplification of tmoA contain mismatches with the TMOA-F/TMOA-R primer set. Together with the 27 clones having negative hybridization and positive amplicon production may contain a tmoA-like gene that is divergent from previously described sequences. Conclusions. The combination of macroarray screening by hybridization and PCR appeared to be a powerful tool to capture all tmoA-like genes in the studied polluted site that might be missed by primers designed on the basis of cultured toluene degraders. Sequencing of these clones is expected to considerably enrich the catalogue of known genes involved in toluene degradation