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Sample records for bioprospecting metagenomes glycosyl

  1. Bioprospecting metagenomes: Glycosyl hydrolases for converting biomass

    Energy Technology Data Exchange (ETDEWEB)

    Li, L.; van der Lelie, D.; McCorkle, S. R.; Monchy, S.; Taghavi, S.

    2009-05-18

    Throughout immeasurable time, microorganisms evolved and accumulated remarkable physiological and functional heterogeneity, and now constitute the major reserve for genetic diversity on earth. Using metagenomics, namely genetic material recovered directly from environmental samples, this biogenetic diversification can be accessed without the need to cultivate cells. Accordingly, microbial communities and their metagenomes, isolated from biotopes with high turnover rates of recalcitrant biomass, such as lignocellulosic plant cell walls, have become a major resource for bioprospecting; furthermore, this material is a major asset in the search for new biocatalytics (enzymes) for various industrial processes, including the production of biofuels from plant feedstocks. However, despite the contributions from metagenomics technologies consequent upon the discovery of novel enzymes, this relatively new enterprise requires major improvements. In this review, we compare function-based metagenome screening and sequence-based metagenome data mining, discussing the advantages and limitations of both methods. We also describe the unusual enzymes discovered via metagenomics approaches, and discuss the future prospects for metagenome technologies.

  2. Bioprospecting potential of the soil metagenome: novel enzymes and bioactivities.

    Science.gov (United States)

    Lee, Myung Hwan; Lee, Seon-Woo

    2013-09-01

    The microbial diversity in soil ecosystems is higher than in any other microbial ecosystem. The majority of soil microorganisms has not been characterized, because the dominant members have not been readily culturable on standard cultivation media; therefore, the soil ecosystem is a great reservoir for the discovery of novel microbial enzymes and bioactivities. The soil metagenome, the collective microbial genome, could be cloned and sequenced directly from soils to search for novel microbial resources. This review summarizes the microbial diversity in soils and the efforts to search for microbial resources from the soil metagenome, with more emphasis on the potential of bioprospecting metagenomics and recent discoveries.

  3. Marine metagenomics as a source for bioprospecting

    KAUST Repository

    Kodzius, Rimantas

    2015-08-12

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  4. Marine metagenomics as a source for bioprospecting.

    Science.gov (United States)

    Kodzius, Rimantas; Gojobori, Takashi

    2015-12-01

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  5. Metagenomics as a preliminary screen for antimicrobial bioprospecting.

    Science.gov (United States)

    Al-Amoudi, Soha; Razali, Rozaimi; Essack, Magbubah; Amini, Mohammad Shoaib; Bougouffa, Salim; Archer, John A C; Lafi, Feras F; Bajic, Vladimir B

    2016-12-15

    Since the composition of soil directs the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies has been focused on sediment types with unique features characteristic of extreme environments. However, not much is known about the potential of microbiomes that inhabit the highly saline and hot Red Sea lagoons. This case study explores mangrove mud and the microbial mat of sediments collected from the Rabigh harbor lagoon and Al Kharrar lagoon for antimicrobial bioprospecting. Rabigh harbor lagoon appears the better location, and the best sediment type for this purpose is mangrove mud. On the other hand, Al Kharrar lagoon displayed increased anaerobic hydrocarbon degradation and an abundance of bacterial DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma- and Delta-proteobacteria), but we also report that bacterial DNA sequences associated with antibiotic synthesis enzymes are derived from Gamma-, Delta- and Alpha-proteobacteria. This suggests that selection pressure associated with hydrocarbon contamination tend to enrich the bacterial classes DNA associated with antibiotic synthesis enzymes. Although Actinobacteria tends to be the common target for research when it comes to antimicrobial bioprospecting, our study suggests that Firmicutes (Bacilli and Clostridia), Bacteroidetes, Cyanobacteria, and Proteobacteria should be antimicrobial bioprospecting targets as well. To the best of our knowledge, this is the first metagenomic study that analyzed the microbiomes in Red Sea lagoons for antimicrobial bioprospecting.

  6. Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.

    Science.gov (United States)

    Adesioye, Fiyinfoluwa A; Makhalanyane, Thulani P; Biely, Peter; Cowan, Don A

    2016-11-01

    Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.

  7. Metagenomics as a preliminary screen for antimicrobial bioprospecting

    KAUST Repository

    Al Amoudi, Soha

    2016-09-16

    Since the composition of soil directs the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies has been focused on sediment types with unique features characteristic of extreme environments. However, not much is known about the potential of microbiomes that inhabit the highly saline and hot Red Sea lagoons. This case study explores mangrove mud and the microbial mat of sediments collected from the Rabigh harbor lagoon and Al Kharrar lagoon for antimicrobial bioprospecting. Rabigh harbor lagoon appears the better location, and the best sediment type for this purpose is mangrove mud. On the other hand, Al Kharrar lagoon displayed increased anaerobic hydrocarbon degradation and an abundance of bacterial DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma-and Delta-proteobacteria), but we also report that bacterial DNA sequences associated with antibiotic synthesis enzymes are derived from Gamma-, Delta-and Alpha-proteobacteria. This suggests that selection pressure associated with hydrocarbon contamination tend to enrich the bacterial classes DNA associated with antibiotic synthesis enzymes. Although Actinobacteria tends to be the common target for research when it comes to antimicrobial bioprospecting, our study suggests that Firmicutes (Bacilli and Clostridia), Bacteroidetes, Cyanobacteria, and Proteobacteria should be antimicrobial bioprospecting targets as well. To the best of our knowledge, this is the first metagenomic study that analyzed the microbiomes in Red Sea lagoons for antimicrobial bioprospecting. (C) 2016 The Authors. Published by Elsevier B.V.

  8. Improved cultivation and metagenomics as new tools for bioprospecting in cold environments

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2015-01-01

    into the laboratory or through metagenomic approaches where culture-independent DNA sequence information can be combined with functional screening. The coupling of these two approaches circumvents the need for pure, cultured isolates and can be used to generate targeted information on communities enriched...... be limited as few hosts are available for expression of genes with extremophilic properties. This review summarizes the methods developed for improved cultivation as well as the metagenomic approaches for bioprospecting with focus on the challenges faced by bioprospecting in cold environments....

  9. Improved cultivation and metagenomics as new tools for bioprospecting in cold environments

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2015-01-01

    Only a small minority of microorganisms from an environmental sample can be cultured in the laboratory leaving the enormous bioprospecting potential of the uncultured diversity unexplored. This resource can be accessed by improved cultivation methods in which the natural environment is brought...... for specific activities or properties. Bioprospecting in extreme environments is often associated with additional challenges such as low biomass, slow cell growth, complex sample matrices, restricted access, and problematic in situ analyses. In addition, the choice of vector system and expression host may...... be limited as few hosts are available for expression of genes with extremophilic properties. This review summarizes the methods developed for improved cultivation as well as the metagenomic approaches for bioprospecting with focus on the challenges faced by bioprospecting in cold environments....

  10. Improved cultivation and metagenomics as new tools for bioprospecting in cold environments.

    Science.gov (United States)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2015-01-01

    Only a small minority of microorganisms from an environmental sample can be cultured in the laboratory leaving the enormous bioprospecting potential of the uncultured diversity unexplored. This resource can be accessed by improved cultivation methods in which the natural environment is brought into the laboratory or through metagenomic approaches where culture-independent DNA sequence information can be combined with functional screening. The coupling of these two approaches circumvents the need for pure, cultured isolates and can be used to generate targeted information on communities enriched for specific activities or properties. Bioprospecting in extreme environments is often associated with additional challenges such as low biomass, slow cell growth, complex sample matrices, restricted access, and problematic in situ analyses. In addition, the choice of vector system and expression host may be limited as few hosts are available for expression of genes with extremophilic properties. This review summarizes the methods developed for improved cultivation as well as the metagenomic approaches for bioprospecting with focus on the challenges faced by bioprospecting in cold environments.

  11. Bioprospecting

    Science.gov (United States)

    Reed, Philip A.

    2004-01-01

    This article introduces the subject of bioprospecting to students. It is a very old biotechnology that involves some very new techniques. Genetic engineering and other processes allow biologists, chemists, and biotechnologists to collect microorganisms and change them in ways that previously were not possible. Organisms that thrive under adverse…

  12. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Li L. L.; van der Lelie D.; Taghavi, S.; McCorkle, S. M.; Zhang, Y.-B.; Blewitt, M. G.; Brunecky, R.; Adney, W. S.; Himmel, M. E.; Brumm, P.; Drinkwater, C.; Mead, D. A.; Tringe, S. G.

    2011-08-01

    To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-{alpha}-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-{beta}-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-{beta}-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  13. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Li Luen-Luen

    2011-08-01

    Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  14. Bioprospecting hot spring metagenome: lipase for the production of biodiesel.

    Science.gov (United States)

    Sahoo, Rajesh Kumar; Kumar, Mohit; Sukla, Lala Behari; Subudhi, Enketeswara

    2017-02-01

    Screening of metagenomic library from Taptapani Hot Spring (Odisha) yielded a positive lipase clone (pUC-lip479). Sequence analysis showed an ORF (RK-lip479) of 416 amino acid residues which was overexpressed in Escherichia coli BL21 (DE3). Optimum pH and temperature of purified lipase RK-lip479 were 8.0 and 65 °C, respectively, and found to be stable over a pH range of 7.0-9.0 and temperatures 55-75 °C. RK-lip479 could hydrolyse a wide range of 4-nitrophenyl esters (4-nitrophenyoctanoate, 4-nitrophenyldodecanoate, 4-nitrophenylpalmitate, 4-nitrophenylmyristate and 4-nitrophenylstearate), and maximum activity was observed with 4-nitrophenyldodecanoate. RK-lip479 was resistant to many organic solvents, especially isopropanol, DMSO, methanol, DMF, ethanol, dichloromethane, acetone, glycerol and ethyl acetate. RK-lip479 also showed activity in the presence of monovalent (Na(+) and K(+)), divalent (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+), Co(2+), Zn(2+) and Ag(2+) ) and trivalent cations (Fe(3+) and Al(3+)). Yield of biodiesel production was in the range of 40-76% using various waste oils with RK-Lip479 under optimized conditions.

  15. A Review of Bioinformatics Tools for Bio-Prospecting from Metagenomic Sequence Data

    Science.gov (United States)

    Roumpeka, Despoina D.; Wallace, R. John; Escalettes, Frank; Fotheringham, Ian; Watson, Mick

    2017-01-01

    The microbiome can be defined as the community of microorganisms that live in a particular environment. Metagenomics is the practice of sequencing DNA from the genomes of all organisms present in a particular sample, and has become a common method for the study of microbiome population structure and function. Increasingly, researchers are finding novel genes encoded within metagenomes, many of which may be of interest to the biotechnology and pharmaceutical industries. However, such “bioprospecting” requires a suite of sophisticated bioinformatics tools to make sense of the data. This review summarizes the most commonly used bioinformatics tools for the assembly and annotation of metagenomic sequence data with the aim of discovering novel genes. PMID:28321234

  16. Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

    Science.gov (United States)

    Sathya, T A; Khan, Mahejibin

    2014-11-01

    Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over. The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application. Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms. Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors. The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed.

  17. Bioprospecting metagenomics of a microbial community on cotton degradation: Mining for new glycoside hydrolases.

    Science.gov (United States)

    Zhang, Guoxiu; Liu, Pei; Zhang, Lei; Wei, Wei; Wang, Xuedong; Wei, Dongzhi; Wang, Wei

    2016-09-20

    Glycoside hydrolases (GHases) of higher performance are immediately needed for efficient degradation of plant biomass into fermentable sugars in industrial processes. The current study represents functional characterization of the enzymatic repertoire involved in crude cotton biomass degradation. Physical contact between cells and substrate is necessary for efficient hydrolysis of cellulose. Cytophagales, which plays a major role in cotton biomass decomposition, was identified as a prevalent community member by 16S rRNA analysis. From the metagenome data, 2058 GHase homologs were identified, of which sixteen were successfully expressed in E. coli. Four enzymes showed activities on p-nitrophenyl-β-d-xylopyranoside, four showed activities on p-nitrophenyl-β-d-glucopyranoside, two had activities against p-nitrophenyl-β-d-glucuronide, one showed activity on laminarin, three had activities against p-nitrophenyl-N-acetyl-β-d-glucosaminide, one had activity towards carboxymethyl cellulose, and one towards p-nitrophenyl-β-d-mannopyranoside. Metagenomics provides a good resource for mining novel biomass degrading enzymes. The sixteen GHases that were cloned may have potential application for biomass conversion and bioproduct production. Functional characterization of the enzymatic repertoire in cotton biomass degradation and analysis of the GHases provide insight into the composition and interaction of enzymes and pathways of plant biomass degradation.

  18. Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends.

    Science.gov (United States)

    Ferrer, Manuel; Martínez-Martínez, Mónica; Bargiela, Rafael; Streit, Wolfgang R; Golyshina, Olga V; Golyshin, Peter N

    2016-01-01

    Recent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53,000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high-throughput expression for subsequent production and characterization. The pre-screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106-fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time-consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine-tuned naïve- and sequence-based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market.

  19. Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases.

    Science.gov (United States)

    Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Zeuner, Birgitte; Łężyk, Mateusz; Difilippo, Elisabetta; Logtenberg, Madelon J; Schols, Henk A; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

    2015-10-01

    This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both β-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors β-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases.

  20. Bioprospecting and Functional Analysis of Neglected Environments

    DEFF Research Database (Denmark)

    Vogt, Josef Korbinian

    of functional relationships in single organisms and environments. The sequencing data can also be mined for novel compounds and enzymes. The process of exploiting biological resources for commercial use is known as bioprospecting. This PhD thesis describes the concept of bioprospecting in the post genomic era...... on Biological Diversity (explained in Chapter 3). Proteolytic enzymes – described in Chapter 4 – are the target for bioprospecting due to their high market value. Section II describes methods used for the analysis of metagenomic and RNA-seq datasets, including Manuscript I, which includes the taxonomic...

  1. Bioprospecting in the genomic age.

    Science.gov (United States)

    Hicks, Michael A; Prather, Kristala L J

    2014-01-01

    The genomic revolution promises great advances in the search for useful biocatalysts. Function-based metagenomic approaches have identified several enzymes with properties that make them useful candidates for a variety of bioprocesses. As DNA sequencing costs continue to decline, the volume of genomic data, along with their corresponding predicted protein sequences, will continue to increase dramatically, necessitating new approaches to leverage this information for gene-based bioprospecting efforts. Additionally, as new functions are discovered and correlated with this sequence information, the knowledge of the often complex relationship between a protein's sequence and function will improve. This in turn will lead to better gene-based bioprospecting approaches and facilitate the tailoring of desired properties through protein engineering projects. In this chapter, we discuss a number of recent advances in bioprospecting within the context of the genomic age.

  2. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures.

    Science.gov (United States)

    Mori, Toshio; Kamei, Ichiro; Hirai, Hirofumi; Kondo, Ryuichiro

    2014-01-01

    To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.

  3. Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.

    Directory of Open Access Journals (Sweden)

    Manuel Ferrer

    Full Text Available Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43, hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyranosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.

  4. Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases

    DEFF Research Database (Denmark)

    Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Zeuner, Birgitte

    2015-01-01

    This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme......-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest...

  5. Ecology and bioprospecting.

    Science.gov (United States)

    Beattie, Andrew J; Hay, Mark; Magnusson, Bill; de Nys, Rocky; Smeathers, James; Vincent, Julian F V

    2011-05-01

    Bioprospecting is the exploration of biodiversity for new resources of social and commercial value. It is carried out by a wide range of established industries such as pharmaceuticals, manufacturing and agriculture as well as a wide range of comparatively new ones such as aquaculture, bioremediation, biomining, biomimetic engineering and nanotechnology. The benefits of bioprospecting have emerged from such a wide range of organisms and environments worldwide that it is not possible to predict what species or habitats will be critical to society, or industry, in the future. The benefits include an unexpected variety of products that include chemicals, genes, metabolic pathways, structures, materials and behaviours. These may provide physical blueprints or inspiration for new designs. Criticism aimed at bioprospecting has been addressed, in part, by international treaties and legal agreements aimed at stopping biopiracy and many activities are now funded by agencies that require capacity-building and economic benefits in host countries. Thus, much contemporary bioprospecting has multiple goals, including the conservation of biodiversity, the sustainable management of natural resources and economic development. Ecologists are involved in three vital ways: first, applying ecological principles to the discovery of new resources. In this context, natural history becomes a vast economic database. Second, carrying out field studies, most of them demographic, to help regulate the harvest of wild species. Third, emphasizing the profound importance of millions of mostly microscopic species to the global economy.

  6. Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

    Science.gov (United States)

    Mai, Zhimao; Su, Hongfei; Zhang, Si

    2016-01-01

    A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of Km and Vmax values toward agarose were 4.6 mg·mL−1 and 967.5 μM·min−1·mg−1, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries. PMID:27548158

  7. Bioprospection of marine microorganisms: biotechnological applications and methods.

    Science.gov (United States)

    Dionisi, Hebe M; Lozada, Mariana; Olivera, Nelda L

    2012-01-01

    Environmental microorganisms constitute an almost inexhaustible reserve of genetic and functional diversity, accumulated during millions of years of adaptive evolution to various selective pressures. In particular, the extent of microbial biodiversity in marine habitats seems to grow larger as new techniques emerge to measure it. This has resulted in novel and more complex approaches for the screening of molecules and activities of biotechnological interest in these environments. In this review, we explore the different partially overlapping biotechnological fields that make use of microorganisms and we describe the different marine habitats that are particularly attractive for bioprospection. In addition, we review the methodological approaches currently used for microbial bioprospection, from the traditional cultivation techniques to state of the art metagenomic approaches, with emphasis in the marine environment.

  8. A novel cold active esterase derived from Colombian high Andean forest soil metagenome

    NARCIS (Netherlands)

    Jiménez, Diego Javier; Montaña, José Salvador; Alvarez, Diana; Baena, Sandra

    2012-01-01

    In order to search new lipolytic enzymes and conduct bioprospecting of microbial communities from high Andean forest soil, a metagenomic library of approximately 20,000 clones was constructed in Escherichia coli using plasmid p-Bluescript II SK+. The library covered 80 Mb of the metagenomic DNA main

  9. Bioprospecting keratinous materials.

    Science.gov (United States)

    Jones, L N; Sinclair, R D; Carver, J; Ecroyd, H; Lui, Y; Bennett, L E

    2010-01-01

    The concept of bioprospecting for bioactive peptides from keratin-containing materials such as wool, hair, skin and feathers presents an exciting opportunity for discovery of novel functional food ingredients and nutraceuticals, while value-adding to cheap and plentiful natural sources. The published literature reports multiple examples of proline-rich peptides with productive bio-activity in models of human disease including tumour formation, hypertension control and Alzheimer's disease. Bioactive peptides have been identified from food and other protein sources however the bioactivity of keratin-related proteins and peptides is largely unknown. Considering the high representation of proline-rich peptides among proven bioactive peptides, the proline-rich character of keratinous proteins supports current research. A selection of mammalian (cow epidermis, sheep wool) and avian (chicken feather) keratinous materials were subjected to enzymatic hydrolysis using established processing methods. A bio-assay of determining inhibition of early stage amyloid aggregation involved using a model fibril-forming protein - reduced and carboxymethylated bovine K-casein (RCMk-CN) and quantitation of fibril development with the amyloid-specific fluorophore, Thioflavin T (ThT). The assay was fully validated for analytical repeatability and used together with appropriate positive controls. Peptide library products derived from chicken feather (n=9), sheep wool (n=9) and bovine epidermis (n=9) were screened in the fibril inhibition assay based on K-casein. 3 of 27 products exhibited interesting levels of bio-activity with regard to fibril inhibition. HPLC profiles provide an indication of the complexity of the assemblage of peptides in the three active products. We conclude the bioprospecting research using keratinous materials shows promise for discovery of useful bioactive peptides.

  10. Red Sea as a source for bioprospecting

    KAUST Repository

    Kodzius, Rimantas

    2015-12-12

    King-Abdullah University of Science and Technology (KAUST) is located on the shores of the Red Sea in Saudi Arabia. The Red Sea is well known for its unique environment, harboring various microbes capable of surviving in salty brines. We collected sediment samples from brine pool adjacent to the Thuwal cold seeps in the Red Sea. The taxonomic analysis showed the diversity and abundance of bacterial and archaeal operational taxonomic units (OUT). Recently we established in the laboratory a microdroplet technology to encapsulate single cells. This technology enables us to analyze single-cell genomes and perform the high-throughput screening. The genomes of both cultivable and uncultivable organisms can be analyzed. We envision the collection of complimentary data, obtained by various techniques, such as single-cell genomics, metagenomics, and transcriptomics. That will enable us not only to understand the environment and microorganism communities but also will allow to discover the previously unknown genes, pathways, and whole genomes. These data will facilitate the enhancement of biological and chemical producers, and pave the way for bioprospecting.

  11. Functional metagenomics of extreme environments.

    Science.gov (United States)

    Mirete, Salvador; Morgante, Verónica; González-Pastor, José Eduardo

    2016-04-01

    The bioprospecting of enzymes that operate under extreme conditions is of particular interest for many biotechnological and industrial processes. Nevertheless, there is a considerable limitation to retrieve novel enzymes as only a small fraction of microorganisms derived from extreme environments can be cultured under standard laboratory conditions. Functional metagenomics has the advantage of not requiring the cultivation of microorganisms or previous sequence information to known genes, thus representing a valuable approach for mining enzymes with new features. In this review, we summarize studies showing how functional metagenomics was employed to retrieve genes encoding for proteins involved not only in molecular adaptation and resistance to extreme environmental conditions but also in other enzymatic activities of biotechnological interest.

  12. The microbial diversity of Polar environments is a fertile ground for bioprospecting.

    Science.gov (United States)

    de Pascale, Donatella; De Santi, Concetta; Fu, Juan; Landfald, Bjarne

    2012-12-01

    The term bioprospecting has been adopted for systematic searches in nature for new bioactive compounds, genes, proteins, microorganisms and other products with potential for commercial use. Much effort has been focused on microorganisms able to thrive under harsh conditions, including the Polar environments. Both the lipid and protein cellular building blocks of Polar microorganisms are shaped by their adaptation to the permanently low temperatures. In addition, strongly differing environments, such as permafrost, glaciers and sea ice, have contributed to additional functional diversity. Emerging massive-parallel sequencing technologies have revealed the existence of a huge, hitherto unseen diversity of low-abundance phylotypes--the rare biosphere--even in the Polar environments. This realization has further strengthened the need to employ cultivation-independent approaches, including metagenomics and single-cell genomic sequencing, to get comprehensive access to the genetic diversity of microbial communities for bioprospecting purposes. In this review, we present an updated snapshot of recent findings on the molecular basis for adaptation to the cold and the phylogenetic diversities of different Polar environments. Novel approaches in bioprospecting are presented and we conclude by showing recent bioprospecting outcomes in terms of new molecules patented or applied by some biotech companies.

  13. Biodiversity and chemodiversity: future perspectives in bioprospecting.

    Science.gov (United States)

    Ramesha, B T; Gertsch, Jürg; Ravikanth, G; Priti, V; Ganeshaiah, K N; Uma Shaanker, R

    2011-10-01

    Biological diversity and its constituent chemical diversity have served as one of the richest sources of bioprospecting leading to the discovery of some of the most important bioactive molecules for mankind. Despite this excellent record, in the recent past, however, bioprospecting of biological resources has met with little success; there has been a perceptible decline in the discovery of novel bioactive compounds. Several arguments have been proposed to explain the current poor success in bioprospecting. Among them, it has been argued that to bioprospect more biodiversity may not necessarily be productive, considering that chemical and functional diversity might not scale with biological diversity. In this paper, we offer a critique on the current perception of biodiversity and chemodiversity and ask to what extent it is relevant in the context of bioprospecting. First, using simple models, we analyze the relation among biodiversity, chemodiversity and functional redundancies in chemical plans of plants and argue that the biological space for exploration might still be wide open. Second, in the context of future bioprospecting, we argue that brute-force high throughput screening approaches alone are insufficient and cost ineffective in realizing bioprospecting success. Therefore, intelligent or non-random approaches to bioprospecting need to be adopted. We review here few examples of such approaches and show how these could be further developed and used in the future to accelerate the pace of discovery.

  14. Bioprospecting Sediments from Red Sea Coastal Lagoons for Microorganisms and Their Antimicrobial Potential

    KAUST Repository

    Al-Amoudi, Soha

    2016-12-08

    Since the soils nutrient composition along with the associated biotic and abiotic factors direct the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies have been focused on sediment types with unique features characteristic of extreme environments. Red Sea lagoon ecosystems are environments with such unique features as they are highly saline. However, not much is known about the potential of their microbiomes to produce bioactive compounds. Here, we explored sediment types such as mangrove mud, microbial mat, and barren soil collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL) as sources for antibiotic bioprospecting. Our antibiotic bioprospecting process started with a metagenomic study that provides a more precise view of the microbial community inhabiting these sites and serves as a preliminary screen for potential antibiotics. Taking the outcomes of the metagenomic screening into account, the next step we established a library of culturable strains from the analyzed samples. We screened each strain from that library for antibiotic activity against four target strains (Staphylococcus aureus ATCC 25923, Escherichia coli dh5 α, Pseudomonas syringae pv. tomato dc3000 and Salmonella typhimurium dt2) and for the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes known to support synthesis of secondary metabolites that act like antimicrobial agents. The metagenomics study showed a shift in dominant phyla consistent with a historical exposure to hydrocarbon contamination and that AKL unexpectedly displayed more contamination than RHL. This may be due to dominant phyla in AKL being consistent with early hydrocarbon exposure (when contamination levels are still high) and the dominant phyla in RHL being consistent with late hydrocarbon exposure (when contamination levels are lower as a result of an extended period of hydrocarbon degradation). Additionally, RHL samples

  15. Bioprospecting plant-associated microbiomes.

    Science.gov (United States)

    Müller, Christina A; Obermeier, Melanie M; Berg, Gabriele

    2016-10-10

    There is growing demand for new bioactive compounds and biologicals for the pharmaceutical, agro- and food industries. Plant-associated microbes present an attractive and promising source to this end, but are nearly unexploited. Therefore, bioprospecting of plant microbiomes is gaining more and more attention. Due to their highly specialized and co-evolved genetic pool, plant microbiomes host a rich secondary metabolism. This article highlights the potential detection and use of secondary metabolites and enzymes derived from plant-associated microorganisms in biotechnology. As an example we summarize the findings from the moss microbiome with special focus on the genus Sphagnum and its biotechnological potential for the discovery of novel microorganisms and bioactive molecules. The selected examples illustrate unique and yet untapped properties of plant-associated microbiomes, which are an immense treasure box for future research.

  16. Marine metagenomics, a valuable tool for enzymes and bioactive compounds discovery

    Directory of Open Access Journals (Sweden)

    Rosalba eBarone

    2014-09-01

    Full Text Available The enormous potential in diversity of the marine life is still not fully exploited due to the difficulty in culturing many of the microorganisms under laboratory conditions. In this mini-review we underlined the importance of using an omic technique, such as metagenomics, to access the uncultured majority of microbial communities. We report examples of several hydrolytic enzymes and natural products isolated by functional sequenced-based and function- screening strategies assisted by new high-throughput DNA sequencing technology and recent bioinformatics tools. This article ends with an overview of the potential future perspectives of the metagenomics in bioprospecting novel biocatalysts and bioactive compounds from marine sources.

  17. Bioprospecting of Brevibacillus brevis isolated from soil.

    Science.gov (United States)

    Samrot, Antony V; Prasad, Rejin J J; Rio, Antony J; Sneha, Sahaya J

    2015-01-01

    It is obviously essential to find new compounds that possess industrial and medicinal importance and potential to improve the lifestyle of human population. Bioprospecting these compounds from natural resources has additional benefits since they are less toxic as well as biocompatible. In this study, Brevibacillus brevis was isolated from soil and its enzymes production, antibacterial activity and anticancer activity were assessed. The organism was found to be a promising source of amylase enzymes, antibacterial as well as anticancer compounds.

  18. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  19. BIO-PROSPECTING, IPR, TRADITIONAL KNOWLEDGE AND ITS TRADE SECRETS

    Directory of Open Access Journals (Sweden)

    Lad Meenal Deepak

    2012-10-01

    Full Text Available Bio-prospecting involves searching for collecting, and deriving genetic material from samples of bio-diversity that can be used in commercialized pharmaceutical, agricultural, industrial, or chemical processing end products. Salient features of Bio-prospecting are: Conservation and sustainable use of biological diversity, Rights of traditional, indigenous knowledge holders; fair and equitable sharing of benefits arising from the research, indigenous knowledge, intellectual property, or application of biological resources. The meaningful participation in these processes by traditional, indigenous knowledge holders.As far as Bio-prospecting & final commercial manufacturing company is concerned certain larger pharmaceutical companies operate their own Bio-prospecting units. They can develop a commercial drug from an idea to Bio-prospecting for genetic material, clinical trial, manufacturing & marketing.Herbal medicines are not marketed as pharmaceuticals & are not regulated by FDA. These drugs lack in quality control & mostly sold as dietary supplements. Pharmaceutical industries ordinarily synthesize compounds from a limited amount of natural products & the Herbal medicine industries usually don’t attempt to do so because of high cost involved for the relatively low return. Pharmaceutical Bio-prospecting involves high returns & requires low volume harvesting.The convential biological diversities are an international protocol which gives expression to the novel idea that the Bio-prospecting process could yield conservation & develop benefits. The Trade-Related Aspects of Intellectual Property Rights (TRIPS Agreement under the World Trade Organization (WTO is the pre-eminent international agreement governing intellectual property rights. It covers a broad range of intellectual property rights, including copyright, trademarks, geographical indications, trade secrets, and patents. Along with TRIPS a focuses given to rights of traditional indigenous

  20. Metagenomic studies of the Red Sea.

    Science.gov (United States)

    Behzad, Hayedeh; Ibarra, Martin Augusto; Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    the bio-prospecting potential of the Red Sea microbiota. Furthermore, we discuss the limitations of the previous studies and the need for generating a large and representative metagenomic database of the Red Sea to help establish a dynamic model of the Red Sea microbiota.

  1. Phylogenies reveal predictive power of traditional medicine in bioprospecting

    DEFF Research Database (Denmark)

    Saslis-Lagoudakis, C Haris; Savolainen, Vincent; Williamson, Elizabeth M

    2012-01-01

    There is controversy about whether traditional medicine can guide drug discovery, and investment in bioprospecting informed by ethnobotanical data has fluctuated. One view is that traditionally used medicinal plants are not necessarily efficacious and there are no robust methods for distinguishing...... that phylogenetic cross-cultural comparisons can focus screening efforts on a subset of traditionally used plants that are richer in bioactive compounds, and could revitalize the use of traditional knowledge in bioprospecting....

  2. Bioprospection: From the Economics of Contracts to Reflexive Governance

    OpenAIRE

    Dedeurwaerdere, Tom

    2004-01-01

    Bioprospection practices have proliferated as biotechnological and pharmaceutical companies engage in the collection and genetic screening of biological and genetic resources throughout the world. The purpose of this article is to examine the competing proposals for the institutional framing of bioprospection based on the provisions of access and benefit-sharing embodied in the Convention on Biological Diversity. In particular, through evaluating the contribution of neo-institutionalist and e...

  3. Bioprospecting thermophiles for cellulase production: a review.

    Science.gov (United States)

    Acharya, Somen; Chaudhary, Anita

    2012-07-01

    Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

  4. Bioprospecting thermophiles for cellulase production: a review

    Directory of Open Access Journals (Sweden)

    Somen Acharya

    2012-09-01

    Full Text Available Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

  5. Swine Fecal Metagenomics

    Science.gov (United States)

    Metagenomic approaches are providing rapid and more robust means to investigate the composition and functional genetic potential of complex microbial communities. In this study, we utilized a metagenomic approach to further understand the functional diversity of the swine gut. To...

  6. Beyond the Thumbrule Approach: Regulatory Innovations for Bioprospecting in India

    Directory of Open Access Journals (Sweden)

    Kabir Sanjay Bavikatte and Morten Walløe Tvedt

    2015-06-01

    Full Text Available In the wake of the Nagoya Protocol on Access and Benefit Sharing (ABS, there is unprecedented attention on good Green Governance, which implies the development of resource efficient, rights based and effective domestic frameworks to regulate bioprospecting. India has been pioneering in this regard due to its ABS legislation in 2002 that long preceded the Nagoya Protocol. However much has changed since 2002 and while India has learnt a great deal from its ‘learning by doing’ method, there are valuable lessons that can be learnt from innovations in the ABS frameworks of other countries. The innovations of these countries are a great resource for National Biodiversity Authority (NBA in India that is seeking to make the processing of the bioprospecting applications in India more optimal. The current paper highlights the challenges faced by the NBA in processing the high volumes of bioprospecting applications, analyses the reasons for such challenges and proposes solutions for the same.

  7. Approaching marine bioprospecting in hexacorals by RNA deep sequencing.

    Science.gov (United States)

    Johansen, Steinar D; Emblem, Ase; Karlsen, Bård Ove; Okkenhaug, Siri; Hansen, Hilde; Moum, Truls; Coucheron, Dag H; Seternes, Ole Morten

    2010-07-31

    RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-generation sequencing platforms have recently been developed for de novo whole transcriptome analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics (sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential identification of all expressed genes in a sample. When combined to high-throughput bioinformatics and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral transcriptomes with the focus on cold-water sea anemones and related organisms.

  8. Bioprospecting Archaea: Focus on Extreme Halophiles

    KAUST Repository

    Antunes, André

    2016-12-12

    In 1990, Woese et al. divided the Tree of Life into three separate domains: Eukarya, Bacteria, and Archaea. Archaea were originally perceived as little more than “odd bacteria” restricted to extreme environmental niches, but later discoveries challenged this assumption. Members of this domain populate a variety of unexpected environments (e.g. soils, seawater, and human bodies), and we currently witness ongoing massive expansions of the archaeal branch of the Tree of Life. Archaea are now recognized as major players in the biosphere and constitute a significant fraction of the earth’s biomass, yet they remain underexplored. An ongoing surge in exploration efforts is leading to an increase in the (a) number of isolated strains, (b) associated knowledge, and (c) utilization of Archaea in biotechnology. They are increasingly employed in fields as diverse as biocatalysis, biocomputing, bioplastic production, bioremediation, bioengineering, food, pharmaceuticals, and nutraceuticals. This chapter provides a general overview on bioprospecting Archaea, with a particular focus on extreme halophiles. We explore aspects such as diversity, ecology, screening techniques and biotechnology. Current and future trends in mining for applications are discussed.

  9. Bioprospecting of Moringa (Moringaceae: Microbiological Perspective

    Directory of Open Access Journals (Sweden)

    Harpreet Kaur

    2013-03-01

    Full Text Available Plants produce primary and secondary metabolites which encompass a wide array of functions. Some of these have been subsequently exploited by humans for their beneficial role in a diverse array of applications. However, out of 750,000 species available on earth, only 1 to 10 % is being potentially used. Moringa is one such genus belonging to the family of Moringaceae, a monotypic family of single genera with around 33 species. Most of these species have not been explored fully despite the enormous bioactivity reports concerning various potentials such as: cardiac and circulatory stimulants; anti-tumor; antipyretic; antiepileptic; anti-inflammatory; antiulcer; antispasmodic; diuretic antihypertensive; cholesterol lowering; antioxidant; antidiabetic; hepato protective; antibacterial and antifungal activities. They are claimed to treat different ailments in the indigenous system of medicine. Surprisingly, some of the species have been reported to be extinct from the face of earth before their exploration and exploitation for economic benefits. This review focuses on the bio-prospects of Moringa particularly on relatively little explored area of their microbiological applications.

  10. Les contrats de bio-prospection, des outils pour le développement durable ?

    OpenAIRE

    Munoz Urena, Hugo,

    2012-01-01

    6 pages.; International audience; Bio-prospection contracts are consensual legal instruments, in which the parties to the bio-prospection relationship define their rights and duties. These contracts are vaguely regulated by general principles established in the Biological Diversity Convention (International Law instrument). Currently there is much debate about the extent of these principles and about the perspectives for the development of these contracts.; Les contrats de bio-prospection son...

  11. Livermore Metagenomics Analysis Toolkit

    Energy Technology Data Exchange (ETDEWEB)

    2012-10-01

    LMAT is designed to take as input a collection of raw metagenomic sequencer reads, and search each read against a reference genome database and assign a taxonomic label and confidence value to each read and report a summary of the predicted taxonomic contents of the metagenomic sample.

  12. Metagenomics at Grass Roots

    Indian Academy of Sciences (India)

    Sudeshna Mazumdar-Leighton; Vivek K Choudhary

    2017-03-01

    Metagenomics is a robust, interdisciplinary approach for studyingmicrobial community composition, function, and dynamics.It typically involves a core of molecular biology, microbiology,ecology, statistics, and computational biology. Excitingoutcomes anticipated from these studies include unravelingof complex interactions that characterize the ecologicalmilieu of microbial communities. Diverse habitats fromwhich metagenomes have been reported include human guts,caterpillar guts, thermal vents in oceans, ore deposits, polarcaps, and even soils that adhere to plant roots. Knowledgegenerated from metagenomic projects has tremendous potentialto benefit human health, agriculture, and ecosystemfunctions. This article provides a brief history of technicaladvances in metagenomics, including DNA sequencing methods,and some case studies. A specific example is providedof microbial metagenomes found at the roots of native grassspecies (family Poaceae) that can grow on degraded lands undergoingrevegetation.

  13. [Mini review] metagenomic studies of the Red Sea

    KAUST Repository

    Behzad, Hayedeh

    2015-10-23

    Red Sea, and the bio-prospecting potential of the Red Sea microbiota. Furthermore, we discuss the limitations of the previous studies and the need for generating a large and representative metagenomic database of the Red Sea to help establish a dynamic model of the Red Sea microbiota.

  14. Metagenomics and antibiotics.

    Science.gov (United States)

    Garmendia, L; Hernandez, A; Sanchez, M B; Martinez, J L

    2012-07-01

    Most of the bacterial species that form part of the biosphere have never been cultivated. In this situation, a comprehensive study of bacterial communities requires the utilization of non-culture-based methods, which have been named metagenomics. In this paper we review the use of different metagenomic techniques for understanding the effect of antibiotics on microbial communities, to synthesize new antimicrobial compounds and to analyse the distribution of antibiotic resistance genes in different ecosystems. These techniques include functional metagenomics, which serves to find new antibiotics or new antibiotic resistance genes, and descriptive metagenomics, which serves to analyse changes in the composition of the microbiota and to track the presence and abundance of already known antibiotic resistance genes in different ecosystems.

  15. Ocean microbial metagenomics

    Science.gov (United States)

    Kerkhof, Lee J.; Goodman, Robert M.

    2009-09-01

    Technology for accessing the genomic DNA of microorganisms, directly from environmental samples without prior cultivation, has opened new vistas to understanding microbial diversity and functions. Especially as applied to soils and the oceans, environments on Earth where microbial diversity is vast, metagenomics and its emergent approaches have the power to transform rapidly our understanding of environmental microbiology. Here we explore select recent applications of the metagenomic suite to ocean microbiology.

  16. A primer on metagenomics.

    Directory of Open Access Journals (Sweden)

    John C Wooley

    2010-02-01

    Full Text Available Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

  17. A primer on metagenomics.

    Science.gov (United States)

    Wooley, John C; Godzik, Adam; Friedberg, Iddo

    2010-02-26

    Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

  18. Surface glycosylation of polymeric membranes

    Institute of Scientific and Technical Information of China (English)

    DAI ZhengWei; WAN LingShu; XU ZhiKang

    2008-01-01

    Surface glycosylation of polymeric membranes has been inspired by the structure of natural biomembranes. It refers to that glycosyl groups are introduced onto the membrane surface by various strategies, which combine the separation function of the membrane with the biological function of the saccharides in one system. In this review, progress in the surface glycosylation of polymeric membranes is highlighted in two aspects, i.e. the glycosylation methods and the potential applications of the surface-glycosylated membranes.

  19. The YNP metagenome project

    DEFF Research Database (Denmark)

    Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.

    2013-01-01

    , or lake habitats and therefore offer a tremendous opportunity for studying the structure and function of different model microbial communities using environmental metagenomics. One of the broader goals of this study was to establish linkages among microbial distribution, metabolic potential......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3......) archaeal-dominated sediments. The metagenomes were analyzed using a suite of complementary and integrative bioinformatic tools, including phylogenetic and functional analysis of both individual sequence reads and assemblies of predominant phylotypes.This volume identifies major environmental determinants...

  20. Metagenomics of extreme environments.

    Science.gov (United States)

    Cowan, D A; Ramond, J-B; Makhalanyane, T P; De Maayer, P

    2015-06-01

    Whether they are exposed to extremes of heat or cold, or buried deep beneath the Earth's surface, microorganisms have an uncanny ability to survive under these conditions. This ability to survive has fascinated scientists for nearly a century, but the recent development of metagenomics and 'omics' tools has allowed us to make huge leaps in understanding the remarkable complexity and versatility of extremophile communities. Here, in the context of the recently developed metagenomic tools, we discuss recent research on the community composition, adaptive strategies and biological functions of extremophiles.

  1. Glycosylation of solute carriers

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Carlsson, Michael C; Pedersen, Stine Helene Falsig

    2016-01-01

    as their posttranslational regulation, but only relatively little is known about the role of SLC glycosylation. Glycosylation is one of the most abundant posttranslational modifications of animal proteins and through recent advances in our understanding of protein-glycan interactions, the functional roles of SLC......Solute carriers (SLCs) are one of the largest groups of multi-spanning membrane proteins in mammals and include ubiquitously expressed proteins as well as proteins with highly restricted tissue expression. A vast number of studies have addressed the function and organization of SLCs as well...

  2. Solid-Phase Random Glycosylation

    DEFF Research Database (Denmark)

    Agoston, K.; Kröger, Lars; Dekany, Gyula;

    2009-01-01

    Two different approaches were employed to study solid phase random glycosylations to obtain oligosaccharide libraries. In approach I, Wang resin esters were attached to the acceptors structures. Following their glycosylation and resin cleavage, the peracetylated components of the oligosaccharide ...

  3. Phylogenies reveal predictive power of traditional medicine in bioprospecting.

    Science.gov (United States)

    Saslis-Lagoudakis, C Haris; Savolainen, Vincent; Williamson, Elizabeth M; Forest, Félix; Wagstaff, Steven J; Baral, Sushim R; Watson, Mark F; Pendry, Colin A; Hawkins, Julie A

    2012-09-25

    There is controversy about whether traditional medicine can guide drug discovery, and investment in bioprospecting informed by ethnobotanical data has fluctuated. One view is that traditionally used medicinal plants are not necessarily efficacious and there are no robust methods for distinguishing those which are most likely to be bioactive when selecting species for further testing. Here, we reconstruct a genus-level molecular phylogenetic tree representing the 20,000 species found in the floras of three disparate biodiversity hotspots: Nepal, New Zealand, and the Cape of South Africa. Borrowing phylogenetic methods from community ecology, we reveal significant clustering of the 1,500 traditionally used species, and provide a direct measure of the relatedness of the three medicinal floras. We demonstrate shared phylogenetic patterns across the floras: related plants from these regions are used to treat medical conditions in the same therapeutic areas. This finding strongly indicates independent discovery of plant efficacy, an interpretation corroborated by the presence of a significantly greater proportion of known bioactive species in these plant groups than in random samples. We conclude that phylogenetic cross-cultural comparisons can focus screening efforts on a subset of traditionally used plants that are richer in bioactive compounds, and could revitalize the use of traditional knowledge in bioprospecting.

  4. Bioprospecting for bioactives from seaweeds: potential, obstacles and alternatives

    Directory of Open Access Journals (Sweden)

    Renato C. Pereira

    2012-08-01

    Full Text Available Seaweeds are potential sources of high biotechnological interest due to production of a great diversity of compounds exhibiting a broad spectrum of biological activities. On the other hand, there is an urgent need for management options for a sustainable approach to the use of marine organisms as a source of bioactive compounds. This review discusses the bioprospection for bioactive seaweed compounds as pharmaceuticals and antifouling agents, encompassing their potential and possible obstacles and alternatives. In spite of their potential, research on pharmaceuticals and antifouling agents from seaweeds includes mainly the search for molecules that exhibit these biological activities, but lacks of consideration of fundamental and limiting aspects such as the development of alternatives to sustainable supply. However, for the complete development of pharmaceuticals and antifouling compounds in Brazil, marine bioprospection should be more comprehensive, associating the search for molecules with an analysis of their supply. In this way, it is possible to promote sustainable development and conservation of biodiversity, as well as to assert the economic development of Brazil.

  5. Recent progresses in metagenomics

    Science.gov (United States)

    Metagenomics addresses the collective genetic structure and functional composition of a microbial community at its native habitat. This approach has emerged as a powerful tool to study the structure and function of the microbiota for the past few years and is revolutionizing studies of microbial ec...

  6. Glycosylation with Disarmed Glycosyl Bromides Promoted by Iodonium Ions

    DEFF Research Database (Denmark)

    Lanz, Gyrithe; Madsen, Robert

    2016-01-01

    Iodonium ions have been developed for activating glycosyl bromides in the coupling to glycosyl acceptors. The iodonium ions are generated from N-iodosuccinimide and a protic acid such as camphorsulfonic acid or triflic acid, where the latter gives the most reactive promoter system. The couplings...... occur with the release of iodine monobromide, and the best results are obtained with benzoylated glycosyl donors and acceptors. In this way, disarmed glycosyl bromides can serve as glycosyl donors without the use of heavy-metal salts....

  7. Ethnobotany/ethnopharmacology and mass bioprospecting: issues on intellectual property and benefit-sharing.

    Science.gov (United States)

    Soejarto, D D; Fong, H H S; Tan, G T; Zhang, H J; Ma, C Y; Franzblau, S G; Gyllenhaal, C; Riley, M C; Kadushin, M R; Pezzuto, J M; Xuan, L T; Hiep, N T; Hung, N V; Vu, B M; Loc, P K; Dac, L X; Binh, L T; Chien, N Q; Hai, N V; Bich, T Q; Cuong, N M; Southavong, B; Sydara, K; Bouamanivong, S; Ly, H M; Thuy, Tran Van; Rose, W C; Dietzman, G R

    2005-08-22

    Ethnobotany/ethnopharmacology has contributed to the discovery of many important plant-derived drugs. Field explorations to seek and document indigenous/traditional medical knowledge (IMK/TMK), and/or the biodiversity with which the IMK/TMK is attached, and its conversion into a commercialized product is known as bioprospecting or biodiversity prospecting. When performed in a large-scale operation, the effort is referred to as mass bioprospecting. Experiences from the mass bioprospecting efforts undertaken by the United States National Cancer Institute, the National Cooperative Drug Discovery Groups (NCDDG) and the International Cooperative Biodiversity Groups (ICBG) programs demonstrate that mass bioprospecting is a complex process, involving expertise from diverse areas of human endeavors, but central to it is the Memorandum of Agreement (MOA) that recognizes issues on genetic access, prior informed consent, intellectual property and the sharing of benefits that may arise as a result of the effort. Future mass bioprospecting endeavors must take heed of the lessons learned from past and present experiences in the planning for a successful mass bioprospecting venture.

  8. Soil metagenomics and tropical soil productivity

    OpenAIRE

    Karen A Garrett

    2009-01-01

    This presentation summarizes research in the soil metagenomics cross cutting research activity. Soil metagenomics studies soil microbial communities as contributors to soil health.C CCRA-4 (Soil Metagenomics)

  9. Surface glycosylation of polymeric membranes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Surface glycosylation of polymeric membranes has been inspired by the structure of natural biomem-branes. It refers to that glycosyl groups are introduced onto the membrane surface by various strate-gies, which combine the separation function of the membrane with the biological function of the sac-charides in one system. In this review, progress in the surface glycosylation of polymeric membranes is highlighted in two aspects, i.e. the glycosylation methods and the potential applications of the sur-face-glycosylated membranes.

  10. Gram-Negative Flagella Glycosylation

    Directory of Open Access Journals (Sweden)

    Susana Merino

    2014-02-01

    Full Text Available Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.

  11. Hot Spring Metagenomics

    Directory of Open Access Journals (Sweden)

    Olalla López-López

    2013-04-01

    Full Text Available Hot springs have been investigated since the XIX century, but isolation and examination of their thermophilic microbial inhabitants did not start until the 1950s. Many thermophilic microorganisms and their viruses have since been discovered, although the real complexity of thermal communities was envisaged when research based on PCR amplification of the 16S rRNA genes arose. Thereafter, the possibility of cloning and sequencing the total environmental DNA, defined as metagenome, and the study of the genes rescued in the metagenomic libraries and assemblies made it possible to gain a more comprehensive understanding of microbial communities—their diversity, structure, the interactions existing between their components, and the factors shaping the nature of these communities. In the last decade, hot springs have been a source of thermophilic enzymes of industrial interest, encouraging further study of the poorly understood diversity of microbial life in these habitats.

  12. The YNP metagenome project

    DEFF Research Database (Denmark)

    Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.;

    2013-01-01

    The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3...

  13. Plant protein glycosylation

    Science.gov (United States)

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  14. Systematics-guided bioprospecting for bioactive microbial natural products.

    Science.gov (United States)

    Liu, Xueting; Bolla, Krishna; Ashforth, Elizabeth Jane; Zhuo, Ying; Gao, Hong; Huang, Pei; Stanley, Sarah A; Hung, Deborah T; Zhang, Lixin

    2012-01-01

    Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.

  15. Genome-based bioprospecting of microbes for new therapeutics.

    Science.gov (United States)

    Zotchev, Sergey B; Sekurova, Olga N; Katz, Leonard

    2012-12-01

    Bioprospecting of natural sources for new medicines has a long and successful history, exemplified by the fact that over 50% of all drugs currently on the market are either derived from or inspired by natural products. However, development of new natural product-based therapeutics has been on the decline over the past 20 years, mainly owing to frequent re-discovery of already known compounds coupled with high costs for screening, characterization and development. With the onset of the genomic era allowing rapid sequencing and analysis of bacterial and fungal genomes, it became evident that these organisms possess 'hidden treasures' in the form of gene clusters potentially governing biosynthesis of novel biologically active compounds. This review highlights current progress in mining for and expression of these gene clusters, which may revolutionize the drug discovery pipelines in the near future.

  16. The botanical explorer's legacy: a promising bioprospecting tool.

    Science.gov (United States)

    Helmstädter, Axel

    2016-11-16

    Records about the traditional uses of medicinal plants can be considered useful in bioprospecting (i.e., the search for new active agents or lead structures in nature). Several sources like Egyptian papyri, early modern herbals and pharmacopoeias have been studied in this respect. It is proposed to use recordings of botanically interested explorers of the 19th and early 20th centuries as well. Some of them give detailed information about traditionally used medicinal plants and analysis shows that a considerable number of these have never been scientifically investigated. Existing studies, however, are confirming the traditional uses described to a great extent. Thus, the explorer's writings should not be neglected while looking for starting points for plant screening; success seems more likely than with screening at random.

  17. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    OpenAIRE

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-01-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carb...

  18. Databases of the marine metagenomics

    KAUST Repository

    Mineta, Katsuhiko

    2015-10-28

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  19. Databases of the marine metagenomics.

    Science.gov (United States)

    Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  20. Microbial metagenomics: beyond the genome.

    Science.gov (United States)

    Gilbert, Jack A; Dupont, Christopher L

    2011-01-01

    Metagenomics literally means "beyond the genome." Marine microbial metagenomic databases presently comprise approximately 400 billion base pairs of DNA, only approximately 3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  1. Microbial Metagenomics: Beyond the Genome

    Science.gov (United States)

    Gilbert, Jack A.; Dupont, Christopher L.

    2011-01-01

    Metagenomics literally means “beyond the genome.” Marine microbial metagenomic databases presently comprise ˜400 billion base pairs of DNA, only ˜3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  2. Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2016-03-01

    Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

  3. The glycosylation of myeloperoxidase

    DEFF Research Database (Denmark)

    Ravnsborg, Tina; Houen, Gunnar; Højrup, Peter

    2010-01-01

    The enzyme myeloperoxidase (MPO) is an important part of the neutrophil immune reaction and can be found in alfa granula. The presence of MPO can be used to distinguish acute myelogenous leukemia from acute lymphocytic leukemia. However, the methods employed to do so, such as flow cytometry...... and immunohistochemistry rely on antibody recognition, and therefore the characterization of the mature MPO, including post-translational modifications, must be considered as important as epitope mapping. MPO has 5 N-linked glycosylation sites, occupied by both high mannose and complex glycan structures. In this study we...

  4. Acetobixan, an inhibitor of cellulose synthesis identified by microbial bioprospecting.

    Directory of Open Access Journals (Sweden)

    Ye Xia

    Full Text Available In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI, named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.

  5. Acetobixan, an inhibitor of cellulose synthesis identified by microbial bioprospecting.

    Science.gov (United States)

    Xia, Ye; Lei, Lei; Brabham, Chad; Stork, Jozsef; Strickland, James; Ladak, Adam; Gu, Ying; Wallace, Ian; DeBolt, Seth

    2014-01-01

    In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.

  6. A guide to successful bioprospecting: informed by actinobacterial systematics.

    Science.gov (United States)

    Goodfellow, Michael; Fiedler, Hans-Peter

    2010-08-01

    New structurally diverse natural products are discovered when novel screening procedures are introduced or when high quality biological materials from new sources are examined in existing screens, hence it is important to foster these two aspects of novelty in drug discovery programmes. Amongst prokaryotes, actinomycetes, notably streptomycetes, remain a rich source of new natural products though it has become increasingly difficult to find such metabolites from common actinomycetes as screening 'old friends' leads to the costly rediscovery of known compounds. The bioprospecting strategy which is the subject of this review is based upon the premise that new secondary metabolites can be found by screening relatively small numbers of dereplicated, novel actinomycetes isolated from marine sediments. The success of the strategy is exemplified by the discovery of a range of novel bioactive compounds, notably atrop-abyssomicin C and proximicins A, B and C from Verrucosispora strains isolated from sediment samples taken from the Sea of Japan and the Raune Fjord, respectively, and the dermacozines derived from Dermacoccus strains isolated from the Challenger Deep of the Mariana Trench in the Pacific Ocean. The importance of current advances in prokaryotic systematics in work of this nature is stressed and a plea made that resources be sought to train, support and employ the next generation of actinobacterial systematists.

  7. Bioprospecting of Red Sea Sponges for Novel Antiviral Pharmacophores

    KAUST Repository

    O'Rourke, Aubrie

    2015-05-01

    Natural products offer many possibilities for the treatment of disease. More than 70% of the Earth’s surface is ocean, and recent exploration and access has allowed for new additions to this catalog of natural treasures. The Central Red Sea off the coast of Saudi Arabia serves as a newly accessible location, which provides the opportunity to bioprospect marine sponges with the purpose of identifying novel antiviral scaffolds. Antivirals are underrepresented in present day clinical trials, as well as in the academic screens of marine natural product libraries. Here a high-throughput pipeline was initiated by prefacing the antiviral screen with an Image-based High-Content Screening (HCS) technique in order to identify candidates with antiviral potential. Prospective candidates were tested in a biochemical or cell-based assay for the ability to inhibit the NS3 protease of the West Nile Virus (WNV NS protease) as well as replication and reverse transcription of the Human Immunodeficiency Virus 1 (HIV-1). The analytical chemistry techniques of High-Performance Liquid Chromatograpy (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR) where used in order to identify the compounds responsible for the characteristic antiviral activity of the selected sponge fractions. We have identified a 3-alkyl pyridinium from Amphimedon chloros as the causative agent of the observed WNV NS3 protease inhibition in vitro. Additionally, we identified debromohymenialdisine, hymenialdisine, and oroidin from Stylissa carteri as prospective scaffolds capable of HIV-1 inhibition.

  8. Open resource metagenomics: a model for sharing metagenomic libraries.

    Science.gov (United States)

    Neufeld, J D; Engel, K; Cheng, J; Moreno-Hagelsieb, G; Rose, D R; Charles, T C

    2011-11-30

    Both sequence-based and activity-based exploitation of environmental DNA have provided unprecedented access to the genomic content of cultivated and uncultivated microorganisms. Although researchers deposit microbial strains in culture collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences from the hits retrieved from specific screens. Physical metagenomic libraries, conceptually similar to entire sequence datasets, are usually not straightforward to obtain by interested parties subsequent to publication. In order to facilitate unrestricted distribution of metagenomic libraries, we propose the adoption of open resource metagenomics, in line with the trend towards open access publishing, and similar to culture- and mutant-strain collections that have been the backbone of traditional microbiology and microbial genetics. The concept of open resource metagenomics includes preparation of physical DNA libraries, preferably in versatile vectors that facilitate screening in a diversity of host organisms, and pooling of clones so that single aliquots containing complete libraries can be easily distributed upon request. Database deposition of associated metadata and sequence data for each library provides researchers with information to select the most appropriate libraries for further research projects. As a starting point, we have established the Canadian MetaMicroBiome Library (CM(2)BL [1]). The CM(2)BL is a publicly accessible collection of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning multiple biomes. The libraries were constructed such that the cloned DNA can be easily transferred to Gateway® compliant vectors, facilitating functional screening in virtually any surrogate microbial host for which there are available plasmid vectors. The libraries, which we are placing in the public domain, will be distributed upon request without restriction to members of both the

  9. Multispectral sorter for rapid, nondestructive optical bioprospecting for algae biofuels

    Science.gov (United States)

    Davis, Ryan W.; Wu, Hauwen; Singh, Seema

    2014-03-01

    Microalgal biotechnology is a nascent yet burgeoning field for developing the next generation of sustainable feeds, fuels, and specialty chemicals. Among the issues facing the algae bioproducts industry, the lack of efficient means of cultivar screening and phenotype selection represents a critical hurdle for rapid development and diversification. To address this challenge, we have developed a multi-modal and label-free optical tool which simultaneously assesses the photosynthetic productivity and biochemical composition of single microalgal cells, and provides a means for actively sorting attractive specimen (bioprospecting) based on the spectral readout. The device integrates laser-trapping micro-Raman spectroscopy and pulse amplitude modulated (PAM) fluorometry of microalgal cells in a flow cell. Specifically, the instrument employs a dual-purpose epi-configured IR laser for single-cell trapping and Raman spectroscopy, and a high-intensity VISNIR trans-illumination LED bank for detection of variable photosystem II (PSII) fluorescence. Micro-Raman scatter of single algae cells revealed vibrational modes corresponding to the speciation and total lipid content, as well as other major biochemical pools, including total protein, carbohydrates, and carotenoids. PSII fluorescence dynamics provide a quantitative estimate of maximum photosynthetic efficiency and regulated and non-regulated non-photochemical quenching processes. The combined spectroscopic readouts provide a set of metrics for subsequent optical sorting of the cells by the laser trap for desirable biomass properties, e.g. the combination of high lipid productivity and high photosynthetic yield. Thus the device provides means for rapid evaluation and sorting of algae cultures and environmental samples for biofuels development.

  10. Web Resources for Metagenomics Studies

    Institute of Scientific and Technical Information of China (English)

    Pravin Dudhagara; Sunil Bhavsar; Chintan Bhagat; Anjana Ghelani; Shreyas Bhatt; Rajesh Patel

    2015-01-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  11. Web Resources for Metagenomics Studies

    Directory of Open Access Journals (Sweden)

    Pravin Dudhagara

    2015-10-01

    Full Text Available The development of next-generation sequencing (NGS platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  12. Web Resources for Metagenomics Studies.

    Science.gov (United States)

    Dudhagara, Pravin; Bhavsar, Sunil; Bhagat, Chintan; Ghelani, Anjana; Bhatt, Shreyas; Patel, Rajesh

    2015-10-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  13. Metagenomic analysis of microbial communities and beyond

    DEFF Research Database (Denmark)

    Schreiber, Lars

    2014-01-01

    From small clone libraries to large next-generation sequencing datasets – the field of community genomics or metagenomics has developed tremendously within the last years. This chapter will summarize some of these developments and will also highlight pitfalls of current metagenomic analyses....... It will illustrate the general workflow of a metagenomic study and introduce the three different metagenomic approaches: (1) the random shotgun approach that focuses on the metagenome as a whole, (2) the targeted approach that focuses on metagenomic amplicon sequences, and (3) the function-driven approach that uses...... heterologous expression of metagenomic DNA fragments to discover novel metabolic functions. Lastly, the chapter will shortly discuss the meta-analysis of gene expression of microbial communities, more precisely metatranscriptomics and metaproteomics....

  14. Functional importance of PAI-1 glycosylation

    DEFF Research Database (Denmark)

    Christensen, Anni; Naessens, Dominik; Skottrup, Peter Durand

    Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...... attached at the 2 sites. Analysing the susceptibility of glycosylated and non-glycosylated PAI-1 to activity neutralisation by monoclonal antibodies, we found that the IC50-values for neutralisation by some monoclonal antibodies differed strongly between glycosylated and non-glycosylated PAI-1. The most...

  15. Functional importance of PAI-1 glycosylation

    DEFF Research Database (Denmark)

    Christensen, Anni; Naessens, Dominik; Skottrup, Peter

    2001-01-01

    Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...... attached at the 2 sites. Analysing the susceptibility of glycosylated and non-glycosylated PAI-1 to activity neutralisation by monoclonal antibodies, we found that the IC50-values for neutralisation by some monoclonal antibodies differed strongly between glycosylated and non-glycosylated PAI-1. The most...

  16. Functional Intestinal Metagenomics (Chapter 18)

    NARCIS (Netherlands)

    Bogert, van den B.; Leimena, M.M.; Vos, de W.M.; Zoetendal, E.G.; Kleerebezem, M.

    2011-01-01

    The premiere two-volume reference on revelations from studying complex microbial communities in many distinct habitats Metagenomics is an emerging field that has changed the way microbiologists study microorganisms. It involves the genomic analysis of microorganisms by extraction and cloning of DNA

  17. Multivariate analysis of functional metagenomes

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Dinsdale

    2013-04-01

    Full Text Available Metagenomics is a primary tool for the description of microbial and viral communities. The sheer magnitude of the data generated in each metagenome makes identifying key differences in the function and taxonomy between communities difficult to elucidate. Here we discuss the application of seven different data mining and statistical analyses by comparing and contrasting the metabolic functions of 212 microbial metagenomes within and between 10 environments. Not all approaches are appropriate for all questions, and researchers should decide which approach addresses their questions. This work demonstrated the use of each approach: for example, random forests provided a robust and enlightening description of both the clustering of metagenomes and the metabolic processes that were important in separating microbial communities from different environments. All analyses identified that the presence of phage genes within the microbial community was a predictor of whether the microbial community was host associated or free living. Several analyses identified the subtle differences that occur with environments, such as those seen in different regions of the marine environment.

  18. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  19. Chemoenzymatic Fc Glycosylation via Engineered Aldehyde Tags

    OpenAIRE

    2014-01-01

    Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that ...

  20. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    Science.gov (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  1. Epigenetic regulation of protein glycosylation.

    Science.gov (United States)

    Zoldoš, Vlatka; Grgurević, Srđana; Lauc, Gordan

    2010-10-01

    Protein N-glycosylation is an ancient metabolic pathway that still exists in all three domains of life (Archaea, Bacteria and Eukarya). The covalent addition of one or more complex oligosaccharides (glycans) to protein backbones greatly diversifies their structures and makes the glycoproteome several orders of magnitude more complex than the proteome itself. Contrary to polypeptides, which are defined by a sequence of nucleotides in the corresponding genes, the glycan part of glycoproteins are encoded in a complex dynamic network of hundreds of proteins, whereby activity is defined by both genetic sequence and the regulation of gene expression. Owing to the complex nature of their biosynthesis, glycans are particularly versatile and apparently a large part of human variation derives from differences in protein glycosylation. Composition of the individual glycome appears to be rather stable, and thus differences in the pattern of glycan synthesis between individuals could originate either from genetic polymorphisms or from stable epigenetic regulation of gene expression in different individuals. Studies of epigenetic modification of genes involved in protein glycosylation are still scarce, but their results indicate that this process might be very important for the regulation of protein glycosylation.

  2. Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2015-04-01

    Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway engineering and evolutionary engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast.

  3. Glycosylation regulates prestin cellular activity.

    Science.gov (United States)

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  4. An application of statistics to comparative metagenomics

    Directory of Open Access Journals (Sweden)

    Rohwer Forest

    2006-03-01

    Full Text Available Abstract Background Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. Results Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that were overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. Conclusion The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

  5. [Meta-Mesh: metagenomic data analysis system].

    Science.gov (United States)

    Su, Xiaoquan; Song, Baoxing; Wang, Xuetao; Ma, Xinle; Xu, Jian; Ning, Kang

    2014-01-01

    With the current accumulation of metagenome data, it is possible to build an integrated platform for processing of rigorously selected metagenomic samples (also referred as "metagenomic communities" here) of interests. Any metagenomic samples could then be searched against this database to find the most similar sample(s). However, on one hand, current databases with a large number of metagenomic samples mostly serve as data repositories but not well annotated database, and only offer few functions for analysis. On the other hand, the few available methods to measure the similarity of metagenomic data could only compare a few pre-defined set of metagenome. It has long been intriguing scientists to effectively calculate similarities between microbial communities in a large repository, to examine how similar these samples are and to find the correlation of the meta-information of these samples. In this work we propose a novel system, Meta-Mesh, which includes a metagenomic database and its companion analysis platform that could systematically and efficiently analyze, compare and search similar metagenomic samples. In the database part, we have collected more than 7 000 high quality and well annotated metagenomic samples from the public domain and in-house facilities. The analysis platform supplies a list of online tools which could accept metagenomic samples, build taxonomical annotations, compare sample in multiple angle, and then search for similar samples against its database by a fast indexing strategy and scoring function. We also used case studies of "database search for identification" and "samples clustering based on similarity matrix" using human-associated habitat samples to demonstrate the performance of Meta-Mesh in metagenomic analysis. Therefore, Meta-Mesh would serve as a database and data analysis system to quickly parse and identify similar

  6. Identification and Quantification of Protein Glycosylation

    Directory of Open Access Journals (Sweden)

    Ziv Roth

    2012-01-01

    Full Text Available Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated. Glycosylation plays a role in protein folding, interaction, stability, and mobility, as well as in signal transduction. Thus, by regulating protein activity, glycosylation is involved in the normal functioning of the cell and in the development of diseases. Indeed, in the past few decades there has been a growing realization of the importance of protein glycosylation, as aberrant glycosylation has been implicated in metabolic, neurodegenerative, and neoplastic diseases. Thus, the identification and quantification of protein-borne oligosaccharides have become increasingly important both in the basic sciences of biochemistry and glycobiology and in the applicative sciences, particularly biomedicine and biotechnology. Here, we review the state-of-the-art methodologies for the identification and quantification of oligosaccharides, specifically N- and O-glycosylated proteins.

  7. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, T.M.; Squina, F.M. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Paixao, D.A.A.; Franco Cairo, J.P.L.; Buchli, F.; Ruller, R. [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Campinas, SP (Brazil); Prade, R. [Oklahoma State University, Sillwater, OK (United States)

    2012-07-01

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  8. Exploration of noncoding sequences in metagenomes.

    Directory of Open Access Journals (Sweden)

    Fabián Tobar-Tosse

    Full Text Available Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C content, Codon Usage (Cd, Trinucleotide Usage (Tn, and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

  9. Metagenomic Assembly: Overview, Challenges and Applications

    Science.gov (United States)

    Ghurye, Jay S.; Cepeda-Espinoza, Victoria; Pop, Mihai

    2016-01-01

    Advances in sequencing technologies have led to the increased use of high throughput sequencing in characterizing the microbial communities associated with our bodies and our environment. Critical to the analysis of the resulting data are sequence assembly algorithms able to reconstruct genes and organisms from complex mixtures. Metagenomic assembly involves new computational challenges due to the specific characteristics of the metagenomic data. In this survey, we focus on major algorithmic approaches for genome and metagenome assembly, and discuss the new challenges and opportunities afforded by this new field. We also review several applications of metagenome assembly in addressing interesting biological problems. PMID:27698619

  10. Emerging trends in genomic approaches for microbial bioprospecting.

    Science.gov (United States)

    Akondi, K B; Lakshmi, V V

    2013-02-01

    Microorganisms constitute two out of the three domains of life on earth. They exhibit vast biodiversity and metabolic versatility. This enables the microorganisms to inhabit and thrive in even the most extreme environmental conditions, making them all pervading. The magnitude of biodiversity observed among microorganisms substantially supersedes that exhibited by the eukaryotes. These characteristics make the microbial world a very lucrative and inexhaustible resource for prospecting novel bioactive molecules. Despite their vast potential, over 99% of the microbial world still remains to be explored. The primary reason for this is that the culture-dependent methods used in the laboratories are grossly insufficient, as they support the growth of under 1% of the microorganisms found in nature. This limitation necessitated the development of techniques to circumvent culture dependency and gain access to the outstanding majority of the microorganisms. The development of culture-independent techniques has essentially reshaped the study of microbial diversity and community dynamics. Application of genomic and metagenomic approaches is contributing substantially towards characterization of the real microbial diversity. The amenability of these techniques to high throughput has opened the doors to explore the vast number of "uncultivable" microbial forms in substantially lesser time. The present article provides an update on the recent technological advances and emerging trends in exploring microbial community.

  11. [Ethical and legal principles for the activities of bioprospection in relation to human beings and the human genome].

    Science.gov (United States)

    Romeo Casabona, Carlos María

    2012-01-01

    During recent decades, bioprospecting has become an important field of research, which looks for development alternatives, entry into global (environmental) markets, and the subsequent obtention of benefits under sustainable development principles. However, there is still so much to discuss regarding the social and environmental impacts produced by this activity, as well as its main limitations. To this end, the Forum/round-table discussion, entitled "Bioprospección, Etica y Sociedad" was organised to take place on 28 March 2012 at the National University of Colombia. Its main objective was to enrich our knowledge on bioprospecting considering the ethical considerations that involve society. The presentation given by Professor ROMEO CASABONA, regarding the connection between bioprospecting and the human genome deserves special attention and is presented below.

  12. Subtractive assembly for comparative metagenomics, and its application to type 2 diabetes metagenomes.

    Science.gov (United States)

    Wang, Mingjie; Doak, Thomas G; Ye, Yuzhen

    2015-11-02

    Comparative metagenomics remains challenging due to the size and complexity of metagenomic datasets. Here we introduce subtractive assembly, a de novo assembly approach for comparative metagenomics that directly assembles only the differential reads that distinguish between two groups of metagenomes. Using simulated datasets, we show it improves both the efficiency of the assembly and the assembly quality of the differential genomes and genes. Further, its application to type 2 diabetes (T2D) metagenomic datasets reveals clear signatures of the T2D gut microbiome, revealing new phylogenetic and functional features of the gut microbial communities associated with T2D.

  13. Exploring neighborhoods in the metagenome universe.

    Science.gov (United States)

    Aßhauer, Kathrin P; Klingenberg, Heiner; Lingner, Thomas; Meinicke, Peter

    2014-07-14

    The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of the available sequence data alone. We have studied efficient sequence-based methods for large-scale identification of similar metagenomes within a database retrieval context. In a broad comparison of different profiling methods we found that vector-based distance measures are well-suitable for the detection of metagenomic neighbors. Our evaluation on more than 1700 publicly available metagenomes indicates that for a query metagenome from a particular habitat on average nine out of ten nearest neighbors represent the same habitat category independent of the utilized profiling method or distance measure. While for well-defined labels a neighborhood accuracy of 100% can be achieved, in general the neighbor detection is severely affected by a natural overlap of manually annotated categories. In addition, we present results of a novel visualization method that is able to reflect the similarity of metagenomes in a 2D scatter plot. The visualization method shows a similarly high accuracy in the reduced space as compared with the high-dimensional profile space. Our study suggests that for inspection of metagenome neighborhoods the profiling methods and distance measures can be chosen to provide a convenient interpretation of results in terms of the underlying features. Furthermore, supplementary metadata of metagenome samples in the future needs to comply with readily available ontologies for fine-grained and standardized annotation. To make profile-based k-nearest-neighbor search and the 2D-visualization of the metagenome universe available to the research community, we included the proposed methods in our CoMet-Universe server for comparative metagenome analysis.

  14. N-glycosylation in sugarcane

    Directory of Open Access Journals (Sweden)

    Maia Ivan G.

    2001-01-01

    Full Text Available The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

  15. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.

    Directory of Open Access Journals (Sweden)

    Mateusz Lezyk

    Full Text Available This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29. Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc as the substrate. In contrast, one fucosidase (Mfuc6 exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.

  16. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.

    Science.gov (United States)

    Lezyk, Mateusz; Jers, Carsten; Kjaerulff, Louise; Gotfredsen, Charlotte H; Mikkelsen, Maria D; Mikkelsen, Jørn D

    2016-01-01

    This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.

  17. Current and future resources for functional metagenomics.

    Science.gov (United States)

    Lam, Kathy N; Cheng, Jiujun; Engel, Katja; Neufeld, Josh D; Charles, Trevor C

    2015-01-01

    Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries-physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

  18. Current and future resources for functional metagenomics

    Directory of Open Access Journals (Sweden)

    Kathy Nguyen Lam

    2015-10-01

    Full Text Available Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries – physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

  19. Metagenomic data analysis : computational methods and applications

    NARCIS (Netherlands)

    Gori, F.

    2013-01-01

    Metagenomics is the study of the genomic content of microbial communities, acquired through DNA sequencing technology. The main advantage of metagenomics is that it can overcome the limitations of individual genome sequencing, that can work only on the few culturable microbes. Unfortunately, the an

  20. Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios

    DEFF Research Database (Denmark)

    Karlsson, Oskar Erik; Hansen, Trine; Knutsson, Rickard

    2013-01-01

    of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics...

  1. Hot and sweet: protein glycosylation in Crenarchaeota.

    Science.gov (United States)

    Meyer, Benjamin H; Albers, Sonja-Verena

    2013-02-01

    Every living cell is covered with a dense and complex array of covalently attached sugars or sugar chains. The majority of these glycans are linked to proteins via the so-called glycosylation process. Protein glycosylation is found in all three domains of life: Eukarya, Bacteria and Archaea. However, on the basis of the limit in analytic tools for glycobiology and genetics in Archaea, only in the last few years has research on archaeal glycosylation pathways started mainly in the Euryarchaeota Haloferax volcanii, Methanocaldococcus maripaludis and Methanococcus voltae. Recently, major steps of the crenarchaeal glycosylation process of the thermoacidophilic archaeon Sulfolobus acidocaldarius have been described. The present review summarizes the proposed N-glycosylation pathway of S. acidocaldarius, describing the phenotypes of the mutants disrupted in N-glycan biosynthesis as well as giving insights into the archaeal O-linked and glycosylphosphatidylinositol anchor glycosylation process.

  2. [Advances of metagenomics in discovering novel biocatalysts].

    Science.gov (United States)

    Wang, Kui; Wang, Sidi; Huang, Rui; Liu, Yuhuan

    2012-04-01

    Microorganisms contain a large number of biocatalysts, which are of great potential in industrial applications. However, the traditional cultural approaches can obtain only less than 1% of microorganisms. As a culture-independent method, metagenomics is an advanced solution by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes. It serves as an effective tool for studying these uncultured microorganisms. Therefore, mining novel biocatalysts from metagenome has drawn the attention of researchers in the world. In this paper, environment sample category, genomic DNA extraction, library construction and screening strategies were reviewed. Recent examples of isolated biocatalysts from metagenomic libraries were presented. Future research directions of metagenomics were also discussed.

  3. Exploring the viral world through metagenomics.

    Science.gov (United States)

    Rosario, Karyna; Breitbart, Mya

    2011-10-01

    Viral metagenomics, or shotgun sequencing of purified viral particles, has revolutionized the field of environmental virology by allowing the exploration of viral communities in a variety of sample types throughout the biosphere. The introduction of viral metagenomics has demonstrated that dominant viruses in environmental communities are not well-represented by the cultured viruses in existing sequence databases. Viral metagenomic studies have provided insights into viral ecology by elucidating the genetic potential, community structure, and biogeography of environmental viruses. In addition, viral metagenomics has expanded current knowledge of virus-host interactions by uncovering genes that may allow viruses to manipulate their hosts in unexpected ways. The intrinsic potential for virus discovery through viral metagenomics can help advance a wide array of disciplines including evolutionary biology, pathogen surveillance, and biotechnology.

  4. Metagenomics using next-generation sequencing.

    Science.gov (United States)

    Bragg, Lauren; Tyson, Gene W

    2014-01-01

    Traditionally, microbial genome sequencing has been restricted to the small number of species that can be grown in pure culture. The progressive development of culture-independent methods over the last 15 years now allows researchers to sequence microbial communities directly from environmental samples. This approach is commonly referred to as "metagenomics" or "community genomics". However, the term metagenomics is applied liberally in the literature to describe any culture-independent analysis of microbial communities. Here, we define metagenomics as shotgun ("random") sequencing of the genomic DNA of a sample taken directly from the environment. The metagenome can be thought of as a sampling of the collective genome of the microbial community. We outline the considerations and analyses that should be undertaken to ensure the success of a metagenomic sequencing project, including the choice of sequencing platform and methods for assembly, binning, annotation, and comparative analysis.

  5. Glycosylation and Activities of Natural Products.

    Science.gov (United States)

    Huang, Gangliang; Lv, Meijiao; Hu, Jinchuan; Huang, Kunlin; Xu, Hong

    2016-01-01

    Natural products are widely found in nature, their number and variety are numerous, the structures are complex and diverse. These natural products have many physiological and pharmacological activities. Glycosylation can increase the diversity of structure and function of natural product, it has become the focus of drug research and development. The impacts of glycosylation of natural products to water solubility, pharmacological activities, bioavailability, or others were described in this review, which provides a reference for the development and application of glycosylated natural products.

  6. Functional importance of PAI-1 glycosylation

    DEFF Research Database (Denmark)

    Christensen, Anni; Naessens, Dominik; Skottrup, Peter

    Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N-linked glyc......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N...

  7. Bioprospecting of antimicrobial activity of extracts of endophytic fungi from Bauhinia guianensis.

    Science.gov (United States)

    Pinheiro, Eduardo A A; Pina, Jeferson R S; Feitosa, André O; Carvalho, Josiwander M; Borges, Fábio C; Marinho, Patrícia S B; Marinho, Andrey M R

    Antibiotic resistance results in higher medical costs, prolonged hospital stays and increased mortality and is rising to dangerously high levels in all parts of the world. Therefore, this study aims to search for new antimicrobial agents through bioprospecting of extracts of endophytic fungi from Bauhinia guianensis, a typical Amazonian plant used in combating infections. Seventeen (17) fungi were isolated and as result the methanolic extract of the fungus Exserohilum rostratum showed good activity against the bacteria tested. The polyketide monocerin was isolated by the chromatographic technique, identified by NMR and MS, showing broad antimicrobial spectrum.

  8. Marine Vibrionaceae as a reservoir for bioprospecting and ecology studies

    DEFF Research Database (Denmark)

    Giubergia, Sonia

    The exploration of biodiversity (“bioprospecting”) provides mankind with an immense pool of novel organisms, molecules and information, which can be exploited for the development of innovative biotechnological processes and new ways to treat diseases. In the past decades, the marine environment...... environment. Studying microbial eco-physiology is important not only for the development of ecological models, but also as foundation for bioprospecting studies, where this knowledge may be used, for example, to elicit silent biosynthetic gene clusters during natural product discovery....

  9. Metagenomics and future perspectives in virus discovery.

    Science.gov (United States)

    Mokili, John L; Rohwer, Forest; Dutilh, Bas E

    2012-02-01

    Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. Identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. Traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. However, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. Viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity. In the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. Phylogenetically, some of these viruses are distantly related to previously discovered viruses. In addition, 60-99% of the sequences generated in different viral metagenomic studies are not homologous to known viruses. In this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. We discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. In addition, we propose a new model of the Koch's postulates named the 'Metagenomic Koch's Postulates'. Unlike the original Koch's postulates and the Molecular Koch's postulates as formulated by Falkow, the metagenomic Koch's postulates focus on the identification of metagenomic traits in disease cases. The metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen.

  10. The future of skin metagenomics.

    Science.gov (United States)

    Mathieu, Alban; Vogel, Timothy M; Simonet, Pascal

    2014-01-01

    Metagenomics, the direct exploitation of environmental microbial DNA, is complementary to traditional culture-based approaches for deciphering taxonomic and functional microbial diversity in a plethora of ecosystems, including those related to the human body such as the mouth, saliva, teeth, gut or skin. DNA extracted from human skin analyzed by sequencing the PCR-amplified rrs gene has already revealed the taxonomic diversity of microbial communities colonizing the human skin ("skin microbiome"). Each individual possesses his/her own skin microbial community structure, with marked taxonomic differences between different parts of the body and temporal evolution depending on physical and chemical conditions (sweat, washing etc.). However, technical limitations due to the low bacterial density at the surface of the human skin or contamination by human DNA still has inhibited extended use of the metagenomic approach for investigating the skin microbiome at a functional level. These difficulties have been overcome in part by the new generation of sequencing platforms that now provide sequences describing the genes and functions carried out by skin bacteria. These methodological advances should help us understand the mechanisms by which these microorganisms adapt to the specific chemical composition of each skin and thereby lead to a better understanding of bacteria/human host interdependence. This knowledge will pave the way for more systemic and individualized pharmaceutical and cosmetic applications.

  11. Glycosylation of a Newly Functionalized Orthoester Derivative

    Directory of Open Access Journals (Sweden)

    Kohei Kawa

    2014-02-01

    Full Text Available Tandem glycosylation of the 6-O-Fmoc-substituted benzyl orthoester derivative 2a was carried out in moderate yields by electrogenerated acid (EGA. The Fmoc group was effectively removed under mild basic conditions, and the product was submitted to the subsequent glycosylation.

  12. PROTEIN N-GLYCOSYLATION OF GASTROPODS

    OpenAIRE

    2009-01-01

    Glycosylation plays an important role in several types of recognition processes associated with fertilisation and development, allergies, pathological events and cell death. Whereas the amino acid sequence of a protein is fixed by the DNA, the glycosylation abilities depend on enzymes and substrates currently present in the cell.

  13. EBI metagenomics--a new resource for the analysis and archiving of metagenomic data.

    Science.gov (United States)

    Hunter, Sarah; Corbett, Matthew; Denise, Hubert; Fraser, Matthew; Gonzalez-Beltran, Alejandra; Hunter, Christopher; Jones, Philip; Leinonen, Rasko; McAnulla, Craig; Maguire, Eamonn; Maslen, John; Mitchell, Alex; Nuka, Gift; Oisel, Arnaud; Pesseat, Sebastien; Radhakrishnan, Rajesh; Rocca-Serra, Philippe; Scheremetjew, Maxim; Sterk, Peter; Vaughan, Daniel; Cochrane, Guy; Field, Dawn; Sansone, Susanna-Assunta

    2014-01-01

    Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive.

  14. Comparative metagenomics of the Red Sea

    KAUST Repository

    Mineta, Katsuhiko

    2016-01-26

    Metagenome produces a tremendous amount of data that comes from the organisms living in the environments. This big data enables us to examine not only microbial genes but also the community structure, interaction and adaptation mechanisms at the specific location and condition. The Red Sea has several unique characteristics such as high salinity, high temperature and low nutrition. These features must contribute to form the unique microbial community during the evolutionary process. Since 2014, we started monthly samplings of the metagenomes in the Red Sea under KAUST-CCF project. In collaboration with Kitasato University, we also collected the metagenome data from the ocean in Japan, which shows contrasting features to the Red Sea. Therefore, the comparative metagenomics of those data provides a comprehensive view of the Red Sea microbes, leading to identify key microbes, genes and networks related to those environmental differences.

  15. Toward Accurate and Quantitative Comparative Metagenomics

    Science.gov (United States)

    Nayfach, Stephen; Pollard, Katherine S.

    2016-01-01

    Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

  16. [Pathology and viral metagenomics, a recent history].

    Science.gov (United States)

    Bernardo, Pauline; Albina, Emmanuel; Eloit, Marc; Roumagnac, Philippe

    2013-05-01

    Human, animal and plant viral diseases have greatly benefited from recent metagenomics developments. Viral metagenomics is a culture-independent approach used to investigate the complete viral genetic populations of a sample. During the last decade, metagenomics concepts and techniques that were first used by ecologists progressively spread into the scientific field of viral pathology. The sample, which was first for ecologists a fraction of ecosystem, became for pathologists an organism that hosts millions of microbes and viruses. This new approach, providing without a priori high resolution qualitative and quantitative data on the viral diversity, is now revolutionizing the way pathologists decipher viral diseases. This review describes the very last improvements of the high throughput next generation sequencing methods and discusses the applications of viral metagenomics in viral pathology, including discovery of novel viruses, viral surveillance and diagnostic, large-scale molecular epidemiology, and viral evolution.

  17. Metagenome Fragment Classification Using -Mer Frequency Profiles

    OpenAIRE

    Gail Rosen; Elaine Garbarine; Diamantino Caseiro; Robi Polikar; Bahrad Sokhansanj

    2008-01-01

    A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique -mer frequency profiles of 635 microbial genomes publicly available as of February 2008....

  18. Snowball: Strain aware gene assembly of Metagenomes

    OpenAIRE

    Gregor, I.; Schönhuth, A.; McHardy, A. C.

    2015-01-01

    Gene assembly is an important step in functional analysis of shotgun metagenomic data. Nonetheless, strain aware assembly remains a challenging task, as current assembly tools often fail to distinguish among strain variants or require closely related reference genomes of the studied species to be available. We have developed Snowball, a novel strain aware and reference-free gene assembler for shotgun metagenomic data. It uses profile hidden Markov models (HMMs) of gene domains of interest to ...

  19. Screening and expression of genes from metagenomes.

    Science.gov (United States)

    Leis, Benedikt; Angelov, Angel; Liebl, Wolfgang

    2013-01-01

    Microorganisms are the most abundant and widely spread organisms on earth. They colonize a huge variety of natural and anthropogenic environments, including very specialized ecological niches and even extreme habitats, which are made possible by the immense metabolic diversity and genetic adaptability of microbes. As most of the organisms from environmental samples defy cultivation, cultivation-independent metagenomics approaches have been applied since more than one decade to access and characterize the phylogenetic diversity in microbial communities as well as their metabolic potential and ecological functions. Thereby, metagenomics has fully emerged as an own scientific field for mining new biocatalysts for many industrially relevant processes in biotechnology and pharmaceutics. This review summarizes common metagenomic approaches ranging from sampling, isolation of nucleic acids, construction of metagenomic libraries and their evaluation. Sequence-based screenings implement next-generation sequencing platforms, microarrays or PCR-based methods, while function-based analysis covers heterologous expression of metagenomic libraries in diverse screening setups. Major constraints and advantages of each strategy are described. The importance of alternative host-vector systems is discussed, and in order to underline the role of phylogenetic and physiological distance from the gene donor and the expression host employed, a case study is presented that describes the screening of a genomic library from an extreme thermophilic bacterium in both Escherichia coli and Thermus thermophilus. Metatranscriptomics, metaproteomics and single-cell-based methods are expected to complement metagenomic screening efforts to identify novel biocatalysts from environmental samples.

  20. Challenges and Opportunities of Airborne Metagenomics

    KAUST Repository

    Behzad, H.

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  1. Challenges and opportunities of airborne metagenomics.

    Science.gov (United States)

    Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  2. Synthetic glycosylated natural products have satisfactory activities.

    Science.gov (United States)

    Huang, Gangliang; Mei, Xinya

    2014-01-01

    Many natural products contain sugar residues, which are essential components for great medicinal importance. The sugar moieties can improve water-solubility of natural products and decrease their toxicity. At the same time, the glycosidic residues are crucial for the activities of natural products. Much effort has been expended over the past decades in developing novel and efficient methodologies to synthesize the glycosylated natural products. This review highlights recent developments in the synthesis of glycosylated natural products. The structure-activity relationships of some of these glycosylated natural products, together with the structure characteristics of their interaction with the biological targets, are also involved.

  3. Rapid Synthesis of Glycosyl Bromides by Ultrasound Irradiation

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jin-zhong; ZHANG Xue-qin; WU Xin; XING Ze-bing; YUE Ai-qin; SHAO Hua-wu

    2013-01-01

    A convenient and environmentally friendly reactor for the synthesis of glycosyl bromides via ultrasound irradiation was designed.Peracetylated glycosyl bromides were synthesized from free saccharides by means of a one-pot method.Benzoylated and 6-subsituted glycosyl bromides were prepared from protected saccharides.The glycosyl bromides were obtained in isolated yields of 83% to 96%.

  4. 21 CFR 864.7470 - Glycosylated hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section... Glycosylated hemoglobin assay. (a) Identification. A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a, A1b, and A1c) in a patient's blood by a column...

  5. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes

    Directory of Open Access Journals (Sweden)

    Stevens R

    2008-09-01

    Full Text Available Abstract Background Random community genomes (metagenomes are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. Results A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats. Conclusion The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis – the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org

  6. Undetectable Glycosylated Hemoglobin in Autoimmune Hemolytic Anemia

    OpenAIRE

    Mitani, Noriyuki; Taguchi, Akihiko; Sakuragi, Shizu; Matsui, Kumiko; Tanaka, Yoshinori; Matsuda, Kazuhiro; Shinohara, Kenji

    2005-01-01

    We encountered two cases of autoimmune hemolytic anemia (AIHA) with undetectable glycosylated hemoglobin (HbA1C) level at diagnosis. Hemolytic anemia improved by administration of prednisolone (PSL) and HbA1C became measurable after response.

  7. Interactive metagenomic visualization in a Web browser

    Directory of Open Access Journals (Sweden)

    Phillippy Adam M

    2011-09-01

    Full Text Available Abstract Background A critical output of metagenomic studies is the estimation of abundances of taxonomical or functional groups. The inherent uncertainty in assignments to these groups makes it important to consider both their hierarchical contexts and their prediction confidence. The current tools for visualizing metagenomic data, however, omit or distort quantitative hierarchical relationships and lack the facility for displaying secondary variables. Results Here we present Krona, a new visualization tool that allows intuitive exploration of relative abundances and confidences within the complex hierarchies of metagenomic classifications. Krona combines a variant of radial, space-filling displays with parametric coloring and interactive polar-coordinate zooming. The HTML5 and JavaScript implementation enables fully interactive charts that can be explored with any modern Web browser, without the need for installed software or plug-ins. This Web-based architecture also allows each chart to be an independent document, making them easy to share via e-mail or post to a standard Web server. To illustrate Krona's utility, we describe its application to various metagenomic data sets and its compatibility with popular metagenomic analysis tools. Conclusions Krona is both a powerful metagenomic visualization tool and a demonstration of the potential of HTML5 for highly accessible bioinformatic visualizations. Its rich and interactive displays facilitate more informed interpretations of metagenomic analyses, while its implementation as a browser-based application makes it extremely portable and easily adopted into existing analysis packages. Both the Krona rendering code and conversion tools are freely available under a BSD open-source license, and available from: http://krona.sourceforge.net.

  8. Ethnodiagnostic skills of the Digo community for malaria: a lead to traditional bioprospecting.

    Directory of Open Access Journals (Sweden)

    Joseph Mwanzia Nguta

    2011-06-01

    Full Text Available Malaria is a major public health problem that is presently complicated by the development of resistance by Plasmodium falciparum to the mainstay drugs. Thus, new drugs with unique structures and mechanism of action are required to treat drug-resistant strains of malaria. Historically, compounds containing a novel structure from natural origin represent a major source for the discovery and development of new drugs for several diseases. This paper presents ethnophytotherapeutic remedies, ethnodiagnostic skills and related traditional knowledge utilized by the Digo community of the Kenyan Coast to diagnose malaria as a lead to traditional bioprospecting. The current study was carried out in three Digo villages of Diani sub-location between May 2009 and December 2009. Data was collected using semi-structured interviews, and open and close-ended questionnaires. A total of sixty (60 respondents (34 men and 26 women provided the targeted information. The results show that the indigenous knowledge of Digo community on malaria encompasses not only the symptoms of malaria but also the factors that are responsible for causing malaria, attributes favoring the breeding of mosquitoes and practices employed to guard against mosquito bites or to protect households against malaria. This knowledge is closely in harmony with scientific approaches to the treatment and control of the disease. The Digo community uses sixty (60 medicinal plants distributed in fifty two (52 genera and twenty seven(27 families to treat malaria. The most frequently mentioned symptoms were fever, joint pains and vomiting while the most frequently mentioned practices employed to guard against mosquito bites and/or to protect households against malaria was burning of herbal plants such as Ocimum suave and ingestion of herbal decoctions and concoctions. The Digo community has abundant ethnodiagnostic skills for malaria which forms the basis of their traditional bioprospecting techniques.

  9. Ethnodiagnostic skills of the digo community for malaria: a lead to traditional bioprospecting.

    Science.gov (United States)

    Nguta, Joseph Mwanzia; Mbaria, James M; Gathumbi, Peter K; Gakuya, Daniel; Kabasa, John David; Kiama, Stephen Gitahi

    2011-01-01

    Malaria is a major public health problem that is presently complicated by the development of resistance by Plasmodium falciparum to the mainstay drugs. Thus, new drugs with unique structures and mechanism of action are required to treat drug-resistant strains of malaria. Historically, compounds containing a novel structure from natural origin represent a major source for the discovery and development of new drugs for several diseases. This paper presents ethnophytotherapeutic remedies, ethnodiagnostic skills, and related traditional knowledge utilized by the Digo community of the Kenyan Coast to diagnose malaria as a lead to traditional bioprospecting. The current study was carried out in three Digo villages of Diani sub-location between May 2009 and December 2009. Data was collected using semi-structured interviews, and open and close-ended questionnaires. A total of 60 respondents (34 men and 26 women) provided the targeted information. The results show that the indigenous knowledge of Digo community on malaria encompasses not only the symptoms of malaria but also the factors that are responsible for causing malaria, attributes favoring the breeding of mosquitoes and practices employed to guard against mosquito bites or to protect households against malaria. This knowledge is closely in harmony with scientific approaches to the treatment and control of the disease. The Digo community uses 60 medicinal plants distributed in 52 genera and 27 families to treat malaria. The most frequently mentioned symptoms were fever, joint pains, and vomiting while the most frequently mentioned practices employed to guard against mosquito bites and/or to protect households against malaria was burning of herbal plants such as Ocimum suave and ingestion of herbal decoctions and concoctions. The Digo community has abundant ethnodiagnostic skills for malaria which forms the basis of their traditional bioprospecting techniques.

  10. Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils.

    Science.gov (United States)

    Sprocati, Anna Rosa; Alisi, Chiara; Tasso, Flavia; Fiore, Alessia; Marconi, Paola; Langella, Francesca; Haferburg, Götz; Nicoara, Andrei; Neagoe, Aurora; Kothe, Erika

    2014-01-01

    The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon's index (alpha diversity) and Sørensen's Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

  11. Digital Marine Bioprospecting: Mining New Neurotoxin Drug Candidates from the Transcriptomes of Cold-Water Sea Anemones

    Directory of Open Access Journals (Sweden)

    Åse Emblem

    2012-10-01

    Full Text Available Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.

  12. Digital marine bioprospecting: mining new neurotoxin drug candidates from the transcriptomes of cold-water sea anemones.

    Science.gov (United States)

    Urbarova, Ilona; Karlsen, Bård Ove; Okkenhaug, Siri; Seternes, Ole Morten; Johansen, Steinar D; Emblem, Ase

    2012-10-01

    Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.

  13. Molecular Detection and Environment-Specific Diversity of Glycosyl Hydrolase Family 1 β-Glucosidase in Different Habitats

    Science.gov (United States)

    Tiwari, Rameshwar; Kumar, Kanika; Singh, Surender; Nain, Lata; Shukla, Pratyoosh

    2016-01-01

    β-glucosidase is a crucial element of the microbial cellulose multienzyme complex since it is responsible for the regulation of the entire cellulose hydrolysis process. Therefore, the aim of the present work was to explore the diversity and distribution of glycosyl hydrolase family 1 β-glucosidase genes in three different environmental niches including, Himalayan soil, cow dung and compost by metagenomic approach. Preliminary evaluation through metabolic profiling using BIOLOG based utilization patterns of carbon, nitrogen, phosphorus and sulfur revealed the environment and substrate specific nature of the indigenous microbial population. Furthermore, clonal library selection, screening and sequence analysis revealed that most of the GH1 β-glucosidase proteins had low identities with the available database. Analysis of the distribution of GH1 β-glucosidase gene fragments and β-glucosidase producing microbial community revealed the environment specific nature. The OTUs obtained from Himalayan soil and compost metagenomic libraries were grouped into 19 different genera comprising 6 groups. The cow dung sample displayed the least diversity of GH1 β-glucosidase sequences, with only 14 genera, distributed among three groups- Bacteroidetes, Firmicutes, and Actinobacteria. The metagenomic study coupled with metabolic profiling of GH1 β-glucosidase illustrated the existence of intricate relationship between the geochemical environmental factors and inherent microbial community.

  14. Molecular detection and environment-specific diversity of glycosyl hydrolase family 1 β-glucosidase in different habitats

    Directory of Open Access Journals (Sweden)

    Rameshwar Tiwari

    2016-10-01

    Full Text Available β-glucosidase is a crucial element of the microbial cellulose multienzyme complex since it is responsible for the regulation of the entire cellulose hydrolysis process. Therefore, the aim of the present work was to explore the diversity and distribution of glycosyl hydrolase family 1 β-glucosidase genes in three different environmental niches including, Himalayan soil, cow dung and compost by metagenomic approach. Preliminary evaluation through metabolic profiling using BIOLOG based utilization patterns of carbon, nitrogen, phosphorus and sulphur revealed the environment and substrate specific nature of the indigenous microbial population. Furthermore, clonal library selection, screening and sequence analysis revealed that most of the GH1 β-glucosidase proteins had low identities with the available database. Analysis of the distribution of GH1 β-glucosidase gene fragments and β-glucosidase producing microbial community revealed the environment specific nature. The OTUs obtained from Himalayan soil and compost metagenomic libraries were grouped into 19 different genera comprising 6 groups. The cow dung sample displayed the least diversity of GH1 β-glucosidase sequences, with only 14 genera, distributed among three groups- Bacteroidetes, Firmicutes and Actinobacteria. The metagenomic study coupled with metabolic profiling of GH1 β-glucosidase illustrated the existence of intricate relationship between the geochemical environmental factors and inherent microbial community.

  15. Shotgun metagenomic data streams: surfing without fear

    Energy Technology Data Exchange (ETDEWEB)

    Berendzen, Joel R [Los Alamos National Laboratory

    2010-12-06

    Timely information about bio-threat prevalence, consequence, propagation, attribution, and mitigation is needed to support decision-making, both routinely and in a crisis. One DNA sequencer can stream 25 Gbp of information per day, but sampling strategies and analysis techniques are needed to turn raw sequencing power into actionable knowledge. Shotgun metagenomics can enable biosurveillance at the level of a single city, hospital, or airplane. Metagenomics characterizes viruses and bacteria from complex environments such as soil, air filters, or sewage. Unlike targeted-primer-based sequencing, shotgun methods are not blind to sequences that are truly novel, and they can measure absolute prevalence. Shotgun metagenomic sampling can be non-invasive, efficient, and inexpensive while being informative. We have developed analysis techniques for shotgun metagenomic sequencing that rely upon phylogenetic signature patterns. They work by indexing local sequence patterns in a manner similar to web search engines. Our methods are laptop-fast and favorable scaling properties ensure they will be sustainable as sequencing methods grow. We show examples of application to soil metagenomic samples.

  16. Preliminary High-Throughput Metagenome Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

    2007-03-26

    Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

  17. Viral Metagenomics: MetaView Software

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, C; Smith, J

    2007-10-22

    The purpose of this report is to design and develop a tool for analysis of raw sequence read data from viral metagenomics experiments. The tool should compare read sequences of known viral nucleic acid sequence data and enable a user to attempt to determine, with some degree of confidence, what virus groups may be present in the sample. This project was conducted in two phases. In phase 1 we surveyed the literature and examined existing metagenomics tools to educate ourselves and to more precisely define the problem of analyzing raw read data from viral metagenomic experiments. In phase 2 we devised an approach and built a prototype code and database. This code takes viral metagenomic read data in fasta format as input and accesses all complete viral genomes from Kpath for sequence comparison. The system executes at the UNIX command line, producing output that is stored in an Oracle relational database. We provide here a description of the approach we came up with for handling un-assembled, short read data sets from viral metagenomics experiments. We include a discussion of the current MetaView code capabilities and additional functionality that we believe should be added, should additional funding be acquired to continue the work.

  18. Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

    Directory of Open Access Journals (Sweden)

    Yamakoshi Fumiko

    2011-02-01

    Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

  19. Glucose-tolerant β-glucosidase retrieved from the metagenome

    Directory of Open Access Journals (Sweden)

    Taku eUchiyama

    2015-06-01

    Full Text Available β-glucosidases (BGLs hydrolyze cellooligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (~mM concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (approximately 10,000 colonies and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7 was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 µM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose and high Vmax (91 µmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 µmol min-1 mg-1 with cellobiose among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

  20. One-pot conversion reactions of glycosyl boranophosphates into glycosyl phosphate derivatives via acyl phosphite intermediates.

    Science.gov (United States)

    Sato, Kazuki; Wada, Takeshi

    2016-11-29

    A one-pot synthesis of glycosyl phosphates and their P-modified analogs from glycosyl boranophosphates under mild basic conditions has been conducted. (31)P NMR monitoring of the reaction mixture revealed that the key intermediates of these reactions were acyl phosphites, which could not be formed from the corresponding H-phosphonate diesters.

  1. FANTOM: Functional and taxonomic analysis of metagenomes

    Directory of Open Access Journals (Sweden)

    Sanli Kemal

    2013-02-01

    Full Text Available Abstract Background Interpretation of quantitative metagenomics data is important for our understanding of ecosystem functioning and assessing differences between various environmental samples. There is a need for an easy to use tool to explore the often complex metagenomics data in taxonomic and functional context. Results Here we introduce FANTOM, a tool that allows for exploratory and comparative analysis of metagenomics abundance data integrated with metadata information and biological databases. Importantly, FANTOM can make use of any hierarchical database and it comes supplied with NCBI taxonomic hierarchies as well as KEGG Orthology, COG, PFAM and TIGRFAM databases. Conclusions The software is implemented in Python, is platform independent, and is available at http://www.sysbio.se/Fantom.

  2. Pathway-Based Functional Analysis of Metagenomes

    Science.gov (United States)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  3. Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens.

    Science.gov (United States)

    Aylward, Frank O; Burnum, Kristin E; Scott, Jarrod J; Suen, Garret; Tringe, Susannah G; Adams, Sandra M; Barry, Kerrie W; Nicora, Carrie D; Piehowski, Paul D; Purvine, Samuel O; Starrett, Gabriel J; Goodwin, Lynne A; Smith, Richard D; Lipton, Mary S; Currie, Cameron R

    2012-09-01

    Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.

  4. [Metagenomics in studying gastrointestinal tract microorganism].

    Science.gov (United States)

    Xu, Bo; Yang, Yunjuan; Li, Junjun; Tang, Xianghua; Mu, Yuelin; Huang, Zunxi

    2013-12-01

    Animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. The gut microbial community of humans and animals has received significant attention from researchers because of its association with health and disease. The application of metagenomics technology enables researchers to study not only the microbial composition but also the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in studying gastrointestinal tract microorganism with metagenomics and the bioinformatics technology.

  5. METAREP: JCVI metagenomics reports—an open source tool for high-performance comparative metagenomics

    OpenAIRE

    Goll, Johannes; Rusch, Douglas B; Tanenbaum, David M.; Thiagarajan, Mathangi; Li, Kelvin; Methé, Barbara A.; Yooseph, Shibu

    2010-01-01

    Summary: JCVI Metagenomics Reports (METAREP) is a Web 2.0 application designed to help scientists analyze and compare annotated metagenomics datasets. It utilizes Solr/Lucene, a high-performance scalable search engine, to quickly query large data collections. Furthermore, users can use its SQL-like query syntax to filter and refine datasets. METAREP provides graphical summaries for top taxonomic and functional classifications as well as a GO, NCBI Taxonomy and KEGG Pathway Browser. Users can ...

  6. The metagenomic approach and causality in virology

    Directory of Open Access Journals (Sweden)

    Silvana Beres Castrignano

    2015-01-01

    Full Text Available Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease.

  7. Snowball: Strain aware gene assembly of Metagenomes

    NARCIS (Netherlands)

    Gregor, I.; Schönhuth, A.; McHardy, A.C.

    2015-01-01

    Gene assembly is an important step in functional analysis of shotgun metagenomic data. Nonetheless, strain aware assembly remains a challenging task, as current assembly tools often fail to distinguish among strain variants or require closely related reference genomes of the studied species to be av

  8. Biomolecular and metagenomic analyses of biofouling communities

    Science.gov (United States)

    Despite the decades of research that have focused on understanding the formation of biofouling communities, relatively little is known about the soft fouling consortia that are responsible for their formation and function. In this study, we used PhyloChip microbial profiling, metagenomic DNA sequenc...

  9. Assembly of viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2014-01-01

    textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow r

  10. Assembly of viral genomes from metagenomes

    Directory of Open Access Journals (Sweden)

    Saskia L Smits

    2014-12-01

    Full Text Available Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

  11. A catalog of the mouse gut metagenome

    DEFF Research Database (Denmark)

    Xiao, Liang; Feng, Qiang; Liang, Suisha;

    2015-01-01

    We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing laborato...

  12. Reference databases for taxonomic assignment in metagenomics.

    Science.gov (United States)

    Santamaria, Monica; Fosso, Bruno; Consiglio, Arianna; De Caro, Giorgio; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Marzano, Marinella; Alonso-Alemany, Daniel; Valiente, Gabriel; Pesole, Graziano

    2012-11-01

    Metagenomics is providing an unprecedented access to the environmental microbial diversity. The amplicon-based metagenomics approach involves the PCR-targeted sequencing of a genetic locus fitting different features. Namely, it must be ubiquitous in the taxonomic range of interest, variable enough to discriminate between different species but flanked by highly conserved sequences, and of suitable size to be sequenced through next-generation platforms. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the ribosomal DNA operon and one or more hyper-variable regions of 16S ribosomal RNA gene are typically used to identify fungal and bacterial species, respectively. In this context, reliable reference databases and taxonomies are crucial to assign amplicon sequence reads to the correct phylogenetic ranks. Several resources provide consistent phylogenetic classification of publicly available 16S ribosomal DNA sequences, whereas the state of ribosomal internal transcribed spacers reference databases is notably less advanced. In this review, we aim to give an overview of existing reference resources for both types of markers, highlighting strengths and possible shortcomings of their use for metagenomics purposes. Moreover, we present a new database, ITSoneDB, of well annotated and phylogenetically classified ITS1 sequences to be used as a reference collection in metagenomic studies of environmental fungal communities. ITSoneDB is available for download and browsing at http://itsonedb.ba.itb.cnr.it/.

  13. Functional metagenomics: recent advances and future challenges.

    Science.gov (United States)

    Chistoserdovai, Ludmila

    2010-01-01

    Metagenomics is a relatively new but fast growing field within environmental biology directed at obtaining knowledge on genomes of environmental microbes as well as of entire microbial communities. With the sequencing technologies improving steadily, generating large amounts of sequence is becoming routine. However, it remains difficult to connect specific microbial phyla to specific functions in the environment. A number of 'functional metagenomics' approaches have been implemented in the recent years that allow high-resolution genomic analysis of uncultivated microbes, connecting them to specific functions in the environment. These include analysis of niche-specialized low complexity communities, reactor enrichments, and the use labeling technologies. Metatranscriptomics and metaproteomics are the newest sub-disciplines within the metagenomics field that provide further levels of resolution for functional analysis of uncultivated microbes and communities. The recent emergence of new (next generation) sequencing technologies, resulting in higher sequence output and dramatic drop in the price of sequencing, will be defining a new era in metagenomics. At this time the sequencing effort will be taken to a new level to allow addressing new, previously unattainable biological questions as well as accelerating genome-based discovery for medical and biotechnological applications.

  14. The metagenomic approach and causality in virology

    Science.gov (United States)

    Castrignano, Silvana Beres; Nagasse-Sugahara, Teresa Keico

    2015-01-01

    Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease. PMID:25902566

  15. C-Glycosyl furans I: a synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Baneza Sanz, J.M.; Galisteo Gonzalez, D.; Lopez Sastre, J.A.; Rodriguez Amo, J.F.; Romero Avila Garcia, M.C.; Santos Garcia, M.; Sanz Tejedor, M.A. [Departamento de Quimica Organica, E.T.S.I.I., Universidad de Valladolid (Spain)

    1995-12-31

    The C-glycosyl heterocycles joint their interest as potential antiviral and Anti bacterial agents to their nature as C-glycoside precursors through different heterocycle transformation. With respect to C-glycosyl furans, the reaction of sugar condensation with furan, produces better results when it is carried out with Dziewszek`s method than with Suzuki`s one (n-BuLi in THF) and yields higher quantities of the isomer having the same configuration at the new asymmetric center than that of the nearest chiral carbon atom. (Author) 19 refs.

  16. Extreme sweetness: protein glycosylation in archaea.

    Science.gov (United States)

    Eichler, Jerry

    2013-03-01

    Although N-glycosylation was first reported in archaea almost 40 years ago, detailed insights into this process have become possible only recently, with the availability of complete genome sequences for almost 200 archaeal species and the development of appropriate molecular tools. As a result of these advances, recent efforts have not only succeeded in delineating the pathways involved in archaeal N-glycosylation, but also begun to reveal how such post-translational protein modification helps archaea to survive in some of the harshest environments on the planet.

  17. Glycosyl Thioimidates as Versatile Building Blocks for Organic Synthesis

    Science.gov (United States)

    Hasty, S. J.

    2013-01-01

    This review discusses the synthesis and application of glycosyl thioimidates in chemical glycosylation and oligosaccharide assembly. Although glycosyl thioimidates include a broad range of compounds, the discussion herein centers on S-benzothiazolyl (SBaz), S-benzoxazolyl (SBox), S-thiazolinyl (STaz), and S-benzimidazolyl (SBiz) glycosides. These heterocyclic moieties have recently emerged as excellent anomeric leaving groups that express unique characteristics for highly diastereoselective glycosylation and help to provide the streamlined access to oligosaccharides. PMID:24288416

  18. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter;

    2003-01-01

    glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC(50)-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosylated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes...

  19. Control of mucin-type O-glycosylation

    DEFF Research Database (Denmark)

    Bennett, Eric P; Mandel, Ulla; Clausen, Henrik;

    2012-01-01

    Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine ...

  20. The Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni;

    of these sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627...

  1. The Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni;

    2002-01-01

    of these sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627...

  2. Is glycosylated haemoglobin a marker of fertility?

    DEFF Research Database (Denmark)

    Hjollund, N H; Jensen, Tina Kold; Bonde, Jens Peter;

    1999-01-01

    We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...

  3. Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

    DEFF Research Database (Denmark)

    Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

    2003-01-01

    Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry...

  4. Bioprospecting of Turbinaria Macroalgae as a Potential Source of Health Protective Compounds.

    Science.gov (United States)

    Stranska-Zachariasova, Milena; Kurniatanty, Isma; Gbelcova, Helena; Jiru, Monika; Rubert, Josep; Nindhia, Tjokorda Gde Tirta; D'Acunto, Cosimo Walter; Sumarsono, Sony Heru; Tan, Marselina Irasonia; Hajslova, Jana; Ruml, Tomas

    2017-02-01

    The present study aims to focus on the bioprospecting of marine macroalgae of Turbinaria species, plenteous biomass of the world ocean. Three types of solvents, i.e., H2 O, MeOH/H2 O (80:20, v/v) and hexane/i-PrOH (50:50, v/v), were used for extraction. Both the biological activity and the pattern of present chemicals were characterized. For the cell proliferation assay, the human embryonic kidney 293 cells, cervix/breast/pancreatic adenocarcinoma, and osteosarcoma cells were used. For the antioxidant activity determination, both intracellular assay with human embryonic kidney and cervix adenocarcinoma cells, as well as the biochemical DPPH test, were employed. To complete the information about macroalgae composition, organic compounds were characterized by the liquid chromatography coupled with high resolution tandem mass spectrometry. Attention was concentrated mainly on the lipidomic profile characterization. In spite the fact that any significant antiproliferative effect was not observed for cancer cells, both the Turbinaria species were shown to be good protectors against the oxidative stress of the non-cancer cells. Most of the antioxidants were determined in the hexane/i-PrOH extract. As regards the lipids identified, most of them belonged to the triacylglycerols followed by sphingomyelins, diacylglycerols, and polar (lyso)phospholipids. Additionally to fatty acids with 14, 16 and 18 carbons, also those with odd carbon numbers were frequently present.

  5. Bioprospecting of marine invertebrates for new natural products - a chemical and zoogeographical perspective.

    Science.gov (United States)

    Leal, Miguel Costa; Madeira, Carolina; Brandão, Cláudio Alexandre; Puga, João; Calado, Ricardo

    2012-08-16

    Bioprospecting for new marine natural products (NPs) has increased significantly over the last decades, leading to an unprecedented discovery of new molecules. Marine invertebrates have been the most important source of these NPs, with researchers commonly targeting particular taxonomic groups, marine regions and/or molecules from specific chemical groups. The present review focuses on new NPs identified from marine invertebrates between 2000 and 2009, and performs a detailed analysis on: (1) the chemical groups of these NPs; (2) the association of particular chemical groups to specific marine invertebrate taxa; and (3) the yielding of molecules from the same chemical group from organisms occurring in a particular geographic region. Our survey revealed an increasing number of new terpenoids being discovered between 2000 and 2009, contrasting with the decreasing trend in the discovery of new alkaloids and aliphatic molecules. Overall, no particular association was identified between marine invertebrate taxa and chemical groups of new NPs. Nonetheless, it is worth noting that most NPs recorded from cnidarians and mollusks were terpenoids, while most NPs identified in echinoderms were aliphatic compounds or carbohydrates. The geographical trends observed in our study do not support the idea of particular chemical groups of new NPs being associated with marine invertebrates from any specific geographical region, as NPs from different chemical groups were commonly distributed worldwide.

  6. Bioprospecting around Arctic islands: Marine bacteria as rich source of biocatalysts.

    Science.gov (United States)

    De Santi, Concetta; Altermark, Bjørn; de Pascale, Donatella; Willassen, Nils-Peder

    2016-03-01

    We have investigated the biotechnological potential of Arctic marine bacteria for their ability to produce a broad spectrum of cold-active enzymes. Marine bacteria exhibiting these features are of great interest for both fundamental research and industrial applications. Macrobiota, water and sediment samples have been collected during 2010 and 2011 expeditions around the Lofoten and Svalbard islands. Bacteria were isolated from this material and identified through 16S rRNA gene sequence analysis for the purpose of establishing a culture collection of marine Arctic bacteria. Herein, we present the functional screening for different extracellular enzymatic activities from 100 diversely chosen microbial isolates incubated at 4 and 20 °C. The production of esterase/lipase, DNase, and protease activities were revealed in 67, 53, and 56% of the strains, respectively, while 41, 23, 9, and 7% of the strains possessed amylase, chitinase, cellulase, and xylanase activities, respectively. Our findings show that phylogenetically diverse bacteria, including many new species, could be cultured from the marine arctic environment. The Arctic polar environment is still an untapped reservoir of biodiversity for bioprospecting.

  7. Bioprospecting of Marine Invertebrates for New Natural Products — A Chemical and Zoogeographical Perspective

    Directory of Open Access Journals (Sweden)

    Ricardo Calado

    2012-08-01

    Full Text Available Bioprospecting for new marine natural products (NPs has increased significantly over the last decades, leading to an unprecedented discovery of new molecules. Marine invertebrates have been the most important source of these NPs, with researchers commonly targeting particular taxonomic groups, marine regions and/or molecules from specific chemical groups. The present review focuses on new NPs identified from marine invertebrates between 2000 and 2009, and performs a detailed analysis on: (1 the chemical groups of these NPs; (2 the association of particular chemical groups to specific marine invertebrate taxa; and (3 the yielding of molecules from the same chemical group from organisms occurring in a particular geographic region. Our survey revealed an increasing number of new terpenoids being discovered between 2000 and 2009, contrasting with the decreasing trend in the discovery of new alkaloids and aliphatic molecules. Overall, no particular association was identified between marine invertebrate taxa and chemical groups of new NPs. Nonetheless, it is worth noting that most NPs recorded from cnidarians and mollusks were terpenoids, while most NPs identified in echinoderms were aliphatic compounds or carbohydrates. The geographical trends observed in our study do not support the idea of particular chemical groups of new NPs being associated with marine invertebrates from any specific geographical region, as NPs from different chemical groups were commonly distributed worldwide.

  8. Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production.

    Science.gov (United States)

    Mutanda, T; Ramesh, D; Karthikeyan, S; Kumari, S; Anandraj, A; Bux, F

    2011-01-01

    Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.

  9. Rhodolith beds in Brazil: a new potential habitat for marine bioprospection

    Directory of Open Access Journals (Sweden)

    Gilberto M. Amado-Filho

    2012-08-01

    Full Text Available Rhodoliths are the free-living forms of a number of nongeniculate coralline algae. Rhodolith beds are a common feature of subtidal environments and have been recognized as important carbonate producers and paleoenvironmental indicators, as well as recognized as habitat-forming species. The rhodolith structure provides a hard three-dimensional substrate serving as microhabitat for a wide range of biodiversity, including commercially importance species. The largest known latitudinal occurrence range of rhodolith beds ais on the Brazilian coastal shelf from 2°N to 25°S. Despite their importance for the Brazilian benthic communities, only in the last decade sampling efforts allowed a more comprehensive understanding of the beds' distribution, their structure and associated communities, as well as data concerning the influence of environmental factors on rhodolith bed structure and dynamics. In this work, we review the available information on the biodiversity associated with the recently described Brazilian rhodolith beds of the continental shelf and oceanic islands, focusing on the associated organisms with potential for bioprospection research.

  10. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  11. Glycosylation of hemoglobin and plasma proteins in petrochemical plant workers

    Energy Technology Data Exchange (ETDEWEB)

    Unrug, A.; Tomaszewski, L.

    1985-01-01

    The concentration of glycosylated hemoglobin and (plasma) proteins has been measured in 111 workers of 6 MZRiP departments in Plock and in 54 healthy people. In all subjects the mean concentrations of glycosylated hemoglobin and glycosylated plasma proteins have been in so called wide range of normal values. Significant shifts of glycosylated Hb concentrations have been found in two departments--those of ethylenederivatives and distillation. The concentration of glycosylated plasma proteins has been elevated only in workers of the Catalytic Processes Department.

  12. Current and future trends in metagenomics : Development of knowledge bases

    Science.gov (United States)

    Mori, Hiroshi; Yamada, Takuji; Kurokawa, Ken

    Microbes are essential for every part of life on Earth. Numerous microbes inhabit the biosphere, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects against surrounding environments. Metagenome analysis provides a radically new way of examining such complex microbial community without isolation or cultivation of individual bacterial community members. In this article, we present a brief discussion about a metagenomics and the development of knowledge bases, and also discuss about the future trends in metagenomics.

  13. An Experimental Metagenome Data Management and AnalysisSystem

    Energy Technology Data Exchange (ETDEWEB)

    Markowitz, Victor M.; Korzeniewski, Frank; Palaniappan, Krishna; Szeto, Ernest; Ivanova, Natalia N.; Kyrpides, Nikos C.; Hugenholtz, Philip

    2006-03-01

    The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity of microbial community, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context.

  14. Algal biogeography: metagenomics shows distribution of a picoplanktonic pelagophyte.

    Science.gov (United States)

    Raven, John A

    2012-09-11

    How can we determine the distribution of uncultured marine microorganisms? Targeted metagenomics has provided the complete chloroplast genome sequence, and the distribution, for a picoplanktonic pelagophyte alga.

  15. Insights into antibiotic resistance through metagenomic approaches.

    Science.gov (United States)

    Schmieder, Robert; Edwards, Robert

    2012-01-01

    The consequences of bacterial infections have been curtailed by the introduction of a wide range of antibiotics. However, infections continue to be a leading cause of mortality, in part due to the evolution and acquisition of antibiotic-resistance genes. Antibiotic misuse and overprescription have created a driving force influencing the selection of resistance. Despite the problem of antibiotic resistance in infectious bacteria, little is known about the diversity, distribution and origins of resistance genes, especially for the unculturable majority of environmental bacteria. Functional and sequence-based metagenomics have been used for the discovery of novel resistance determinants and the improved understanding of antibiotic-resistance mechanisms in clinical and natural environments. This review discusses recent findings and future challenges in the study of antibiotic resistance through metagenomic approaches.

  16. Genomics and metagenomics in medical microbiology.

    Science.gov (United States)

    Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre-Edouard

    2013-12-01

    Over the last two decades, sequencing tools have evolved from laborious time-consuming methodologies to real-time detection and deciphering of genomic DNA. Genome sequencing, especially using next generation sequencing (NGS) has revolutionized the landscape of microbiology and infectious disease. This deluge of sequencing data has not only enabled advances in fundamental biology but also helped improve diagnosis, typing of pathogen, virulence and antibiotic resistance detection, and development of new vaccines and culture media. In addition, NGS also enabled efficient analysis of complex human micro-floras, both commensal, and pathological, through metagenomic methods, thus helping the comprehension and management of human diseases such as obesity. This review summarizes technological advances in genomics and metagenomics relevant to the field of medical microbiology.

  17. Construction and screening of marine metagenomic libraries.

    Science.gov (United States)

    Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka; Schmitz, Ruth

    2010-01-01

    Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

  18. Metagenomics: advances in ecology and biotechnology.

    Science.gov (United States)

    Steele, Helen L; Streit, Wolfgang R

    2005-06-15

    This review highlights the significant advances which have been made in prokaryotic ecology and biotechnology due to the application of metagenomic techniques. It is now possible to link processes to specific microorganisms in the environment, such as the detection of a new phototrophic process in marine bacteria, and to characterise the metabolic cooperation which takes place in mixed species biofilms. The range of prokaryote derived products available for biotechnology applications is increasing rapidly. The knowledge gained from analysis of biosynthetic pathways provides valuable information about enzymology and allows engineering of biocatalysts for specific processes. The expansion of metagenomic techniques to include alternative heterologous hosts for gene expression and the development of sophisticated assays which enable screening of thousands of clones offers the possibility to find out even more valuable information about the prokaryotic world.

  19. Metagenomic analysis of soil microbial communities

    Directory of Open Access Journals (Sweden)

    Đokić Lidija

    2010-01-01

    Full Text Available Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity. .

  20. A retrospective metagenomics approach to studying Blastocystis.

    Science.gov (United States)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn; Stensvold, Christen Rune

    2015-07-01

    Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in coabundance gene groups, which are groups of genes that covary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew's correlation coefficient = -0.25, P metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities.

  1. Generating viral metagenomes from the coral holobiont

    Directory of Open Access Journals (Sweden)

    Karen Dawn Weynberg

    2014-05-01

    Full Text Available Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

  2. Involvement of Aberrant Glycosylation in Thyroid Cancer

    Directory of Open Access Journals (Sweden)

    Eiji Miyoshi

    2010-01-01

    Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

  3. A novel functional role of collagen glycosylation

    DEFF Research Database (Denmark)

    Jürgensen, Henrik J; Madsen, Daniel H; Ingvarsen, Signe

    2011-01-01

    , the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose...... receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens....... By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate...

  4. Protein glycosylation in Archaea: sweet and extreme.

    Science.gov (United States)

    Calo, Doron; Kaminski, Lina; Eichler, Jerry

    2010-09-01

    While each of the three domains of life on Earth possesses unique traits and relies on characteristic biological strategies, some processes are common to Eukarya, Bacteria and Archaea. Once believed to be restricted to Eukarya, it is now clear that Bacteria and Archaea are also capable of performing N-glycosylation. However, in contrast to Bacteria, where this posttranslational modification is still considered a rare event, numerous species of Archaea, isolated from a wide range of environments, have been reported to contain proteins bearing Asn-linked glycan moieties. Analysis of the chemical composition of the Asn-linked polysaccharides decorating archaeal proteins has, moreover, revealed the use of a wider variety of sugar subunits than seen in either eukaryal or bacterial glycoproteins. Still, although first reported some 30 years ago, little had been known of the steps or components involved in the archaeal version of this universal posttranslational modification. Now, with the availability of sufficient numbers of genome sequences and the development of appropriate experimental tools, molecular analysis of archaeal N-glycosylation pathways has become possible. Accordingly using halophilic, methanogenic and thermophilic model species, insight into the biosynthesis and attachment of N-linked glycans decorating archaeal glycoproteins is starting to amass. In this review, current understanding of N-glycosylation in Archaea is described.

  5. Diversity in protein glycosylation among insect species.

    Directory of Open Access Journals (Sweden)

    Gianni Vandenborre

    Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

  6. Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues; Pedezzi, Rafael; Souto, Tatiane Beltramini

    2017-04-01

    Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

  7. Bioprospecting from marine sediments of New Brunswick, Canada: exploring the relationship between total bacterial diversity and actinobacteria diversity.

    Science.gov (United States)

    Duncan, Katherine; Haltli, Bradley; Gill, Krista A; Kerr, Russell G

    2014-02-13

    Actinomycetes are an important resource for the discovery of natural products with therapeutic properties. Bioprospecting for actinomycetes typically proceeds without a priori knowledge of the bacterial diversity present in sampled habitats. In this study, we endeavored to determine if overall bacterial diversity in marine sediments, as determined by 16S rDNA amplicon pyrosequencing, could be correlated with culturable actinomycete diversity, and thus serve as a powerful tool in guiding future bioprospecting efforts. Overall bacterial diversity was investigated in eight marine sediments from four sites in New Brunswick, Canada, resulting in over 44,000 high quality sequences (x = 5610 per sample). Analysis revealed all sites exhibited significant diversity (H' = 5.4 to 6.7). Furthermore, statistical analysis of species level bacterial communities (D = 0.03) indicated community composition varied according to site and was strongly influenced by sediment physiochemical composition. In contrast, cultured actinomycetes (n = 466, 98.3% Streptomyces) were ubiquitously distributed among all sites and distribution was not influenced by sediment composition, suggesting that the biogeography of culturable actinomycetes does not correlate with overall bacterial diversity in the samples examined. These actinomycetes provide a resource for future secondary metabolite discovery, as exemplified by the antimicrobial activity observed from preliminary investigation.

  8. Bioprospecting optimal phenology for bioactive molecules in native golden yellow Pleurotus citrinopileatus Singer

    Institute of Scientific and Technical Information of China (English)

    Godfrey Nattoh; Erastus Gatebe; Fredrick Musieba; Julius Mathara

    2016-01-01

    Objective: To bioprospect optimal phenological phases as source of novel molecules from native golden yellow Pleurotus citrinopileatus across four phenologies in both aqueous and ethanol extracts, and identify novel molecules responsible for these activities.Methods: Standard qualitative assay, Folin–Ciocalteu assay; aluminium chloride spectrophotometric, 2, 2-diphenyl-1-picrylhydrazyl, 2, 20-azinobis(3-ethylbenzothiazoline-6-suslfonic acid, ferricyanide reducing antioxidant power were used to determine total flavonoid, polyphenols, radical scavenging, and reducing power. Spectrophotometric methods were used for lycopene, b-carotene, and total carotenoids, while liquid chromatography quadrupole time of flight mass spectrometry was used for identification and comparative quantitation of polyphenols and flavonoids across the four phenological states. Chem Spider? database was used for the identification of compounds based on their empirical formula, accurate mass and literature review of previously reported compounds in mushroom.Results: Primordial phases exhibited higher contents of secondary metabolites than mature basidiocarps. Polyphenols content differed across physiological phases with primordials exhibiting significant high contents(P < 0.05) [(13.803 ± 0.797) mg gallic acid equivalent/g dry weight]. Distribution of total flavonoids was significantly different(P < 0.05) across physiological states and ranged from(3.311 ± 0.730) to(14.824 ± 0.890) mg quercetin equivalent g dry weight. Ten polyphenol acids and seven flavonoids compounds identified varied across these phases with primordials exhibiting relatively high peak areas. Total antioxidant activities showed a positive correlation with total polyphenols(r = 0.969;P < 0.05) and total flavonoids(r = 0.960; P < 0.05) across these phenologies.Conclusions: These findings provide evidence that primordials of golden yellow mushroom as opposed to their fruiting bodies are potent sources of bioactive health

  9. SmashCommunity: A metagenomic annotation and analysis tool

    DEFF Research Database (Denmark)

    Arumugam, Manimozhiyan; Harrington, Eoghan D; Foerstner, Konrad U;

    2010-01-01

    the quantitative phylogenetic and functional compositions of metagenomes, to compare compositions of multiple metagenomes and to produce intuitive visual representations of such analyses. AVAILABILITY: SmashCommunity is freely available at http://www.bork.embl.de/software/smash CONTACT: bork@embl.de....

  10. Unlocking the potential of metagenomics through replicated experimental design

    NARCIS (Netherlands)

    Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; Van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

    2012-01-01

    Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal s

  11. Genomic standards consortium workshop: metagenomics, metadata and metaanalysis (M3).

    Science.gov (United States)

    Sterk, Peter; Hirschman, Lynette; Field, Dawn; Wooley, John

    2010-01-01

    The M3 workshop has, as its primary focus, the rapidly growing area of metagenomics, including the metadata standards and the meta-analysis approaches needed to organize, process and interpret metagenomics data. The PSB Workshop builds on the first M3 meeting, a Special Interest Group (SIG) meeting at ISMB 2009, organized by the Genomics Standards Consortium.

  12. Functional assignment of metagenomic data: challenges and applications.

    Science.gov (United States)

    Prakash, Tulika; Taylor, Todd D

    2012-11-01

    Metagenomic sequencing provides a unique opportunity to explore earth's limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely 'In a given environment, among the microbes present, what are they doing, and how are they doing it?' Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.

  13. Antibiotic Resistome: Improving Detection and Quantification Accuracy for Comparative Metagenomics.

    Science.gov (United States)

    Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

    2016-04-01

    The unprecedented rise of life-threatening antibiotic resistance (AR), combined with the unparalleled advances in DNA sequencing of genomes and metagenomes, has pushed the need for in silico detection of the resistance potential of clinical and environmental metagenomic samples through the quantification of AR genes (i.e., genes conferring antibiotic resistance). Therefore, determining an optimal methodology to quantitatively and accurately assess AR genes in a given environment is pivotal. Here, we optimized and improved existing AR detection methodologies from metagenomic datasets to properly consider AR-generating mutations in antibiotic target genes. Through comparative metagenomic analysis of previously published AR gene abundance in three publicly available metagenomes, we illustrate how mutation-generated resistance genes are either falsely assigned or neglected, which alters the detection and quantitation of the antibiotic resistome. In addition, we inspected factors influencing the outcome of AR gene quantification using metagenome simulation experiments, and identified that genome size, AR gene length, total number of metagenomics reads and selected sequencing platforms had pronounced effects on the level of detected AR. In conclusion, our proposed improvements in the current methodologies for accurate AR detection and resistome assessment show reliable results when tested on real and simulated metagenomic datasets.

  14. Online semi-supervised learning: algorithm and application in metagenomics

    NARCIS (Netherlands)

    S. Imangaliyev; B. Keijser; W. Crielaard; E. Tsivtsivadze

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key role in metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm an

  15. Online Semi-Supervised Learning: Algorithm and Application in Metagenomics

    NARCIS (Netherlands)

    Imangaliyev, S.; Keijser, B.J.F.; Crielaard, W.; Tsivtsivadze, E.

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key rolein metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm and

  16. Recovering full-length viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2015-01-01

    textabstractInfectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral

  17. Cross-cutting activities: Soil quality and soil metagenomics

    OpenAIRE

    Peter P. Motavalli; Garrett, Karen A.

    2008-01-01

    This presentation reports on the work of the SANREM CRSP cross-cutting activities "Assessing and Managing Soil Quality for Sustainable Agricultural Systems" and "Soil Metagenomics to Construct Indicators of Soil Degradation." The introduction gives an overview of the extensiveness of soil degradation globally and defines soil quality. The objectives of the soil quality cross cutting activity are: CCRA-4 (Soil Metagenomics)

  18. Which Microbial Communities Are Present? Sequence-Based Metagenomics

    Science.gov (United States)

    Caffrey, Sean M.

    The use of metagenomic methods that directly sequence environmental samples has revealed the extraordinary microbial diversity missed by traditional culture-based methodologies. Therefore, to develop a complete and representative model of an environment's microbial community and activities, metagenomic analysis is an essential tool.

  19. MetaSim: a sequencing simulator for genomics and metagenomics.

    Directory of Open Access Journals (Sweden)

    Daniel C Richter

    Full Text Available BACKGROUND: The new research field of metagenomics is providing exciting insights into various, previously unclassified ecological systems. Next-generation sequencing technologies are producing a rapid increase of environmental data in public databases. There is great need for specialized software solutions and statistical methods for dealing with complex metagenome data sets. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the development and improvement of metagenomic tools and the planning of metagenomic projects, we introduce a sequencing simulator called MetaSim. Our software can be used to generate collections of synthetic reads that reflect the diverse taxonomical composition of typical metagenome data sets. Based on a database of given genomes, the program allows the user to design a metagenome by specifying the number of genomes present at different levels of the NCBI taxonomy, and then to collect reads from the metagenome using a simulation of a number of different sequencing technologies. A population sampler optionally produces evolved sequences based on source genomes and a given evolutionary tree. CONCLUSIONS/SIGNIFICANCE: MetaSim allows the user to simulate individual read datasets that can be used as standardized test scenarios for planning sequencing projects or for benchmarking metagenomic software.

  20. Metagenomics: Retrospect and Prospects in High Throughput Age

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2015-01-01

    Full Text Available In recent years, metagenomics has emerged as a powerful tool for mining of hidden microbial treasure in a culture independent manner. In the last two decades, metagenomics has been applied extensively to exploit concealed potential of microbial communities from almost all sorts of habitats. A brief historic progress made over the period is discussed in terms of origin of metagenomics to its current state and also the discovery of novel biological functions of commercial importance from metagenomes of diverse habitats. The present review also highlights the paradigm shift of metagenomics from basic study of community composition to insight into the microbial community dynamics for harnessing the full potential of uncultured microbes with more emphasis on the implication of breakthrough developments, namely, Next Generation Sequencing, advanced bioinformatics tools, and systems biology.

  1. Glycosylation in HIV-1 envelope glycoprotein and its biological implications

    KAUST Repository

    Ho, Yung Shwen

    2013-08-01

    Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

  2. N-glycosylation in Haloferax volcanii: adjusting the sweetness

    OpenAIRE

    Eichler, Jerry; Arbiv, Adi; Cohen-Rosenzweig, Chen; Kaminski, Lina; Kandiba, Lina; Konrad, Zvia

    2013-01-01

    Long believed to be restricted to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target protein asparagine residues. Still, it is only in the last decade that pathways of N-glycosylation in Archaea have been delineated. In the haloarchaeon Haloferax volcanii, a series of Agl (archaeal glycosylation) proteins is responsible for the addition of an N-linked pentasaccharide to modified proteins, including the ...

  3. N-glycosylation in Haloferax volcanii: Adjusting the sweetness

    OpenAIRE

    Jerry eEichler; Adi eArbiv; Chen eCohen-Rosenzweig; Lina eKaminski; Lina eKandiba; Zvia eKonrad

    2013-01-01

    Long believed to be restricted to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target protein asparagine residues. Still, it is only in the last decade that pathways of N-glycosylation in Archaea have been delineated. In the haloarchaeon Haloferax volcanii, a series of Agl (archaeal glycosylation) proteins is responsible for the addition of an N-linked pentasaccharide to modified proteins, including the ...

  4. [Congenital disorder of glycosylation type Ia (CDG Ia) - underdiagnosed entity?].

    Science.gov (United States)

    Sätilä, Heli; Kuusela, Anna-Leena; Pietilä, Kati; Niinikoski, Harri; Keskinen, Päivi

    2016-01-01

    Congenital disorders of glycosylation (CDG) are a relatively recently identified group of multisystem disorders caused by defective glycosylation of N-glycosylated proteins. They mainly involve the central and peripheral nervous system, but other organ systems are involved as well. Type CDG Ia accounts for over 80% of cases, characterized by decreased activity of the enzyme phosphomannomutase caused by mutations in chromosome 16 PMM2 gene. Treatment of CDG Ia remains symptomatic.

  5. Links between CD147 Function, Glycosylation, and Caveolin-1

    OpenAIRE

    Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

    2004-01-01

    Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

  6. Cloning, expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene (Mxyl retrieved from compost-soil metagenome.

    Directory of Open Access Journals (Sweden)

    Digvijay Verma

    Full Text Available BACKGROUND: The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. METHODOLOGY/PRINCIPAL FINDINGS: Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1 was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids displayed homology with glycosyl hydrolase (GH family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3. The recombinant xylanase (rMxyl exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. CONCLUSION/SIGNIFICANCE: This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues.

  7. Metagenomics: an inexhaustible access to nature's diversity.

    Science.gov (United States)

    Langer, Martin; Gabor, Esther M; Liebeton, Klaus; Meurer, Guido; Niehaus, Frank; Schulze, Renate; Eck, Jürgen; Lorenz, Patrick

    2006-01-01

    The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications. Due to a limitation in the cultivability of microbes living in certain habitats, technologies have to be established which give access to the enormous resource of uncultivated microbial diversity. Metagenomics promises to provide new and diverse enzymes and biocatalysts as well as bioactive molecules and has the potential to make industrial biotechnology an economic, sustainable success.

  8. Hemoglobin E disease and glycosylated hemoglobin

    OpenAIRE

    Niharika Yedla; Mohammad Shafi Kuchay; Ambrish Mithal

    2015-01-01

    Glycosylated hemoglobin (HbA1C) is a routinely measured parameter to monitor long-term glycemic control in people with diabetes mellitus. The presence of hemoglobin (Hb) variants can affect the accuracy of HbA1C methods. Hb E variant is the most common Hb variant in South-east Asia and North-east India. In the presence of Hb E, HbA1C may not be detectable by ion-exchange chromatography (high-pressure liquid chromatography), but may be estimated by immunoassay technique and boronate affinity c...

  9. BIOPROSPECTING AS A POSSIBLE DEVELOPMENT MECHANISM FOR COLOMBIA Bioprospección como posible mecanismo de desarrollo para Colombia

    Directory of Open Access Journals (Sweden)

    LUZ MARINA MELGAREJO

    2013-04-01

    Full Text Available Bioprospecting has been on the agenda for discussion by the government, the academic community, businesses and other pertinent actors for many years now; it is an integral topic representing a potential approach to exploit biodiversity to the fullest. This document presents some definitions of what bioprospecting is (some restrictive, others with a broader perspective, depending on different approaches, actors, target markets and countries in the context of a mechanism for development and cooperation concerning the construction of capacities, advantages and disadvantages. It was found that bioprospecting in Colombia has been developing from a broad perspective (systematic study of biological-genetic resources, transformation into a product which has led to strengthening some value chains, small- and/or large-scale marketing and some advances in intellectual property rights and the distribution of benefits in order to provide greater benefits for the nation.La bioprospección ha sido durante varios años tema de discusión por los gobiernos, la comunidad académica, las empresas, y otros actores que se integran en el proceso, como potencial de acercamiento y uso de la diversidad biológica. A través del presente documento se presentan algunas definiciones de lo que es la bioprospección (algunas restrictivas otras de perspectiva amplia, dependientes de diferentes aproximaciones, actores, blanco de mercado, contexto del país, entre otros, la bioprospección como mecanismo de desarrollo y cooperación para la construcción de capacidades, ventajas y desventajas. Se encuentra que en Colombia se ha venido desarrollando bioprospección desde la perspectiva amplia (estudio sistemático del recurso biológico-genético, transformación en un producto lo que ha permitido el fortalecimiento de algunas cadenas de valor, comercialización a pequeña o gran escala y algunos avances en temas de propiedad intelectual y distribución de beneficios lo cual

  10. Metagenomic islands of hyperhalophiles: the case of Salinibacter ruber

    Directory of Open Access Journals (Sweden)

    Rohwer Forest

    2009-12-01

    Full Text Available Abstract Background Saturated brines are extreme environments of low diversity. Salinibacter ruber is the only bacterium that inhabits this environment in significant numbers. In order to establish the extent of genetic diversity in natural populations of this microbe, the genomic sequence of reference strain DSM 13855 was compared to metagenomic fragments recovered from climax saltern crystallizers and obtained with 454 sequencing technology. This kind of analysis reveals the presence of metagenomic islands, i.e. highly variable regions among the different lineages in the population. Results Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurring S. ruber cells. These metagenomic islands showed evidence of extensive genomic corruption with atypically low GC content, low coding density, high numbers of pseudogenes and short hypothetical proteins. A detailed analysis of island gene content showed that the genes in metagenomic island 1 code for cell surface polysaccharides. The strain-specific genes of metagenomic island 2 were found to be involved in biosynthesis of cell wall polysaccharide components. Finally, metagenomic island 3 was rich in DNA related enzymes. Conclusion The genomic organisation of S. ruber variable genomic regions showed a number of convergences with genomic islands of marine microbes studied, being largely involved in variable cell surface traits. This variation at the level of cell envelopes in an environment devoid of grazing pressure probably reflects a global strategy of bacteria to escape phage predation.

  11. [Research in metagenomics and its applications in translational medicine].

    Science.gov (United States)

    Jiahuan, Chen; Zheng, Sun; Xiaojun, Wang; Xiaoquan, Su; Kang, Ning

    2015-07-01

    Humans are born with microbiota, which have accompanied us through our life-span. There is an important symbiotic relationship between us and the microbial communities, thus microbial communities are of great importance to our health. All genomic information within this microbiota is referered to as "metagenomics" (also referred to as "human's second genome"). The analysis of high throughput metagenomic data generated from biomedical experiments would provide new approaches for translational research, and it have several applications in clinics. With the help of next generation sequencing technology and the emerging metagenomic approach (analysis of all genomic information in microbiota as a whole), we can overcome the pitfalls of tedious traditional method of isolation and cultivation of single microbial species. The metagenomic approach can also help us to analyze the whole microbial community efficiently and offer deep insights in human-microbe relationships as well as new ideas on many biomedical problems. In this review, we summarize frontiers in metagenomic research, including new concepts and methods. Then, we focus on the applications of metagenomic research in medical researches and clinical applications in recent years, which would clearly show the importance of metagenomic research in the field of translational medicine.

  12. Bioprospecting finds the toughest biological material: extraordinary silk from a giant riverine orb spider.

    Directory of Open Access Journals (Sweden)

    Ingi Agnarsson

    into the river. This hypothesis predicts that rapid change in material properties of silk co-occurred with ecological shifts within the genus, and can thus be tested by combining material science, behavioral observations, and phylogenetics. Our findings highlight the potential benefits of natural history-informed bioprospecting to discover silks, as well as other materials, with novel and exceptional properties to serve as models in biomimicry.

  13. Bio-prospecting of Plants and Marine Organisms in Saudi Arabia for New Potential Bioactivity

    KAUST Repository

    Hajjar, Dina A.

    2016-12-08

    The natural resources offer a unique opportunity for the discovery of active compounds, due to the complexity and biodiversity of their chemical structures. Natural resources have been used as medicines throughout human history. Saudi Arabia’s natural resources, for instance its terrestrial medicinal plants and the Red Sea sponges, have not been extensively investigated with regard to their biological activities. To better identify the diversity of compounds with bioactive potential, new techniques are also necessary in order to improve the drug discovery path. This study comprises three sections. The first section examines Juniperus phoenicea (Arar), Anastatica hierochuntica (Kaff Maryam) and Citrullus colocynthis (Hanzal); these herbal plants were screened for potential bioactivity using a newly developed pipeline based on a high-content screening technique. We report a new cell-based high-throughput phenotypic screening for the bio-prospecting of unknown natural products from Saudi Arabian plants, in order to reveal their biological activities. The second section investigates Avicennia marina plants, screened for reverse transcriptase anti-HIV bioactivity using biochemical assay. Image-based high-content screening with a set of cellular stains was used to investigate the phenotypic results of toxicity and cell cycle arrest. The third section considers the isolation of Actinomycetes from Red Sea Sponges. Actinomycetes bacterial isolates were tested for bioactivity against West Nile Virus NS3 Protease. Analytical chemical techniques such as liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) were used to gain more understanding of the possible chemical compounds responsible for this bioactivity. Overall, the aim of this work is to investigate the potential bioactive effect of several Saudi Arabian plants and Red Sea sponges against cancer cells and viral infections. Our study

  14. The cystic fibrosis lower airways microbial metagenome

    Science.gov (United States)

    Moran Losada, Patricia; Chouvarine, Philippe; Dorda, Marie; Hedtfeld, Silke; Mielke, Samira; Schulz, Angela; Wiehlmann, Lutz

    2016-01-01

    Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes. PMID:27730195

  15. Identifying personal microbiomes using metagenomic codes.

    Science.gov (United States)

    Franzosa, Eric A; Huang, Katherine; Meadow, James F; Gevers, Dirk; Lemon, Katherine P; Bohannan, Brendan J M; Huttenhower, Curtis

    2015-06-02

    Community composition within the human microbiome varies across individuals, but it remains unknown if this variation is sufficient to uniquely identify individuals within large populations or stable enough to identify them over time. We investigated this by developing a hitting set-based coding algorithm and applying it to the Human Microbiome Project population. Our approach defined body site-specific metagenomic codes: sets of microbial taxa or genes prioritized to uniquely and stably identify individuals. Codes capturing strain variation in clade-specific marker genes were able to distinguish among 100s of individuals at an initial sampling time point. In comparisons with follow-up samples collected 30-300 d later, ∼30% of individuals could still be uniquely pinpointed using metagenomic codes from a typical body site; coincidental (false positive) matches were rare. Codes based on the gut microbiome were exceptionally stable and pinpointed >80% of individuals. The failure of a code to match its owner at a later time point was largely explained by the loss of specific microbial strains (at current limits of detection) and was only weakly associated with the length of the sampling interval. In addition to highlighting patterns of temporal variation in the ecology of the human microbiome, this work demonstrates the feasibility of microbiome-based identifiability-a result with important ethical implications for microbiome study design. The datasets and code used in this work are available for download from huttenhower.sph.harvard.edu/idability.

  16. Metagenome Fragment Classification Using -Mer Frequency Profiles

    Directory of Open Access Journals (Sweden)

    Gail Rosen

    2008-01-01

    Full Text Available A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique -mer frequency profiles of 635 microbial genomes publicly available as of February 2008. These profiles are used to train a naive Bayes classifier (NBC that can be used to identify the genome of any fragment. We show that our method is comparable to BLAST for small 25 bp fragments but does not have the ambiguity of BLAST's tied top scores. We demonstrate that this approach is scalable to identify any fragment from hundreds of genomes. It also performs quite well at the strain, species, and genera levels and achieves strain resolution despite classifying ubiquitous genomic fragments (gene and nongene regions. Cross-validation analysis demonstrates that species-accuracy achieves 90% for highly-represented species containing an average of 8 strains. We demonstrate that such a tool can be used on the Sargasso Sea dataset, and our analysis shows that NBC can be further enhanced.

  17. Large-scale screens of metagenomic libraries.

    Science.gov (United States)

    Pham, Vinh D; Palden, Tsultrim; DeLong, Edward F

    2007-01-01

    Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

  18. Genovo: De Novo Assembly for Metagenomes

    Science.gov (United States)

    Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

    Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

  19. Back to the Future of Soil Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Nesme, Joseph; Achouak, Wafa; Agathos, Spiros N.; Bailey, Mark; Baldrian, Petr; Brunel, Dominique; Frostegård, Åsa; Heulin, Thierry; Jansson, Janet K.; Jurkevitch, Edouard; Kruus, Kristiina L.; Kowalchuk, George A.; Lagares, Antonio; Lappin-Scott, Hilary M.; Lemanceau, Philippe; Le Paslier, Denis; Mandic-Mulec, Ines; Murrell, J. Colin; Myrold, David D.; Nalin, Renaud; Nannipieri, Paolo; Neufeld, Josh D.; O' Gara, Fergal; Parnell, John J.; Pühler, Alfred; Pylro, Victor; Ramos, Juan L.; Roesch, Luiz F. W.; Schloter, Michael; Schleper, Christa; Sczyrba, Alexander; Sessitsch, Angela; Sjöling, Sara; Sørensen, Jan; Sørensen, Søren J.; Tebbe, Christoph C.; Topp, Edward; Tsiamis, George; van Elsas, Jan Dirk; van Keulen, Geertje; Widmer, Franco; Wagner, Michael; Zhang, Tong; Zhang, Xiaojun; Zhao, Liping; Zhu, Yong-Guan; Vogel, Timothy M.; Simonet, Pascal

    2016-02-10

    Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek (Table 1). The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?

  20. Metagenomic search strategies for interactions among plants and multiple microbes

    Directory of Open Access Journals (Sweden)

    Ulrich Karl Melcher

    2014-06-01

    Full Text Available Plants harbor multiple microbes. Metagenomics can facilitate understanding of the significance, for the plant, of the microbes and of the interactions among them. However, current approaches to metagenomic analysis of plants are computationally time-consuming. Efforts to speed the discovery process include improvement of computational speed, condensing the sequencing reads into smaller datasets before BLAST searches, simplifying the target database of BLAST searches, and flipping the roles of metagenomic and reference datasets. The latter is exemplified by the E-probe diagnostic nucleic acid analysis (EDNA approach originally devised for improving analysis during plant quarantine.

  1. Enhancing metagenomics investigations of microbial interactions with biofilm technology.

    Science.gov (United States)

    McLean, Robert J C; Kakirde, Kavita S

    2013-11-11

    Investigations of microbial ecology and diversity have been greatly enhanced by the application of culture-independent techniques. One such approach, metagenomics, involves sample collections from soil, water, and other environments. Extracted nucleic acids from bulk environmental samples are sequenced and analyzed, which allows microbial interactions to be inferred on the basis of bioinformatics calculations. In most environments, microbial interactions occur predominately in surface-adherent, biofilm communities. In this review, we address metagenomics sampling and biofilm biology, and propose an experimental strategy whereby the resolving power of metagenomics can be enhanced by incorporating a biofilm-enrichment step during sample acquisition.

  2. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

  3. The Synthesis of Glycosyl Phosphite-Pt(Ⅱ) Complexes

    Institute of Scientific and Technical Information of China (English)

    Ling Hua CAO; Hong Yun GAO; Chuan Jian ZHOU; Yu Ting LIU

    2004-01-01

    Ethylene glycol phosphorochloridite 1 or catechol phosphorochloridite 2 reacted with isopropylidene derivatives of D-glucose, D-galactose, D-mannose and D-fructose, a series of glycosyl phosphites were obtained. These glycosyl phosphites form optically active complexes with simple Pt (Ⅱ) salts. Pt (Ⅱ) is coordinated to the phosphorus atom, most of the metal complexes are quite stable.

  4. Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

    DEFF Research Database (Denmark)

    Spahn, Philipp N.; Hansen, Anders Holmgaard; Kol, Stefan;

    2016-01-01

    biogenesis. This usually implies that costly and time-consuming experimentation is required for clone identification and optimization of biosimilar glycosylation. Here, we describe a computational method that utilizes a Markov model of glycosylation to predict optimal glycoengineering strategies to obtain...

  5. IgG glycosylation changes and MBL2 polymorphisms

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Jacobsen, Søren; Abrahams, Jodie L;

    2012-01-01

    To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA).......To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA)....

  6. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

    2003-01-01

    specifically on glycosylation of either one or the other of the utilised sites. The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites. Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1......-inactivating compounds of potential clinical importance....

  7. Bioprospection in Brazil and Mexico, a new El Dorado?: Between the Instability of Practices and the Permanence of Representations

    Directory of Open Access Journals (Sweden)

    Geoffroy Filoche

    2011-01-01

    Full Text Available Because of their exceptional biodiversity, Brazil and Mexico seemed to be the ideal candidates for the valorization of their genetic resources, as promoted by the Convention on Biological Diversity (CBD. But almost twenty years after the Rio Conference, the actual results of this valorization are disappointing. It can be explained either by the slowness in adopting domestic laws translating the CBD or by the difficulties in controlling and regulating very complex and unstable practices and objects. This leads to put into question the pertinence of the CDB framework, along with its categories and mechanisms. In spite of the relative obsolescence of this framework and the emergence of new environmental issues, which appear as more important, the question of the access to genetic resources and their exploitation through contracts of bioprospection is still being debated because of the various symbolic issues at stake, such as national sovereignty and the defense of biological and cultural diversity.

  8. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...... important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...

  9. Analysis and comparison of very large metagenomes with fast clustering and functional annotation

    Directory of Open Access Journals (Sweden)

    Li Weizhong

    2009-10-01

    Full Text Available Abstract Background The remarkable advance of metagenomics presents significant new challenges in data analysis. Metagenomic datasets (metagenomes are large collections of sequencing reads from anonymous species within particular environments. Computational analyses for very large metagenomes are extremely time-consuming, and there are often many novel sequences in these metagenomes that are not fully utilized. The number of available metagenomes is rapidly increasing, so fast and efficient metagenome comparison methods are in great demand. Results The new metagenomic data analysis method Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP was developed using an ultra-fast sequence clustering algorithm, fast protein family annotation tools, and a novel statistical metagenome comparison method that employs a unique graphic interface. RAMMCAP processes extremely large datasets with only moderate computational effort. It identifies raw read clusters and protein clusters that may include novel gene families, and compares metagenomes using clusters or functional annotations calculated by RAMMCAP. In this study, RAMMCAP was applied to the two largest available metagenomic collections, the "Global Ocean Sampling" and the "Metagenomic Profiling of Nine Biomes". Conclusion RAMMCAP is a very fast method that can cluster and annotate one million metagenomic reads in only hundreds of CPU hours. It is available from http://tools.camera.calit2.net/camera/rammcap/.

  10. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

    DEFF Research Database (Denmark)

    Lezyk, Mateusz Jakub; Jers, Carsten; Kjaerulff, Louise;

    2016-01-01

    This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil......-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para......-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self...

  11. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    Science.gov (United States)

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-09-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

  12. Isolation and characterization of a non-specific endoglucanase from a metagenomic library of goat rumen.

    Science.gov (United States)

    Cheng, Jianbo; Huang, Shuai; Jiang, Haiqin; Zhang, Yunhai; Li, Lvmu; Wang, Juhua; Fan, Caiyun

    2016-01-01

    A cellulase gene (cel28a) was isolated from a rumen microbial metagenome library of goat rumen microorganisms, cloned into E. coli, and expressed in active form. The gene has a length of 1596 bp obtained using a genome walking Kit and encodes a protein of 509 amino acids with a calculated MW of 55 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolase family 5 (GH5). The expressed protein showed activity toward carboxymethylcellulose (CMC) and xylan, suggesting non-specific endoglucanase activity. The optimal conditions for endoglucanase and xylanase activities were 50 °C and pH 5.0. The metal ions (Ca(2+), Fe(2+), Mn(2+) and Co(2+)) stimulated the cellulase activity of cel28a, while the other metal ions and chemicals (Ni(2+), Mg(2+), Zn(2+), Cu(2+), SDS and EDTA) inhibited the cellulase activity. Further examination of substrate preference showed a higher activity with CMC, oat spelt xylan and birchwood xylan than with filter paper and microcrystalline cellulose, again suggesting that the protein was an endoglucanase with xylanase activity.

  13. Trends in the discovery of new marine natural products from invertebrates over the last two decades--where and what are we bioprospecting?

    Directory of Open Access Journals (Sweden)

    Miguel Costa Leal

    Full Text Available It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990-2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively. Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no

  14. Trends in the discovery of new marine natural products from invertebrates over the last two decades--where and what are we bioprospecting?

    Science.gov (United States)

    Leal, Miguel Costa; Puga, João; Serôdio, João; Gomes, Newton C M; Calado, Ricardo

    2012-01-01

    It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI) over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990-2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively). Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no commercial value

  15. Protein N-glycosylation in Archaea: defining Haloferax volcanii genes involved in S-layer glycoprotein glycosylation.

    Science.gov (United States)

    Abu-Qarn, Mehtap; Eichler, Jerry

    2006-07-01

    In this study, characterization of the N-glycosylation process in the haloarchaea Haloferax volcanii was undertaken. Initially, putative Hfx. volcanii homologues of genes involved in eukaryal or bacterial N-glycosylation were identified by bioinformatics. Reverse transcription polymerase chain reaction (RT-PCR) confirmed that the proposed N-glycosylation genes are transcribed, indicative of true proteins being encoded. Where families of related gene sequences were detected, differential transcription of family members under a variety of physiological and environmental conditions was shown. Gene deletions point to certain genes, like alg11, as being essential yet revealed that others, such as the two versions of alg5, are not. Deletion of alg5-A did, however, lead to slower growth and interfered with surface (S)-layer glycoprotein glycosylation, as detected by modified migration on SDS-PAGE and glycostaining approaches. As deletion of stt3, the only component of the oligosaccharide transferase complex detected in Archaea, did not affect cell viability, it appears that N-glycosylation is not essential in Hfx. volcanii. Deletion of stt3 did, nonetheless, hinder both cell growth and S-layer glycoprotein glycosylation. Thus, with genes putatively involved in Hfx. volcanii protein glycosylation identified and the ability to address the roles played by the encoded polypeptides in modifying a reporter glycoprotein, the steps of the archaeal N-glycosylation pathway can be defined.

  16. Efficient Stereoselective Glycosylations of Alcohols by Sugar Perpivalates: The First Use of 1-O-Pivaloylated Glycosyl Donors

    NARCIS (Netherlands)

    Pukin, A.V.; Zuilhof, H.

    2009-01-01

    1-O-Pivaloyl glycosides were shown to be efficient glycosyl donors by using the perpivaloylated derivatives of lactose, galactose and glucose in the direct ZnCl2-promoted glycosylations of various alcohols. The corresponding glycosides were isolated in good yields and ß-selectivity

  17. Comparative Metagenomics of Freshwater Microbial Communities

    Energy Technology Data Exchange (ETDEWEB)

    Hemme, Chris; Deng, Ye; Tu, Qichao; Fields, Matthew; Gentry, Terry; Wu, Liyou; Tringe, Susannah; Watson, David; He, Zhili; Hazen, Terry; Tiedje, James; Rubin, Eddy; Zhou, Jizhong

    2010-05-17

    Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxin resistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid and less leaky in the healthy groundwater system. FW301 encodes many (~;;160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to be capable of CO2 fixation via the reductive carboxylase (reverse TCA) cycle and possibly acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. FW301 encodes a complete set of B12 biosynthesis pathway at high abundance suggesting the use of sodium gradients for energy production in the healthy groundwater community. Overall

  18. Assessment of metagenomic assembly using simulated next generation sequencing data

    DEFF Research Database (Denmark)

    Mende, Daniel R; Waller, Alison S; Sunagawa, Shinichi;

    2012-01-01

    Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators...... with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved....... For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length...

  19. Identifying Differentially Abundant Metabolic Pathways in Metagenomic Datasets

    Science.gov (United States)

    Liu, Bo; Pop, Mihai

    Enabled by rapid advances in sequencing technology, metagenomic studies aim to characterize entire communities of microbes bypassing the need for culturing individual bacterial members. One major goal of such studies is to identify specific functional adaptations of microbial communities to their habitats. Here we describe a powerful analytical method (MetaPath) that can identify differentially abundant pathways in metagenomic data-sets, relying on a combination of metagenomic sequence data and prior metabolic pathway knowledge. We show that MetaPath outperforms other common approaches when evaluated on simulated datasets. We also demonstrate the power of our methods in analyzing two, publicly available, metagenomic datasets: a comparison of the gut microbiome of obese and lean twins; and a comparison of the gut microbiome of infant and adult subjects. We demonstrate that the subpathways identified by our method provide valuable insights into the biological activities of the microbiome.

  20. Functional metagenomics for the investigation of antibiotic resistance.

    Science.gov (United States)

    Mullany, Peter

    2014-04-01

    Antibiotic resistance is a major threat to human health and well-being. To effectively combat this problem we need to understand the range of different resistance genes that allow bacteria to resist antibiotics. To do this the whole microbiota needs to be investigated. As most bacteria cannot be cultivated in the laboratory, the reservoir of antibiotic resistance genes in the non-cultivatable majority remains relatively unexplored. Currently the only way to study antibiotic resistance in these organisms is to use metagenomic approaches. Furthermore, the only method that does not require any prior knowledge about the resistance genes is functional metagenomics, which involves expressing genes from metagenomic clones in surrogate hosts. In this review the methods and limitations of functional metagenomics to isolate new antibiotic resistance genes and the mobile genetic elements that mediate their spread are explored.

  1. Signal processing for metagenomics: extracting information from the soup.

    Science.gov (United States)

    Rosen, Gail L; Sokhansanj, Bahrad A; Polikar, Robi; Bruns, Mary Ann; Russell, Jacob; Garbarine, Elaine; Essinger, Steve; Yok, Non

    2009-11-01

    Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a great deal of insight into bacterial diversity and coding capacity, several computational challenges remain due to the massive size and complexity of metagenomic sequence data. Current tools and techniques are reviewed in this paper which address challenges in 1) genomic fragment annotation, 2) phylogenetic reconstruction, 3) functional classification of samples, and 4) interpreting complementary metaproteomics and metametabolomics data. Also surveyed are important applications of metagenomic studies, including microbial forensics and the roles of microbial communities in shaping human health and soil ecology.

  2. mmnet: An R Package for Metagenomics Systems Biology Analysis

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2015-01-01

    Full Text Available The human microbiome plays important roles in human health and disease. Previous microbiome studies focused mainly on single pure species function and overlooked the interactions in the complex communities on system-level. A metagenomic approach introduced recently integrates metagenomic data with community-level metabolic network modeling, but no comprehensive tool was available for such kind of approaches. To facilitate these kinds of studies, we developed an R package, mmnet, to implement community-level metabolic network reconstruction. The package also implements a set of functions for automatic analysis pipeline construction including functional annotation of metagenomic reads, abundance estimation of enzymatic genes, community-level metabolic network reconstruction, and integrated network analysis. The result can be represented in an intuitive way and sent to Cytoscape for further exploration. The package has substantial potentials in metagenomic studies that focus on identifying system-level variations of human microbiome associated with disease.

  3. mmnet: An R Package for Metagenomics Systems Biology Analysis.

    Science.gov (United States)

    Cao, Yang; Zheng, Xiaofei; Li, Fei; Bo, Xiaochen

    2015-01-01

    The human microbiome plays important roles in human health and disease. Previous microbiome studies focused mainly on single pure species function and overlooked the interactions in the complex communities on system-level. A metagenomic approach introduced recently integrates metagenomic data with community-level metabolic network modeling, but no comprehensive tool was available for such kind of approaches. To facilitate these kinds of studies, we developed an R package, mmnet, to implement community-level metabolic network reconstruction. The package also implements a set of functions for automatic analysis pipeline construction including functional annotation of metagenomic reads, abundance estimation of enzymatic genes, community-level metabolic network reconstruction, and integrated network analysis. The result can be represented in an intuitive way and sent to Cytoscape for further exploration. The package has substantial potentials in metagenomic studies that focus on identifying system-level variations of human microbiome associated with disease.

  4. Microbial Metagenomics : A Tale of the Dead and the Living

    OpenAIRE

    Zaremba-Niedźwiedzka, Katarzyna

    2013-01-01

    It is a microbial world we live in: microbes outnumber other organisms by several orders of magnitude, and they have great importance for the environment. However, environmental microbes are notoriously difficult to grow in the laboratory, and using culture independent techniques is necessary to expand our view. In this thesis, I apply metagenomics and single-cell genomics to environmental samples from ancient human remains and lakes. First, I used metagenomics to learn about bacteria from a ...

  5. Metagenomes provide valuable comparative information on soil microeukaryotes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Stenbæk, Jonas; Santos, Susana

    2016-01-01

    , providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure...... has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods....

  6. Exploration of Metagenome Assemblies with an Interactive Visualization Tool

    Energy Technology Data Exchange (ETDEWEB)

    Cantor, Michael; Nordberg, Henrik; Smirnova, Tatyana; Andersen, Evan; Tringe, Susannah; Hess, Matthias; Dubchak, Inna

    2014-07-09

    Metagenomics, one of the fastest growing areas of modern genomic science, is the genetic profiling of the entire community of microbial organisms present in an environmental sample. Elviz is a web-based tool for the interactive exploration of metagenome assemblies. Elviz can be used with publicly available data sets from the Joint Genome Institute or with custom user-loaded assemblies. Elviz is available at genome.jgi.doe.gov/viz

  7. Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, Jenna L.; Darling, Aaron E.; Eisen, Jonathan A.

    2009-12-01

    Background: Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. Methodology/Principal Findings: We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. Conclusions/Significance: We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with

  8. Signal Processing for Metagenomics: Extracting Information from the Soup

    OpenAIRE

    Rosen, Gail L.; Sokhansanj, Bahrad A.; Polikar, Robi; Bruns, Mary Ann; Russell, Jacob; Garbarine, Elaine; Essinger, Steve; Yok, Non

    2009-01-01

    Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a ...

  9. Biocatalysts and small molecule products from metagenomic studies.

    Science.gov (United States)

    Iqbal, Hala A; Feng, Zhiyang; Brady, Sean F

    2012-04-01

    The vast majority of bacteria present in environmental samples have never been cultured and therefore have not been exploited for the ability to produce useful biocatalysts or collections of biocatalysts generating interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries.

  10. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn;

    2010-01-01

    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...... gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively....

  11. METAGENassist: a comprehensive web server for comparative metagenomics.

    Science.gov (United States)

    Arndt, David; Xia, Jianguo; Liu, Yifeng; Zhou, You; Guo, An Chi; Cruz, Joseph A; Sinelnikov, Igor; Budwill, Karen; Nesbø, Camilla L; Wishart, David S

    2012-07-01

    With recent improvements in DNA sequencing and sample extraction techniques, the quantity and quality of metagenomic data are now growing exponentially. This abundance of richly annotated metagenomic data and bacterial census information has spawned a new branch of microbiology called comparative metagenomics. Comparative metagenomics involves the comparison of bacterial populations between different environmental samples, different culture conditions or different microbial hosts. However, in order to do comparative metagenomics, one typically requires a sophisticated knowledge of multivariate statistics and/or advanced software programming skills. To make comparative metagenomics more accessible to microbiologists, we have developed a freely accessible, easy-to-use web server for comparative metagenomic analysis called METAGENassist. Users can upload their bacterial census data from a wide variety of common formats, using either amplified 16S rRNA data or shotgun metagenomic data. Metadata concerning environmental, culture, or host conditions can also be uploaded. During the data upload process, METAGENassist also performs an automated taxonomic-to-phenotypic mapping. Phenotypic information covering nearly 20 functional categories such as GC content, genome size, oxygen requirements, energy sources and preferred temperature range is automatically generated from the taxonomic input data. Using this phenotypically enriched data, users can then perform a variety of multivariate and univariate data analyses including fold change analysis, t-tests, PCA, PLS-DA, clustering and classification. To facilitate data processing, users are guided through a step-by-step analysis workflow using a variety of menus, information hyperlinks and check boxes. METAGENassist also generates colorful, publication quality tables and graphs that can be downloaded and used directly in the preparation of scientific papers. METAGENassist is available at http://www.metagenassist.ca.

  12. Hemoglobin E disease and glycosylated hemoglobin

    Directory of Open Access Journals (Sweden)

    Niharika Yedla

    2015-01-01

    Full Text Available Glycosylated hemoglobin (HbA1C is a routinely measured parameter to monitor long-term glycemic control in people with diabetes mellitus. The presence of hemoglobin (Hb variants can affect the accuracy of HbA1C methods. Hb E variant is the most common Hb variant in South-east Asia and North-east India. In the presence of Hb E, HbA1C may not be detectable by ion-exchange chromatography (high-pressure liquid chromatography, but may be estimated by immunoassay technique and boronate affinity chromatography. However, the result may be underestimated when correlated with plasma glucose and serum fructosamine levels. Clinicians should be aware of this limitation of HbA1C estimation in patients with Hb E and other Hb variants.

  13. Multiple comparative metagenomics using multiset k-mer counting

    Directory of Open Access Journals (Sweden)

    Gaëtan Benoit

    2016-11-01

    Full Text Available Background Large scale metagenomic projects aim to extract biodiversity knowledge between different environmental conditions. Current methods for comparing microbial communities face important limitations. Those based on taxonomical or functional assignation rely on a small subset of the sequences that can be associated to known organisms. On the other hand, de novo methods, that compare the whole sets of sequences, either do not scale up on ambitious metagenomic projects or do not provide precise and exhaustive results. Methods These limitations motivated the development of a new de novo metagenomic comparative method, called Simka. This method computes a large collection of standard ecological distances by replacing species counts by k-mer counts. Simka scales-up today’s metagenomic projects thanks to a new parallel k-mer counting strategy on multiple datasets. Results Experiments on public Human Microbiome Project datasets demonstrate that Simka captures the essential underlying biological structure. Simka was able to compute in a few hours both qualitative and quantitative ecological distances on hundreds of metagenomic samples (690 samples, 32 billions of reads. We also demonstrate that analyzing metagenomes at the k-mer level is highly correlated with extremely precise de novo comparison techniques which rely on all-versus-all sequences alignment strategy or which are based on taxonomic profiling.

  14. A Bioinformatician's Guide to Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Kunin, Victor; Copeland, Alex; Lapidus, Alla; Mavromatis, Konstantinos; Hugenholtz, Philip

    2008-08-01

    As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe step-by-step the chain of decisions accompanying a metagenomic project from the viewpoint of a bioinformatician. We guide the reader through a standard workflow for a metagenomic project beginning with pre-sequencing considerations such as community composition and sequence data type that will greatly influence downstream analyses. We proceed with recommendations for sampling and data generation including sample and metadata collection, community profiling, construction of shotgun libraries and sequencing strategies. We then discuss the application of generic sequence processing steps (read preprocessing, assembly, and gene prediction and annotation) to metagenomic datasets by contrast to genome projects. Different types of data analyses particular to metagenomes are then presented including binning, dominant population analysis and gene-centric analysis. Finally data management systems and issues are presented and discussed. We hope that this review will assist bioinformaticians and biologists in making better-informed decisions on their journey during a metagenomic project.

  15. Expanding the marine virosphere using metagenomics.

    Directory of Open Access Journals (Sweden)

    Carolina Megumi Mizuno

    Full Text Available Viruses infecting prokaryotic cells (phages are the most abundant entities of the biosphere and contain a largely uncharted wealth of genomic diversity. They play a critical role in the biology of their hosts and in ecosystem functioning at large. The classical approaches studying phages require isolation from a pure culture of the host. Direct sequencing approaches have been hampered by the small amounts of phage DNA present in most natural habitats and the difficulty in applying meta-omic approaches, such as annotation of small reads and assembly. Serendipitously, it has been discovered that cellular metagenomes of highly productive ocean waters (the deep chlorophyll maximum contain significant amounts of viral DNA derived from cells undergoing the lytic cycle. We have taken advantage of this phenomenon to retrieve metagenomic fosmids containing viral DNA from a Mediterranean deep chlorophyll maximum sample. This method allowed description of complete genomes of 208 new marine phages. The diversity of these genomes was remarkable, contributing 21 genomic groups of tailed bacteriophages of which 10 are completely new. Sequence based methods have allowed host assignment to many of them. These predicted hosts represent a wide variety of important marine prokaryotic microbes like members of SAR11 and SAR116 clades, Cyanobacteria and also the newly described low GC Actinobacteria. A metavirome constructed from the same habitat showed that many of the new phage genomes were abundantly represented. Furthermore, other available metaviromes also indicated that some of the new phages are globally distributed in low to medium latitude ocean waters. The availability of many genomes from the same sample allows a direct approach to viral population genomics confirming the remarkable mosaicism of phage genomes.

  16. Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation.

    Science.gov (United States)

    Pratap, J; Rajamohan, G; Dikshit, K L

    2000-04-01

    Degradation of streptokinase (SK) has been frequently observed during large-scale protein production. An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state. The influence of the carbohydrate moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from alpha-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile at 25 degrees C and 37 degrees C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glycosylation of SK results in 30-40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function.

  17. EBI metagenomics in 2016--an expanding and evolving resource for the analysis and archiving of metagenomic data.

    Science.gov (United States)

    Mitchell, Alex; Bucchini, Francois; Cochrane, Guy; Denise, Hubert; ten Hoopen, Petra; Fraser, Matthew; Pesseat, Sebastien; Potter, Simon; Scheremetjew, Maxim; Sterk, Peter; Finn, Robert D

    2016-01-01

    EBI metagenomics (https://www.ebi.ac.uk/metagenomics/) is a freely available hub for the analysis and archiving of metagenomic and metatranscriptomic data. Over the last 2 years, the resource has undergone rapid growth, with an increase of over five-fold in the number of processed samples and consequently represents one of the largest resources of analysed shotgun metagenomes. Here, we report the status of the resource in 2016 and give an overview of new developments. In particular, we describe updates to data content, a complete overhaul of the analysis pipeline, streamlining of data presentation via the website and the development of a new web based tool to compare functional analyses of sequence runs within a study. We also highlight two of the higher profile projects that have been analysed using the resource in the last year: the oceanographic projects Ocean Sampling Day and Tara Oceans.

  18. Sweet to the extreme: protein glycosylation in Archaea.

    Science.gov (United States)

    Yurist-Doutsch, Sophie; Chaban, Bonnie; VanDyke, David J; Jarrell, Ken F; Eichler, Jerry

    2008-06-01

    Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.

  19. Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

    Science.gov (United States)

    Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

    2005-11-01

    Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

  20. Estimating the extent of horizontal gene transfer in metagenomic sequences

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2008-03-01

    Full Text Available Abstract Background Although the extent of horizontal gene transfer (HGT in complete genomes has been widely studied, its influence in the evolution of natural communities of prokaryotes remains unknown. The availability of metagenomic sequences allows us to address the study of global patterns of prokaryotic evolution in samples from natural communities. However, the methods that have been commonly used for the study of HGT are not suitable for metagenomic samples. Therefore it is important to develop new methods or to adapt existing ones to be used with metagenomic sequences. Results We have created two different methods that are suitable for the study of HGT in metagenomic samples. The methods are based on phylogenetic and DNA compositional approaches, and have allowed us to assess the extent of possible HGT events in metagenomes for the first time. The methods are shown to be compatible and quite precise, although they probably underestimate the number of possible events. Our results show that the phylogenetic method detects HGT in between 0.8% and 1.5% of the sequences, while DNA compositional methods identify putative HGT in between 2% and 8% of the sequences. These ranges are very similar to these found in complete genomes by related approaches. Both methods act with a different sensitivity since they probably target HGT events of different ages: the compositional method mostly identifies recent transfers, while the phylogenetic is more suitable for the detections of older events. Nevertheless, the study of the number of HGT events in metagenomic sequences from different communities shows a consistent trend for both methods: the lower amount is found for the sequences of the Sargasso Sea metagenome, while the higher quantity is found in the whale fall metagenome from the bottom of the ocean. The significance of these observations is discussed. Conclusion The computational approaches that are used to find possible HGT events in complete

  1. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

    NARCIS (Netherlands)

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte M; Fischer, Gerhard; Mohamed, Mark F; Hyvönen, Marko; Morgavi, Diego P; Janssen, Dick B; Hollfelder, Florian

    2015-01-01

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidi

  2. Going deeper: metagenome of a hadopelagic microbial community.

    Directory of Open Access Journals (Sweden)

    Emiley A Eloe

    Full Text Available The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT. A total of 145 Mbp of assembled sequence data was generated and compared to two pelagic deep ocean metagenomes and two representative surface seawater datasets from the Sargasso Sea. In a number of instances, all three deep metagenomes displayed similar trends, but were most magnified in the PRT, including enrichment in functions for two-component signal transduction mechanisms and transcriptional regulation. Overrepresented transporters in the PRT metagenome included outer membrane porins, diverse cation transporters, and di- and tri-carboxylate transporters that matched well with the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. The most dramatic adaptational feature of the PRT microbes appears to be heavy metal resistance, as reflected in the large numbers of transporters present for their removal. As a complement to the metagenome approach, single-cell genomic techniques were utilized to generate partial whole-genome sequence data from four uncultivated cells from members of the dominant phyla within the PRT, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes. The single-cell sequence data provided genomic context for many of the highly abundant functional attributes identified from the PRT metagenome, as well as recruiting heavily the PRT metagenomic sequence data compared to 172 available reference marine genomes. Through these multifaceted sequence approaches, new insights have been provided into the unique functional

  3. Environmental Metagenomics: The Data Assembly and Data Analysis Perspectives

    Science.gov (United States)

    Kumar, Vinay; Maitra, S. S.; Shukla, Rohit Nandan

    2015-01-01

    Novel gene finding is one of the emerging fields in the environmental research. In the past decades the research was focused mainly on the discovery of microorganisms which were capable of degrading a particular compound. A lot of methods are available in literature about the cultivation and screening of these novel microorganisms. All of these methods are efficient for screening of microbes which can be cultivated in the laboratory. Microorganisms which live in extreme conditions like hot springs, frozen glaciers, acid mine drainage, etc. cannot be cultivated in the laboratory, this is because of incomplete knowledge about their growth requirements like temperature, nutrients and their mutual dependence on each other. The microbes that can be cultivated correspond only to less than 1 % of the total microbes which are present in the earth. Rest of the 99 % of uncultivated majority remains inaccessible. Metagenomics transcends the culture requirements of microbes. In metagenomics DNA is directly extracted from the environmental samples such as soil, seawater, acid mine drainage etc., followed by construction and screening of metagenomic library. With the ongoing research, a huge amount of metagenomic data is accumulating. Understanding this data is an essential step to extract novel genes of industrial importance. Various bioinformatics tools have been designed to analyze and annotate the data produced from the metagenome. The Bio-informatic requirements of metagenomics data analysis are different in theory and practice. This paper reviews the tools that are available for metagenomic data analysis and the capability such tools—what they can do and their web availability.

  4. The single-species metagenome: subtyping Staphylococcus aureus core genome sequences from shotgun metagenomic data

    Science.gov (United States)

    Li, Ben; Petit III, Robert A.; Qin, Zhaohui S.; Darrow, Lyndsey

    2016-01-01

    In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city. PMID:27781166

  5. Genetics Home Reference: COG5-congenital disorder of glycosylation

    Science.gov (United States)

    ... Golgi (COG) complex. This complex functions in the Golgi apparatus , which is a cellular structure in which newly ... are modified. One process that occurs in the Golgi apparatus is glycosylation, by which sugar molecules (oligosaccharides) are ...

  6. O-Linked glycosylation in Acanthamoeba polyphaga mimivirus.

    Science.gov (United States)

    Hülsmeier, Andreas J; Hennet, Thierry

    2014-08-01

    Acanthamoeba polyphaga mimivirus is a member of the giant nucleocytoplasmic large DNA viruses, infecting various Acanthamoeba spp. The genomes of giant viruses encode components previously thought to be exclusive to cellular life, such as proteins involved in nucleic acid and protein synthesis. Recent work on enzymes involved in carbohydrate biosynthesis and metabolism show that instead of utilizing host cell resources, Mimivirus produces its own glycosylation machinery. To obtain a more detailed view of glycosylation in Mimivirus, we developed a periodate oxidation-based method to selectively enrich Mimivirus surface glycoproteins. O-Glycosylation in Mimivirus glycoproteins was identified by permethylation and matrix-assisted laser desorption/ionization-mass spectrometry analyses of beta-eliminated glycans. We sequenced 26 previously undescribed O-glycans, most of which contain glucose as their reducing end saccharide. These data will facilitate future studies on the functional significance of glycosylation in Mimivirus.

  7. Stability-increasing effects of anthocyanin glycosyl acylation.

    Science.gov (United States)

    Zhao, Chang-Ling; Yu, Yu-Qi; Chen, Zhong-Jian; Wen, Guo-Song; Wei, Fu-Gang; Zheng, Quan; Wang, Chong-De; Xiao, Xing-Lei

    2017-01-01

    This review comprehensively summarizes the existing knowledge regarding the chemical implications of anthocyanin glycosyl acylation, the effects of acylation on the stability of acylated anthocyanins and the corresponding mechanisms. Anthocyanin glycosyl acylation commonly refers to the phenomenon in which the hydroxyl groups of anthocyanin glycosyls are esterified by aliphatic or aromatic acids, which is synthetically represented by the acylation sites as well as the types and numbers of acyl groups. Generally, glycosyl acylation increases the in vitro and in vivo chemical stability of acylated anthocyanins, and the mechanisms primarily involve physicochemical, stereochemical, photochemical, biochemical or environmental aspects under specific conditions. Additionally, the acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees. This review could provide insight into the optimization of the stability of anthocyanins as well as the application of suitable anthocyanins in food, pharmaceutical and cosmetic industries.

  8. Identification and characterization of N-glycosylated proteins using proteomics

    DEFF Research Database (Denmark)

    Selby, David S; Larsen, Martin R; Calvano, Cosima Damiana;

    2008-01-01

    Glycoproteins constitute a large fraction of the proteome. The fundamental role of protein glycosylation in cellular development, growth, and differentiation, tissue development, and in host-pathogen interactions is by now widely accepted. Proteome-wide characterization of glycoproteins...

  9. Glycosylation and the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Glick Mary Catherine

    2001-08-01

    Full Text Available Abstract The cystic fibrosis transmembrane conductance regulator (CFTR has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wtCFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF, and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

  10. Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

    OpenAIRE

    2012-01-01

    Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biolog...

  11. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    OpenAIRE

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic ge...

  12. Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

    DEFF Research Database (Denmark)

    Grathe, S.; Thygesen, M.B.; Larsen, K.

    2005-01-01

    DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe...... the difficult glycosylation of the 3-OH of a glucosamine derivative....

  13. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Science.gov (United States)

    Mahan, Alison E; Jennewein, Madeleine F; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D; Baden, Lindsey; Barouch, Dan H; Alter, Galit

    2016-03-01

    Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  14. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Directory of Open Access Journals (Sweden)

    Alison E Mahan

    2016-03-01

    Full Text Available Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  15. Deciphering a pathway of Halobacterium salinarum N-glycosylation

    OpenAIRE

    Kandiba, Lina; Eichler, Jerry

    2014-01-01

    Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis...

  16. Controllability analysis of protein glycosylation in CHO cells.

    Directory of Open Access Journals (Sweden)

    Melissa M St Amand

    Full Text Available To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line. However, before expending the significant amount of effort and expense required to develop and implement on-line control strategies to address the problem of glycosylation heterogeneity, it is imperative to assess first the extent to which the very complex process of glycosylation is controllable, thereby establishing what is theoretically achievable prior to any experimental attempts. In this work, we present a novel methodology for assessing the output controllability of glycosylation, a prototypical example of an extremely high-dimensional and very non-linear system. We first discuss a method for obtaining the process gain matrix for glycosylation that involves performing model simulations and data analysis systematically and judiciously according to a statistical design of experiments (DOE scheme and then employing Analysis of Variance (ANOVA to determine the elements of process gain matrix from the resulting simulation data. We then discuss how to use the resulting high-dimensional gain matrix to assess controllability. The utility of this method is demonstrated with a practical example where we assess the controllability of various classes of glycans and of specific glycoforms that are typically found in recombinant biologics produced with Chinese Hamster Ovary (CHO cells. In addition to providing useful insight into the extent to which on-line glycosylation control is achievable in actual manufacturing processes, the results also have important

  17. Controllability analysis of protein glycosylation in CHO cells.

    Science.gov (United States)

    St Amand, Melissa M; Tran, Kevin; Radhakrishnan, Devesh; Robinson, Anne S; Ogunnaike, Babatunde A

    2014-01-01

    To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line. However, before expending the significant amount of effort and expense required to develop and implement on-line control strategies to address the problem of glycosylation heterogeneity, it is imperative to assess first the extent to which the very complex process of glycosylation is controllable, thereby establishing what is theoretically achievable prior to any experimental attempts. In this work, we present a novel methodology for assessing the output controllability of glycosylation, a prototypical example of an extremely high-dimensional and very non-linear system. We first discuss a method for obtaining the process gain matrix for glycosylation that involves performing model simulations and data analysis systematically and judiciously according to a statistical design of experiments (DOE) scheme and then employing Analysis of Variance (ANOVA) to determine the elements of process gain matrix from the resulting simulation data. We then discuss how to use the resulting high-dimensional gain matrix to assess controllability. The utility of this method is demonstrated with a practical example where we assess the controllability of various classes of glycans and of specific glycoforms that are typically found in recombinant biologics produced with Chinese Hamster Ovary (CHO) cells. In addition to providing useful insight into the extent to which on-line glycosylation control is achievable in actual manufacturing processes, the results also have important implications for

  18. Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis.

    Science.gov (United States)

    Sun, Yao; Weng, Yuteng; Zhang, Chenyang; Liu, Yi; Kang, Chen; Liu, Zhongshuang; Jing, Bo; Zhang, Qi; Wang, Zuolin

    2015-12-04

    Proteoglycans play important roles in regulating osteogenesis. Dentin matrix protein 1 (DMP1) is a highly expressed bone extracellular matrix protein that regulates both bone development and phosphate metabolism. After glycosylation, an N-terminal fragment of DMP1 protein was identified as a new proteoglycan (DMP1-PG) in bone matrix. In vitro investigations showed that Ser(89) is the key glycosylation site in mouse DMP1. However, the specific role of DMP1 glycosylation is still not understood. In this study, a mutant DMP1 mouse model was developed in which the glycosylation site S(89) was substituted with G(89) (S89G-DMP1). The glycosylation level of DMP1 was down-regulated in the bone matrix of S89G-DMP1 mice. Compared with wild type mice, the long bones of S89G-DMP1 mice showed developmental changes, including the speed of bone remodeling and mineralization, the morphology and activities of osteocytes, and activities of both osteoblasts and osteoclasts. These findings indicate that glycosylation of DMP1 is a key posttranslational modification process during development and that DMP1-PG functions as an indispensable proteoglycan in osteogenesis.

  19. The great screen anomaly-a new frontier in product discovery through functional metagenomics

    NARCIS (Netherlands)

    Ekkers, David Matthias; Cretoiu, Mariana Silvia; Kielak, Anna Maria; van Elsas, Jan Dirk

    2012-01-01

    Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been hera

  20. The magic and menace of metagenomics: prospects for the study of plant growth-promoting rhizobacteria

    NARCIS (Netherlands)

    Leveau, J.H.J.

    2007-01-01

    This article aims to be a pragmatic primer into the field of metagenomics with special emphasis on the prospective contributions of metagenomics to the study of plant growth-promoting rhizobacteria (PGPR). After an introduction into the concepts and methodologies of metagenomics and a discussion of

  1. Accurate genome relative abundance estimation based on shotgun metagenomic reads.

    Directory of Open Access Journals (Sweden)

    Li C Xia

    Full Text Available Accurate estimation of microbial community composition based on metagenomic sequencing data is fundamental for subsequent metagenomics analysis. Prevalent estimation methods are mainly based on directly summarizing alignment results or its variants; often result in biased and/or unstable estimates. We have developed a unified probabilistic framework (named GRAMMy by explicitly modeling read assignment ambiguities, genome size biases and read distributions along the genomes. Maximum likelihood method is employed to compute Genome Relative Abundance of microbial communities using the Mixture Model theory (GRAMMy. GRAMMy has been demonstrated to give estimates that are accurate and robust across both simulated and real read benchmark datasets. We applied GRAMMy to a collection of 34 metagenomic read sets from four metagenomics projects and identified 99 frequent species (minimally 0.5% abundant in at least 50% of the data-sets in the human gut samples. Our results show substantial improvements over previous studies, such as adjusting the over-estimated abundance for Bacteroides species for human gut samples, by providing a new reference-based strategy for metagenomic sample comparisons. GRAMMy can be used flexibly with many read assignment tools (mapping, alignment or composition-based even with low-sensitivity mapping results from huge short-read datasets. It will be increasingly useful as an accurate and robust tool for abundance estimation with the growing size of read sets and the expanding database of reference genomes.

  2. MetaStorm: A Public Resource for Customizable Metagenomics Annotation

    Science.gov (United States)

    Arango-Argoty, Gustavo; Singh, Gargi; Heath, Lenwood S.; Pruden, Amy; Xiao, Weidong; Zhang, Liqing

    2016-01-01

    Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/), which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution. PMID:27632579

  3. Application of metagenomics in the human gut microbiome.

    Science.gov (United States)

    Wang, Wei-Lin; Xu, Shao-Yan; Ren, Zhi-Gang; Tao, Liang; Jiang, Jian-Wen; Zheng, Shu-Sen

    2015-01-21

    There are more than 1000 microbial species living in the complex human intestine. The gut microbial community plays an important role in protecting the host against pathogenic microbes, modulating immunity, regulating metabolic processes, and is even regarded as an endocrine organ. However, traditional culture methods are very limited for identifying microbes. With the application of molecular biologic technology in the field of the intestinal microbiome, especially metagenomic sequencing of the next-generation sequencing technology, progress has been made in the study of the human intestinal microbiome. Metagenomics can be used to study intestinal microbiome diversity and dysbiosis, as well as its relationship to health and disease. Moreover, functional metagenomics can identify novel functional genes, microbial pathways, antibiotic resistance genes, functional dysbiosis of the intestinal microbiome, and determine interactions and co-evolution between microbiota and host, though there are still some limitations. Metatranscriptomics, metaproteomics and metabolomics represent enormous complements to the understanding of the human gut microbiome. This review aims to demonstrate that metagenomics can be a powerful tool in studying the human gut microbiome with encouraging prospects. The limitations of metagenomics to be overcome are also discussed. Metatranscriptomics, metaproteomics and metabolomics in relation to the study of the human gut microbiome are also briefly discussed.

  4. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow.

    Science.gov (United States)

    Lopatina, Anna; Medvedeva, Sofia; Shmakov, Sergey; Logacheva, Maria D; Krylenkov, Vjacheslav; Severinov, Konstantin

    2016-01-01

    The diversity of bacteria present in surface snow around four Russian stations in Eastern Antarctica was studied by high throughput sequencing of amplified 16S rRNA gene fragments and shotgun metagenomic sequencing. Considerable class- and genus-level variation between the samples was revealed indicating a presence of inter-site diversity of bacteria in Antarctic snow. Flavobacterium was a major genus in one sampling site and was also detected in other sites. The diversity of flavobacterial type II-C CRISPR spacers in the samples was investigated by metagenome sequencing. Thousands of unique spacers were revealed with less than 35% overlap between the sampling sites, indicating an enormous natural variety of flavobacterial CRISPR spacers and, by extension, high level of adaptive activity of the corresponding CRISPR-Cas system. None of the spacers matched known spacers of flavobacterial isolates from the Northern hemisphere. Moreover, the percentage of spacers with matches with Antarctic metagenomic sequences obtained in this work was significantly higher than with sequences from much larger publically available environmental metagenomic database. The results indicate that despite the overall very high level of diversity, Antarctic Flavobacteria comprise a separate pool that experiences pressures from mobile genetic elements different from those present in other parts of the world. The results also establish analysis of metagenomic CRISPR spacer content as a powerful tool to study bacterial populations diversity.

  5. Selection in coastal Synechococcus (cyanobacteria populations evaluated from environmental metagenomes.

    Directory of Open Access Journals (Sweden)

    Vera Tai

    Full Text Available Environmental metagenomics provides snippets of genomic sequences from all organisms in an environmental sample and are an unprecedented resource of information for investigating microbial population genetics. Current analytical methods, however, are poorly equipped to handle metagenomic data, particularly of short, unlinked sequences. A custom analytical pipeline was developed to calculate dN/dS ratios, a common metric to evaluate the role of selection in the evolution of a gene, from environmental metagenomes sequenced using 454 technology of flow-sorted populations of marine Synechococcus, the dominant cyanobacteria in coastal environments. The large majority of genes (98% have evolved under purifying selection (dN/dS1, 77 out of 83 (93% were hypothetical. Notable among annotated genes, ribosomal protein L35 appears to be under positive selection in one Synechococcus population. Other annotated genes, in particular a possible porin, a large-conductance mechanosensitive channel, an ATP binding component of an ABC transporter, and a homologue of a pilus retraction protein had regions of the gene with elevated dN/dS. With the increasing use of next-generation sequencing in metagenomic investigations of microbial diversity and ecology, analytical methods need to accommodate the peculiarities of these data streams. By developing a means to analyze population diversity data from these environmental metagenomes, we have provided the first insight into the role of selection in the evolution of Synechococcus, a globally significant primary producer.

  6. Fast and sensitive taxonomic classification for metagenomics with Kaiju.

    Science.gov (United States)

    Menzel, Peter; Ng, Kim Lee; Krogh, Anders

    2016-04-13

    Metagenomics emerged as an important field of research not only in microbial ecology but also for human health and disease, and metagenomic studies are performed on increasingly larger scales. While recent taxonomic classification programs achieve high speed by comparing genomic k-mers, they often lack sensitivity for overcoming evolutionary divergence, so that large fractions of the metagenomic reads remain unclassified. Here we present the novel metagenome classifier Kaiju, which finds maximum (in-)exact matches on the protein-level using the Burrows-Wheeler transform. We show in a genome exclusion benchmark that Kaiju classifies reads with higher sensitivity and similar precision compared with current k-mer-based classifiers, especially in genera that are underrepresented in reference databases. We also demonstrate that Kaiju classifies up to 10 times more reads in real metagenomes. Kaiju can process millions of reads per minute and can run on a standard PC. Source code and web server are available at http://kaiju.binf.ku.dk.

  7. Metagenomic analysis of permafrost microbial community response to thaw

    Energy Technology Data Exchange (ETDEWEB)

    Mackelprang, R.; Waldrop, M.P.; DeAngelis, K.M.; David, M.M.; Chavarria, K.L.; Blazewicz, S.J.; Rubin, E.M.; Jansson, J.K.

    2011-07-01

    We employed deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes and related this data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows for the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses revealed that during transition from a frozen to a thawed state there were rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5°C, permafrost metagenomes converged to be more similar to each other than while they were frozen. We found that multiple genes involved in cycling of C and nitrogen shifted rapidly during thaw. We also constructed the first draft genome from a complex soil metagenome, which corresponded to a novel methanogen. Methane previously accumulated in permafrost was released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost.

  8. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow

    Directory of Open Access Journals (Sweden)

    Anna eLopatina

    2016-03-01

    Full Text Available The diversity of bacteria present in surface snow around four Russian stations in Eastern Antarctica was studied by high throughput sequencing of amplified 16S rRNA gene fragments and shotgun metagenomic sequencing. Considerable class- and genus-level variation between the samples was revealed indicating a presence of inter-site diversity of bacteria in Antarctic snow. Flavobacterium was a major genus in one sampling site and was also detected in other sites. The diversity of flavobacterial type II-C CRISPR spacers in the samples was investigated by metagenome sequencing. Thousands of unique spacers were revealed with less than 35% overlap between the sampling sites, indicating an enormous natural variety of flavobacterial CRISPR spacers and, by extension, high level of adaptive activity of the corresponding CRISPR-Cas system. None of the spacers matched known spacers of flavobacterial isolates from the Northern hemisphere. Moreover, the percentage of spacers with matches with Antarctic metagenomic sequences obtained in this work was significantly higher than with sequences from much larger publically available environmental metagenomic database. The results indicate that despite the overall very high level of diversity, Antarctic Flavobacteria comprise a separate pool that experiences pressures from mobile genetic elements different from those present in other parts of the world. The results also establish analysis of metagenomic CRISPR spacer content as a powerful tool to study bacterial populations diversity.

  9. Disulfide bonds and glycosylation in fungal peroxidases.

    Science.gov (United States)

    Limongi, P; Kjalke, M; Vind, J; Tams, J W; Johansson, T; Welinder, K G

    1995-01-15

    Four conserved disulfide bonds and N-linked and O-linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1-3, 2-7, 4-5 and 6-8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N-linked glycan composed only of GlcNAc and Man at residue Asn142, and two O-linked glycans near the C-terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N-linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man3 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C-terminal peptide of TvMP2 indicated the presence of five Man residues in O-linked glycans. No phosphate was found in these fungal peroxidases.

  10. Glycosylated hemoglobin and hyperbaric oxygen coverage denials.

    Science.gov (United States)

    Moffat, A D; Worth, E R; Weaver, L K

    2015-01-01

    Some Medicaid and Medicare fiscal intermediaries are denying hyperbaric oxygen (HBO2) therapy for diabetic foot ulcer (DFU) patients if the glycosylated hemoglobin (HbA1c) > 7.0%. We performed multiple PubMed searches for any diabetic wound healing clinical trial that documented HbA1c and had a wound healing endpoint. We scrutinized 30 peer-reviewed clinical trials, representing more than 4,400 patients. The average HbA1c from the intervention side of the studies was 8.6% (7.2% - 9.9%) and the control/sham side was 8.3% (6.0% - 10.6%). Twelve studies made a direct attempt to link HbA1c and wound healing. Four retrospective studies and one prospective cohort study assert that lower HbA1c favors wound healing, but review of the studies reveal design flaws that invalidate these conclusions. In total, 25 studies showed no direct correlation between HbA1c levels and wound healing. There was no randomized controlled trial (RCT) data demonstrating that HbA1c 7.0% is unfounded.

  11. Metagenomic analysis of phosphorus removing sludgecommunities

    Energy Technology Data Exchange (ETDEWEB)

    Garcia Martin, Hector; Ivanova, Natalia; Kunin, Victor; Warnecke,Falk; Barry, Kerrie; McHardy, Alice C.; Yeates, Christine; He, Shaomei; Salamov, Asaf; Szeto, Ernest; Dalin, Eileen; Putnam, Nik; Shapiro, HarrisJ.; Pangilinan, Jasmyn L.; Rigoutsos, Isidore; Kyrpides, Nikos C.; Blackall, Linda Louise; McMahon, Katherine D.; Hugenholtz, Philip

    2006-02-01

    Enhanced Biological Phosphorus Removal (EBPR) is not wellunderstood at the metabolic level despite being one of the best-studiedmicrobially-mediated industrial processes due to its ecological andeconomic relevance. Here we present a metagenomic analysis of twolab-scale EBPR sludges dominated by the uncultured bacterium, "CandidatusAccumulibacter phosphatis." This analysis resolves several controversiesin EBPR metabolic models and provides hypotheses explaining the dominanceof A. phosphatis in this habitat, its lifestyle outside EBPR and probablecultivation requirements. Comparison of the same species from differentEBPR sludges highlights recent evolutionary dynamics in the A. phosphatisgenome that could be linked to mechanisms for environmental adaptation.In spite of an apparent lack of phylogenetic overlap in the flankingcommunities of the two sludges studied, common functional themes werefound, at least one of them complementary to the inferred metabolism ofthe dominant organism. The present study provides a much-needed blueprintfor a systems-level understanding of EBPR and illustrates thatmetagenomics enables detailed, often novel, insights into evenwell-studied biological systems.

  12. Screening metagenomic libraries for laccase activities.

    Science.gov (United States)

    Ferrer, Manuel; Beloqui, Ana; Golyshin, Peter N

    2010-01-01

    Laccases are multi-copper oxidoreductases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) able to oxidise a wide variety of phenolic and non-phenolic compounds. They are useful enzymes for a variety of applications, including bioremediation and craft pulp bio-bleaching as the most significant ones. There is a considerable interest to find new laccases through the exploration of biological diversity. Laccases have been found in plants, insects, and bacteria but predominantly in fungi: these enzymes have been documented in about 60 fungal strains. Microbial diversity constitutes a largely unexplored treasure chest with new laccases with a good potential for basic science and biotechnology. At present, due to our inability to cultivate most microbes, the only means of accessing the resources of the microbial world is to harvest genetic resources ("metagenomes"), which can further on be subjected to extensive screening programs. In this chapter, we provide an overview of screening methods to identify laccase-encoding genes from environmental resources.

  13. Precision Metagenomics: Rapid Metagenomic Analyses for Infectious Disease Diagnostics and Public Health Surveillance

    Science.gov (United States)

    Afshinnekoo, Ebrahim; Chou, Chou; Alexander, Noah; Ahsanuddin, Sofia; Schuetz, Audrey N.; Mason, Christopher E.

    2017-01-01

    Next-generation sequencing (NGS) technologies have ushered in the era of precision medicine, transforming the way we treat cancer patients and diagnose disease. Concomitantly, the advent of these technologies has created a surge of microbiome and metagenomic studies over the last decade, many of which are focused on investigating the host-gene-microbial interactions responsible for the development and spread of infectious diseases, as well as delineating their key role in maintaining health. As we continue to discover more information about the etiology of infectious diseases, the translational potential of metagenomic NGS methods for treatment and rapid diagnosis is becoming abundantly clear. Here, we present a robust protocol for the implementation and application of “precision metagenomics” across various sequencing platforms for clinical samples. Such a pipeline integrates DNA/RNA extraction, library preparation, sequencing, and bioinformatics analyses for taxonomic classification, antimicrobial resistance (AMR) marker screening, and functional analysis (biochemical and metabolic pathway abundance). Moreover, the pipeline has 3 tracks: STAT for results within 24 h; Comprehensive that affords a more in-depth analysis and takes between 5 and 7 d, but offers antimicrobial resistance information; and Targeted, which also requires 5–7 d, but with more sensitive analysis for specific pathogens. Finally, we discuss the challenges that need to be addressed before full integration in the clinical setting.

  14. N-Glycosylation of the archaellum filament is not important for archaella assembly and motility, although N-Glycosylation is essential for motility in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Birich, Anton; Albers, Sonja-Verena

    2015-11-01

    N-Glycosylation is one of the predominant posttranslational modifications, which is found in all three domains of life. N-Glycosylation has been shown to influence many biological aspects of proteins, like protein folding, stability or activity. In this study we demonstrate that the archaellum filament subunit FlaB of Sulfolobus acidocaldarius is N-glycosylated. Each of the six predicted N-Glycosylation sites within FlaB are modified with the attachment of an N-glycan. Although, it has been previously shown that N-Glycosylation is essential for motility in S. acidocaldarius, as defects in the N-Glycosylation process resulted in none or reduced motile cells, strains lacking one to all six N-Glycosylation sites within FlaB still remained motile. Deletion of the first five N-Glycosylation sites in FlaB did not significantly affect the motility, whereas removal of all six N-Glycosylation sites reduced motility by about 40%. Transmission electron microscopy analyses of non glycosylated and glycosylated archaellum filament revealed no structural change in length. Therefore N-Glycosylation does not appear to be important for the stability and assembly of the archaellum filament itself, but plays a role in other parts of the archaellum assembly.

  15. Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost.

    Science.gov (United States)

    Yasir, Muhammad; Khan, Haji; Azam, Syed Sikander; Telke, Amar; Kim, Seon Won; Chung, Young Ryun

    2013-06-01

    In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25-55°C) and pH (5.5-8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.

  16. Quantitative metagenomic analyses based on average genome size normalization

    DEFF Research Database (Denmark)

    Frank, Jeremy Alexander; Sørensen, Søren Johannes

    2011-01-01

    Over the past quarter-century, microbiologists have used DNA sequence information to aid in the characterization of microbial communities. During the last decade, this has expanded from single genes to microbial community genomics, or metagenomics, in which the gene content of an environment can...... by estimating average genome sizes. This normalization can relieve comparative biases introduced by differences in community structure, number of sequencing reads, and sequencing read lengths between different metagenomes. We demonstrate the utility of this approach by comparing metagenomes from two different...... marine sources using both conventional small-subunit (SSU) rRNA gene analyses and our quantitative method to calculate the proportion of genomes in each sample that are capable of a particular metabolic trait. With both environments, to determine what proportion of each community they make up and how...

  17. Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

    Science.gov (United States)

    Kay, Gemma L.; Sergeant, Martin J.; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara

    2014-01-01

    ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. PMID:25028426

  18. Functional Metagenomic Investigations of the Human Intestinal Microbiota

    DEFF Research Database (Denmark)

    Moore, Aimee M.; Munck, Christian; Sommer, Morten Otto Alexander

    2011-01-01

    The human intestinal microbiota encode multiple critical functions impacting human health, including metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity...... microorganisms, but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community, independent of identity to known genes, by subjecting the metagenome to functional assays in a genetically tractable host....... Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex...

  19. Introduction to the analysis of environmental sequences: metagenomics with MEGAN.

    Science.gov (United States)

    Huson, Daniel H; Mitra, Suparna

    2012-01-01

    Metagenomics is the study of microbial organisms using sequencing applied directly to environmental samples. Similarly, in metatranscriptomics and metaproteomics, the RNA and protein sequences of such samples are studied. The analysis of these kinds of data often starts by asking the questions of "who is out there?", "what are they doing?", and "how do they compare?". In this chapter, we describe how these computational questions can be addressed using MEGAN, the MEtaGenome ANalyzer program. We first show how to analyze the taxonomic and functional content of a single dataset and then show how such analyses can be performed in a comparative fashion. We demonstrate how to compare different datasets using ecological indices and other distance measures. The discussion is conducted using a number of published marine datasets comprising metagenomic, metatranscriptomic, metaproteomic, and 16S rRNA data.

  20. Fast and sensitive taxonomic classification for metagenomics with Kaiju

    DEFF Research Database (Denmark)

    Menzel, Peter; Ng, Kim Lee; Krogh, Anders

    2016-01-01

    The constantly decreasing cost and increasing output of current sequencing technologies enable large scale metagenomic studies of microbial communities from diverse habitats. Therefore, fast and accurate methods for taxonomic classification are needed, which can operate on increasingly larger...... datasets and reference databases. Recently, several fast metagenomic classifiers have been developed, which are based on comparison of genomic k-mers. However, nucleotide comparison using a fixed k-mer length often lacks the sensitivity to overcome the evolutionary distance between sampled species...... and genomes in the reference database. Here, we present the novel metagenome classifier Kaiju for fast assignment of reads to taxa. Kaiju finds maximum exact matches on the protein-level using the Borrows-Wheeler transform, and can optionally allow amino acid substitutions in the search using a greedy...

  1. Is metagenomics resolving identification of functions in microbial communities?

    Science.gov (United States)

    Chistoserdova, Ludmila

    2014-01-01

    We are coming up on the tenth anniversary of the broad use of the method involving whole metagenome shotgun sequencing, referred to as metagenomics. The application of this approach has definitely revolutionized microbiology and the related fields, including the realization of the importance of the human microbiome. As such, metagenomics has already provided a novel outlook on the complexity and dynamics of microbial communities that are an important part of the biosphere of the planet. Accumulation of massive amounts of sequence data also caused a surge in the development of bioinformatics tools specially designed to provide pipelines for data analysis and visualization. However, a critical outlook into the field is required to appreciate what could be and what has currently been gained from the massive sequence databases that are being generated with ever-increasing speed.

  2. Application of metagenomics in understanding oral health and disease.

    Science.gov (United States)

    Xu, Ping; Gunsolley, John

    2014-04-01

    Oral diseases including periodontal disease and caries are some of the most prevalent infectious diseases in humans. Different microbial species cohabitate and form a polymicrobial biofilm called dental plaque in the oral cavity. Metagenomics using next generation sequencing technologies has produced bacterial profiles and genomic profiles to study the relationships between microbial diversity, genetic variation, and oral diseases. Several oral metagenomic studies have examined the oral microbiome of periodontal disease and caries. Gene annotations in these studies support the association of specific genes or metabolic pathways with oral health and with specific diseases. The roles of pathogenic species and functions of specific genes in oral disease development have been recognized by metagenomic analysis. A model is proposed in which three levels of interactions occur in the oral microbiome that determines oral health or disease.

  3. Study Bioprospecting of Medicinal Plant Extracts of the Semiarid Northeast: Contribution to the Control of Oral Microorganisms

    Science.gov (United States)

    Silva, Maria Suênia P.; Brandão, Deysiane O.; Chaves, Thiago P.; Formiga Filho, Amaro L. N.; Costa, Edja Maria M. de B.; Santos, Vanda L.; Medeiros, Ana Cláudia D.

    2012-01-01

    Dental pathologies can be caused by plaque-forming bacteria and yeast, which reside in the oral cavity. The bacteria growing in dental plaque, a naturally occurring biofilm, display increased resistance to antimicrobial agents. The objective was the evaluation of a preclinical assay of medicinal plants of the semiarid region from the northeast against oral pathogenic microorganism, aiming at bioprospecting a new product. The selection of plant material for this study was based on the ethnobotanical data on the traditional use of plants from the semiarid region. The thirty extracts were subjected to the determination of antibiofilm activity against gram-positive, gram-negative bacteria and yeast. The hydroalcoholic extract which showed positive antibiofilm activity against most of the microorganisms tested in agar diffusion assay was further tested for the determination of minimum inhibitory concentration (MIC) and Bioassay with Artemia salina. Plant samples tested in this study exhibited good antibiofilm activity for the treatment of oral problems. The Schinopsis brasiliensis showed greater activity for Pseudomonas aeruginosa and Staphylococcus aureus, but toxicity against Artemia salina. PMID:22719786

  4. Study bioprospecting of medicinal plant extracts of the semiarid northeast: contribution to the control of oral microorganisms.

    Science.gov (United States)

    Silva, Maria Suênia P; Brandão, Deysiane O; Chaves, Thiago P; Formiga Filho, Amaro L N; Costa, Edja Maria M de B; Santos, Vanda L; Medeiros, Ana Cláudia D

    2012-01-01

    Dental pathologies can be caused by plaque-forming bacteria and yeast, which reside in the oral cavity. The bacteria growing in dental plaque, a naturally occurring biofilm, display increased resistance to antimicrobial agents. The objective was the evaluation of a preclinical assay of medicinal plants of the semiarid region from the northeast against oral pathogenic microorganism, aiming at bioprospecting a new product. The selection of plant material for this study was based on the ethnobotanical data on the traditional use of plants from the semiarid region. The thirty extracts were subjected to the determination of antibiofilm activity against gram-positive, gram-negative bacteria and yeast. The hydroalcoholic extract which showed positive antibiofilm activity against most of the microorganisms tested in agar diffusion assay was further tested for the determination of minimum inhibitory concentration (MIC) and Bioassay with Artemia salina. Plant samples tested in this study exhibited good antibiofilm activity for the treatment of oral problems. The Schinopsis brasiliensis showed greater activity for Pseudomonas aeruginosa and Staphylococcus aureus, but toxicity against Artemia salina.

  5. Bioprospecting the Curculigoside-Cinnamic Acid-Rich Fraction from Molineria latifolia Rhizome as a Potential Antioxidant Therapeutic Agent

    Directory of Open Access Journals (Sweden)

    Der Jiun Ooi

    2016-06-01

    Full Text Available Increasing evidence from both experimental and clinical studies depicts the involvement of oxidative stress in the pathogenesis of various diseases. Specifically, disruption of homeostatic redox balance in accumulated body fat mass leads to obesity-associated metabolic syndrome. Strategies for the restoration of redox balance, potentially by exploring potent plant bioactives, have thus become the focus of therapeutic intervention. The present study aimed to bioprospect the potential use of the curculigoside-cinnamic acid-rich fraction from Molineria latifolia rhizome as an antioxidant therapeutic agent. The ethyl acetate fraction (EAF isolated from M. latifolia rhizome methanolic extract (RME contained the highest amount of phenolic compounds, particularly curculigoside and cinnamic acid. EAF demonstrated glycation inhibitory activities in both glucose- and fructose-mediated glycation models. In addition, in vitro chemical-based and cellular-based antioxidant assays showed that EAF exhibited high antioxidant activities and a protective effect against oxidative damage in 3T3-L1 preadipocytes. Although the efficacies of individual phenolics differed depending on the structure and concentration, a correlational study revealed strong correlations between total phenolic contents and antioxidant capacities. The results concluded that enriched phenolic contents in EAF (curculigoside-cinnamic acid-rich fraction contributed to the overall better reactivity. Our data suggest that this bioactive-rich fraction warrants therapeutic potential against oxidative stress-related disorders.

  6. Study Bioprospecting of Medicinal Plant Extracts of the Semiarid Northeast: Contribution to the Control of Oral Microorganisms

    Directory of Open Access Journals (Sweden)

    Maria Suênia P. Silva

    2012-01-01

    Full Text Available Dental pathologies can be caused by plaque-forming bacteria and yeast, which reside in the oral cavity. The bacteria growing in dental plaque, a naturally occurring biofilm, display increased resistance to antimicrobial agents. The objective was the evaluation of a preclinical assay of medicinal plants of the semiarid region from the northeast against oral pathogenic microorganism, aiming at bioprospecting a new product. The selection of plant material for this study was based on the ethnobotanical data on the traditional use of plants from the semiarid region. The thirty extracts were subjected to the determination of antibiofilm activity against gram-positive, gram-negative bacteria and yeast. The hydroalcoholic extract which showed positive antibiofilm activity against most of the microorganisms tested in agar diffusion assay was further tested for the determination of minimum inhibitory concentration (MIC and Bioassay with Artemia salina. Plant samples tested in this study exhibited good antibiofilm activity for the treatment of oral problems. The Schinopsis brasiliensis showed greater activity for Pseudomonas aeruginosa and Staphylococcus aureus, but toxicity against Artemia salina.

  7. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.

    Science.gov (United States)

    Fu, Jun; Bian, Xiaoying; Hu, Shengbaio; Wang, Hailong; Huang, Fan; Seibert, Philipp M; Plaza, Alberto; Xia, Liqiu; Müller, Rolf; Stewart, A Francis; Zhang, Youming

    2012-05-01

    Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

  8. Bioprospecting the Curculigoside-Cinnamic Acid-Rich Fraction from Molineria latifolia Rhizome as a Potential Antioxidant Therapeutic Agent.

    Science.gov (United States)

    Ooi, Der Jiun; Chan, Kim Wei; Sarega, Nadarajan; Alitheen, Noorjahan Banu; Ithnin, Hairuszah; Ismail, Maznah

    2016-06-17

    Increasing evidence from both experimental and clinical studies depicts the involvement of oxidative stress in the pathogenesis of various diseases. Specifically, disruption of homeostatic redox balance in accumulated body fat mass leads to obesity-associated metabolic syndrome. Strategies for the restoration of redox balance, potentially by exploring potent plant bioactives, have thus become the focus of therapeutic intervention. The present study aimed to bioprospect the potential use of the curculigoside-cinnamic acid-rich fraction from Molineria latifolia rhizome as an antioxidant therapeutic agent. The ethyl acetate fraction (EAF) isolated from M. latifolia rhizome methanolic extract (RME) contained the highest amount of phenolic compounds, particularly curculigoside and cinnamic acid. EAF demonstrated glycation inhibitory activities in both glucose- and fructose-mediated glycation models. In addition, in vitro chemical-based and cellular-based antioxidant assays showed that EAF exhibited high antioxidant activities and a protective effect against oxidative damage in 3T3-L1 preadipocytes. Although the efficacies of individual phenolics differed depending on the structure and concentration, a correlational study revealed strong correlations between total phenolic contents and antioxidant capacities. The results concluded that enriched phenolic contents in EAF (curculigoside-cinnamic acid-rich fraction) contributed to the overall better reactivity. Our data suggest that this bioactive-rich fraction warrants therapeutic potential against oxidative stress-related disorders.

  9. Multi-Layer and Recursive Neural Networks for Metagenomic Classification.

    Science.gov (United States)

    Ditzler, Gregory; Polikar, Robi; Rosen, Gail

    2015-09-01

    Recent advances in machine learning, specifically in deep learning with neural networks, has made a profound impact on fields such as natural language processing, image classification, and language modeling; however, feasibility and potential benefits of the approaches to metagenomic data analysis has been largely under-explored. Deep learning exploits many layers of learning nonlinear feature representations, typically in an unsupervised fashion, and recent results have shown outstanding generalization performance on previously unseen data. Furthermore, some deep learning methods can also represent the structure in a data set. Consequently, deep learning and neural networks may prove to be an appropriate approach for metagenomic data. To determine whether such approaches are indeed appropriate for metagenomics, we experiment with two deep learning methods: i) a deep belief network, and ii) a recursive neural network, the latter of which provides a tree representing the structure of the data. We compare these approaches to the standard multi-layer perceptron, which has been well-established in the machine learning community as a powerful prediction algorithm, though its presence is largely missing in metagenomics literature. We find that traditional neural networks can be quite powerful classifiers on metagenomic data compared to baseline methods, such as random forests. On the other hand, while the deep learning approaches did not result in improvements to the classification accuracy, they do provide the ability to learn hierarchical representations of a data set that standard classification methods do not allow. Our goal in this effort is not to determine the best algorithm in terms accuracy-as that depends on the specific application-but rather to highlight the benefits and drawbacks of each of the approach we discuss and provide insight on how they can be improved for predictive metagenomic analysis.

  10. Protein glycosylation in Helicobacter pylori: beyond the flagellins?

    Directory of Open Access Journals (Sweden)

    Patrick S Hopf

    Full Text Available Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac, bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori.

  11. deFUME: Dynamic exploration of functional metagenomic sequencing data

    DEFF Research Database (Denmark)

    van der Helm, Eric; Geertz-Hansen, Henrik Marcus; Genee, Hans Jasper

    2015-01-01

    Functional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is ...... to a comprehensive visual data overview that facilitates effortless inspection of gene function, clustering and distribution. The webserver is available at cbs.dtu.dk/services/deFUME/and the source code is distributed at github.com/EvdH0/deFUME....

  12. Evaluating the Quantitative Capabilities of Metagenomic Analysis Software.

    Science.gov (United States)

    Kerepesi, Csaba; Grolmusz, Vince

    2016-05-01

    DNA sequencing technologies are applied widely and frequently today to describe metagenomes, i.e., microbial communities in environmental or clinical samples, without the need for culturing them. These technologies usually return short (100-300 base-pairs long) DNA reads, and these reads are processed by metagenomic analysis software that assign phylogenetic composition-information to the dataset. Here we evaluate three metagenomic analysis software (AmphoraNet--a webserver implementation of AMPHORA2--, MG-RAST, and MEGAN5) for their capabilities of assigning quantitative phylogenetic information for the data, describing the frequency of appearance of the microorganisms of the same taxa in the sample. The difficulties of the task arise from the fact that longer genomes produce more reads from the same organism than shorter genomes, and some software assign higher frequencies to species with longer genomes than to those with shorter ones. This phenomenon is called the "genome length bias." Dozens of complex artificial metagenome benchmarks can be found in the literature. Because of the complexity of those benchmarks, it is usually difficult to judge the resistance of a metagenomic software to this "genome length bias." Therefore, we have made a simple benchmark for the evaluation of the "taxon-counting" in a metagenomic sample: we have taken the same number of copies of three full bacterial genomes of different lengths, break them up randomly to short reads of average length of 150 bp, and mixed the reads, creating our simple benchmark. Because of its simplicity, the benchmark is not supposed to serve as a mock metagenome, but if a software fails on that simple task, it will surely fail on most real metagenomes. We applied three software for the benchmark. The ideal quantitative solution would assign the same proportion to the three bacterial taxa. We have found that AMPHORA2/AmphoraNet gave the most accurate results and the other two software were under

  13. Toward a Standards-Compliant Genomic and Metagenomic Publication Record

    DEFF Research Database (Denmark)

    Garrity, GM; Field, D; Kyrpides, N;

    2008-01-01

    Increasingly, we are aware as a community of the growing need to manage the avalanche of genomic and metagenomic data, in addition to related data types like ribosomal RNA and barcode sequences, in a way that tightly integrates contextual data with traditional literature in a machine-readable way...... is in the midst of a publishing revolution. This revolution is marked by a growing shift away from a traditional dichotomy between "journal articles" and "database entries" and an increasing adoption of hybrid models of collecting and disseminating scientific information. With respect to genomes and metagenomes...

  14. Whither or wither geomicrobiology in the era of 'community metagenomics'

    Science.gov (United States)

    Oremland, R.S.; Capone, D.G.; Stolz, J.F.; Fuhrman, J.

    2005-01-01

    Molecular techniques are valuable tools that can improve our understanding of the structure of microbial communities. They provide the ability to probe for life in all niches of the biosphere, perhaps even supplanting the need to cultivate microorganisms or to conduct ecophysiological investigations. However, an overemphasis and strict dependence on such large information-driven endeavours as environmental metagenomics could overwhelm the field, to the detriment of microbial ecology. We now call for more balanced, hypothesis-driven research efforts that couple metagenomics with classic approaches.

  15. Metagenomic-Based Study of the Phylogenetic and Functional Gene Diversity in Galápagos Land and Marine Iguanas

    KAUST Repository

    Hong, Pei-Ying

    2014-12-19

    In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

  16. Glycosylation: impact, control and improvement during therapeutic protein production.

    Science.gov (United States)

    Costa, Ana Rita; Rodrigues, Maria Elisa; Henriques, Mariana; Oliveira, Rosário; Azeredo, Joana

    2014-12-01

    The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.

  17. N-linked protein glycosylation in a bacterial system.

    Science.gov (United States)

    Nothaft, Harald; Liu, Xin; McNally, David J; Szymanski, Christine M

    2010-01-01

    N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.

  18. N-glycosylation in Haloferax volcanii: Adjusting the sweetness

    Directory of Open Access Journals (Sweden)

    Jerry eEichler

    2013-12-01

    Full Text Available Long believed to be restricted to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target protein asparagine residues. Still, it is only in the last decade that pathways of N-glycosylation in Archaea have been delineated. In the haloarchaeon Haloferax volcanii, a series of Agl (archaeal glycosylation proteins is responsible for the addition of an N-linked pentasaccharide to modified proteins, including the surface (S-layer glycoprotein, the sole component of the surface layer surrounding the cell. The S-layer glycoprotein N-linked glycosylation profile changes, however, as a function of surrounding salinity. Upon growth at different salt concentrations, the S-layer glycoprotein is either decorated by the N-linked pentasaccharide introduced above or by both this pentasaccharide as well as a tetrasaccharide of distinct composition. Recent efforts have identified Agl5-Agl15 as components of a second Hfx. volcanii N-glycosylation pathway responsible for generating the tetrasaccharide attached to S-layer glycoprotein when growth occurs in 1.75 M but not 3.4 M NaCl-containing medium.

  19. N-glycosylation in Haloferax volcanii: adjusting the sweetness.

    Science.gov (United States)

    Eichler, Jerry; Arbiv, Adi; Cohen-Rosenzweig, Chen; Kaminski, Lina; Kandiba, Lina; Konrad, Zvia

    2013-12-24

    Long believed to be restricted to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target protein asparagine residues. Still, it is only in the last decade that pathways of N-glycosylation in Archaea have been delineated. In the haloarchaeon Haloferax volcanii, a series of Agl (archaeal glycosylation) proteins is responsible for the addition of an N-linked pentasaccharide to modified proteins, including the surface (S)-layer glycoprotein, the sole component of the surface layer surrounding the cell. The S-layer glycoprotein N-linked glycosylation profile changes, however, as a function of surrounding salinity. Upon growth at different salt concentrations, the S-layer glycoprotein is either decorated by the N-linked pentasaccharide introduced above or by both this pentasaccharide as well as a tetrasaccharide of distinct composition. Recent efforts have identified Agl5-Agl15 as components of a second Hfx. volcanii N-glycosylation pathway responsible for generating the tetrasaccharide attached to S-layer glycoprotein when growth occurs in 1.75 M but not 3.4 M NaCl-containing medium.

  20. Aberrant Glycosylation as Biomarker for Cancer: Focus on CD43

    Directory of Open Access Journals (Sweden)

    Franca Maria Tuccillo

    2014-01-01

    Full Text Available Glycosylation is a posttranslational modification of proteins playing a major role in cell signalling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands. Aberrant expression of glycan structures as well as occurrence of truncated structures, precursors, or novel structures of glycan may affect ligand-receptor interactions and thus interfere with regulation of cell adhesion, migration, and proliferation. Indeed, aberrant glycosylation represents a hallmark of cancer, reflecting cancer-specific changes in glycan biosynthesis pathways such as the altered expression of glycosyltransferases and glycosidases. Most studies have been carried out to identify changes in serum glycan structures. In most cancers, fucosylation and sialylation are significantly modified. Thus, aberrations in glycan structures can be used as targets to improve existing serum cancer biomarkers. The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. In this review, we discuss the aberrant protein glycosylation associated with human cancer and the identification of protein glycoforms as cancer biomarkers. In particular, we will focus on the aberrant CD43 glycosylation as cancer biomarker and the potential to exploit the UN1 monoclonal antibody (UN1 mAb to identify aberrant CD43 glycoforms.

  1. Advances in the biotechnological glycosylation of valuable flavonoids.

    Science.gov (United States)

    Xiao, Jianbo; Muzashvili, Tamar S; Georgiev, Milen I

    2014-11-01

    The natural flavonoids, especially their glycosides, are the most abundant polyphenols in foods and have diverse bioactivities. The biotransformation of flavonoid aglycones into their glycosides is vital in flavonoid biosynthesis. The main biological strategies that have been used to achieve flavonoid glycosylation in the laboratory involve metabolic pathway engineering and microbial biotransformation. In this review, we summarize the existing knowledge on the production and biotransformation of flavonoid glycosides using biotechnology, as well as the impact of glycosylation on flavonoid bioactivity. Uridine diphosphate glycosyltransferases play key roles in decorating flavonoids with sugars. Modern metabolic engineering and proteomic tools have been used in an integrated fashion to generate numerous structurally diverse flavonoid glycosides. In vitro, enzymatic glycosylation tends to preferentially generate flavonoid 3- and 7-O-glucosides; microorganisms typically convert flavonoids into their 7-O-glycosides and will produce 3-O-glycosides if supplied with flavonoid substrates having a hydroxyl group at the C-3 position. In general, O-glycosylation reduces flavonoid bioactivity. However, C-glycosylation can enhance some of the benefits of flavonoids on human health, including their antioxidant and anti-diabetic potential.

  2. High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing

    Directory of Open Access Journals (Sweden)

    Ross Elizabeth M

    2012-07-01

    Full Text Available Abstract Background Variation of microorganism communities in the rumen of cattle (Bos taurus is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS, that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate “rumen metagenome profiles”, and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. Results Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The “rumen metagenome profile” was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P  Conclusions We have presented a simple and high throughput method of

  3. A broad pH range and processive chitinase from a metagenome library

    Directory of Open Access Journals (Sweden)

    S.S. Thimoteo

    Full Text Available Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4 present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

  4. Wrinkled skin and fat pads in patients with ALG8-CDG: revisiting skin manifestations in congenital disorders of glycosylation

    NARCIS (Netherlands)

    Kouwenberg, D.; Gardeitchik, T.; Mohamed, M.; Lefeber, D.J.; Morava, E.

    2014-01-01

    Glycosylation is the posttranslational coupling of sugar chains to proteins or lipids. Proper glycosylation is essential for normal protein structure, function, and trafficking. Mutations in the glycosylation pathway lead to a phenotypically heterogeneous group of metabolic disorders, the congenital

  5. Virus glycosylation: role in virulence and immune interactions.

    Science.gov (United States)

    Vigerust, David J; Shepherd, Virginia L

    2007-05-01

    The study of N-linked glycosylation as it relates to virus biology has become an area of intense interest in recent years due to its ability to impart various advantages to virus survival and virulence. HIV and influenza, two clear threats to human health, have been shown to rely on expression of specific oligosaccharides to evade detection by the host immune system. Additionally, other viruses such as Hendra, SARS-CoV, influenza, hepatitis and West Nile rely on N-linked glycosylation for crucial functions such as entry into host cells, proteolytic processing and protein trafficking. This review focuses on recent findings on the importance of glycosylation to viral virulence and immune evasion for several prominent human pathogens.

  6. LCMV glycosylation modulates viral fitness and cell tropism.

    Directory of Open Access Journals (Sweden)

    Cyrille J Bonhomme

    Full Text Available The glycoprotein (GP of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage cells reverted to WT sequence. Of the seven stable N-glycan deletion mutants, five of these led to altered viral fitness and cell tropism, assessed as growth in either mouse primary cortical neurons or bone marrow derived macrophages. These results demonstrate that the deletion of N-glycans in LCMV GP may confer an advantage to the virus for infection of neurons but a disadvantage in macrophages.

  7. Polar Glycosylated and Lateral Non-Glycosylated Flagella from Aeromonas hydrophila Strain AH-1 (Serotype O11

    Directory of Open Access Journals (Sweden)

    Kelly M. Fulton

    2015-11-01

    Full Text Available Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11 were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-“like” receptor 5 (TLR5.

  8. Fast and sensitive taxonomic classification for metagenomics with Kaiju

    DEFF Research Database (Denmark)

    Menzel, Peter; Ng, Kim Lee; Krogh, Anders

    2016-01-01

    reads in ten real metagenomes compared to programs based on genomic k-mers. Kaiju can process up to millions of reads per minute, and its memory footprint is below 5 GB of RAM, allowing the analysis on a standard PC. The program is available under the GPL3 license at: github.com/bioinformatics-centre/kaiju...

  9. DIME: a novel framework for de novo metagenomic sequence assembly.

    Science.gov (United States)

    Guo, Xuan; Yu, Ning; Ding, Xiaojun; Wang, Jianxin; Pan, Yi

    2015-02-01

    The recently developed next generation sequencing platforms not only decrease the cost for metagenomics data analysis, but also greatly enlarge the size of metagenomic sequence datasets. A common bottleneck of available assemblers is that the trade-off between the noise of the resulting contigs and the gain in sequence length for better annotation has not been attended enough for large-scale sequencing projects, especially for the datasets with low coverage and a large number of nonoverlapping contigs. To address this limitation and promote both accuracy and efficiency, we develop a novel metagenomic sequence assembly framework, DIME, by taking the DIvide, conquer, and MErge strategies. In addition, we give two MapReduce implementations of DIME, DIME-cap3 and DIME-genovo, on Apache Hadoop platform. For a systematic comparison of the performance of the assembly tasks, we tested DIME and five other popular short read assembly programs, Cap3, Genovo, MetaVelvet, SOAPdenovo, and SPAdes on four synthetic and three real metagenomic sequence datasets with various reads from fifty thousand to a couple million in size. The experimental results demonstrate that our method not only partitions the sequence reads with an extremely high accuracy, but also reconstructs more bases, generates higher quality assembled consensus, and yields higher assembly scores, including corrected N50 and BLAST-score-per-base, than other tools with a nearly theoretical speed-up. Results indicate that DIME offers great improvement in assembly across a range of sequence abundances and thus is robust to decreasing coverage.

  10. Metagenomics and other Methods for Measuring Antibiotic Resistance in Agroecosystems

    Science.gov (United States)

    Background: There is broad concern regarding antibiotic resistance on farms and in fields, however there is no standard method for defining or measuring antibiotic resistance in environmental samples. Methods: We used metagenomic, culture-based, and molecular methods to characterize the amount, t...

  11. Marine Metagenome as A Resource for Novel Enzymes

    KAUST Repository

    Alma’abadi, Amani D.

    2015-11-10

    More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  12. Comparative metagenomics of eight geographically remote terrestrial hot springs

    DEFF Research Database (Denmark)

    Menzel, Peter; Islin, Sóley Ruth; Rike, Anne Gunn

    2015-01-01

    Hot springs are natural habitats for thermophilic Archaea and Bacteria. In this paper, we present the metagenomic analysis of eight globally distributed terrestrial hot springs from China, Iceland, Italy, Russia, and the USA with a temperature range between 61 and 92 (∘)C and pH between 1.8 and 7...

  13. Metagenomic gene annotation by a homology-independent approach

    Energy Technology Data Exchange (ETDEWEB)

    Froula, Jeff; Zhang, Tao; Salmeen, Annette; Hess, Matthias; Kerfeld, Cheryl A.; Wang, Zhong; Du, Changbin

    2011-06-02

    Fully understanding the genetic potential of a microbial community requires functional annotation of all the genes it encodes. The recently developed deep metagenome sequencing approach has enabled rapid identification of millions of genes from a complex microbial community without cultivation. Current homology-based gene annotation fails to detect distantly-related or structural homologs. Furthermore, homology searches with millions of genes are very computational intensive. To overcome these limitations, we developed rhModeller, a homology-independent software pipeline to efficiently annotate genes from metagenomic sequencing projects. Using cellulases and carbonic anhydrases as two independent test cases, we demonstrated that rhModeller is much faster than HMMER but with comparable accuracy, at 94.5percent and 99.9percent accuracy, respectively. More importantly, rhModeller has the ability to detect novel proteins that do not share significant homology to any known protein families. As {approx}50percent of the 2 million genes derived from the cow rumen metagenome failed to be annotated based on sequence homology, we tested whether rhModeller could be used to annotate these genes. Preliminary results suggest that rhModeller is robust in the presence of missense and frameshift mutations, two common errors in metagenomic genes. Applying the pipeline to the cow rumen genes identified 4,990 novel cellulases candidates and 8,196 novel carbonic anhydrase candidates.In summary, we expect rhModeller to dramatically increase the speed and quality of metagnomic gene annotation.

  14. Functional Metagenomic Investigations of the Human Intestinal Microbiota

    Directory of Open Access Journals (Sweden)

    Aimee Marguerite Moore

    2011-10-01

    Full Text Available The human intestinal microbiota encode multiple critical functions impacting human health, including, metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity of this microbial community, its recalcitrance to standard cultivation and the immense diversity of its encoded genes has necessitated the development of novel molecular, microbiological, and genomic tools. Functional metagenomics is one such culture-independent technique used for decades to study environmental microorganisms but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community independent of identity to known genes by subjecting the metagenome to functional assays in a genetically tractable host. Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex community and its human host.

  15. Assembly of a marine viral metagenome after physical fractionation.

    Directory of Open Access Journals (Sweden)

    Jennifer R Brum

    Full Text Available Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne'ohe Bay, Hawai'i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.

  16. Novel tools for the functional expression of metagenomic DNA.

    Science.gov (United States)

    Troeschel, Sonja Christina; Drepper, Thomas; Leggewie, Christian; Streit, Wolfgang R; Jaeger, Karl-Erich

    2010-01-01

    The functional expression of environmental genes in a particular host bacterium is hampered by various limitations including inefficient transcription of target genes as well as improper assembly of the corresponding enzymes. Therefore, the identification of novel enzymes from metagenomic libraries by activity-based screening requires efficient expression and screening systems. In the following chapter, we present two novel tools to improve the functional expression of metagenomic genes. (1) Comparative screenings of metagenomic libraries demonstrated that different enzymes were detected when phylogenetically distinct expression host strains were used. Thus, we have developed a strategy, which comprises library construction using a shuttle vector that allows comparative expression and screening of metagenomic DNA in Escherichia coli, Pseudomonas putida, and Bacillus subtilis. (2) Expression studies have revealed that functional expression of environmental genes in heterologous expression hosts is often limited by insufficient promoter recognition. Therefore, a method is described allowing to enhance the expression capacity of E. coli by using the transposon MuExpress. This recombinant transposon is able to insert randomly into environmental DNA fragments thereby facilitating gene expression from its two inducible promoters.

  17. Marine Metagenome as A Resource for Novel Enzymes

    Directory of Open Access Journals (Sweden)

    Amani D. Alma’abadi

    2015-10-01

    Full Text Available More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to a great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  18. The effects of variable sample biomass on comparative metagenomics.

    Science.gov (United States)

    Chafee, Meghan; Maignien, Loïs; Simmons, Sheri L

    2015-07-01

    Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.

  19. Metagenomic species profiling using universal phylogenetic marker genes

    DEFF Research Database (Denmark)

    Sunagawa, Shinichi; Mende, Daniel R; Zeller, Georg;

    2013-01-01

    To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed...

  20. Metagenomic Analyses of Drinking Water Receiving Different Disinfection Treatments

    Science.gov (United States)

    A metagenome-based approach was utilized for assessing the taxonomic affiliation and function potential of microbial populations in free chlorine (CHL) and monochloramine (CHM) treated drinking water (DW). A total of 1,024, 242 (averaging 544 bp) and 849, 349 (averaging 554 bp) ...

  1. Marine Metagenome as A Resource for Novel Enzymes

    Institute of Scientific and Technical Information of China (English)

    Amani D. Alma’abadi; Takashi Gojobori; Katsuhiko Mineta

    2015-01-01

    More than 99%of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel bio-catalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to a great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metage-nomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we dis-cuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  2. INTERACTION BETWEEN THE SURFACE GLYCOSYLATED POLYPROPYLENE MEMBRANE AND LECTIN

    Institute of Scientific and Technical Information of China (English)

    Qian Yang; Ling-shu Wan; Zhi-kang Xu

    2008-01-01

    A glycopolymer bearing glucose residues was tethered onto the surface of polypropylene microporous membrane by UV-induced graft polymerization of α-allyl glucoside. Concanavalin A (Con A), a glucose recognizing lectin, could be specifically adsorbed to the membrane surface. On the other hand, the membrane surface showed no recognition ability to another lectin peanut agglutinin. Moreover, the recognition complex between the glycosylated membrane surface and Con Acould be inhibited by glucose and mannose solution. This surface glycosylated membrane could be used as affinity membrane for protein separation and purification.

  3. The structural biology of enzymes involved in natural product glycosylation.

    Science.gov (United States)

    Singh, Shanteri; Phillips, George N; Thorson, Jon S

    2012-10-01

    The glycosylation of microbial natural products often dramatically influences the biological and/or pharmacological activities of the parental metabolite. Over the past decade, crystal structures of several enzymes involved in the biosynthesis and attachment of novel sugars found appended to natural products have emerged. In many cases, these studies have paved the way to a better understanding of the corresponding enzyme mechanism of action and have served as a starting point for engineering variant enzymes to facilitate to production of differentially-glycosylated natural products. This review specifically summarizes the structural studies of bacterial enzymes involved in biosynthesis of novel sugar nucleotides.

  4. Getting the glycosylation right: implications for the biotechnology industry.

    Science.gov (United States)

    Jenkins, N; Parekh, R B; James, D C

    1996-08-01

    Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

  5. Engineering controlled mammalian type O-Glycosylation in plant cells

    DEFF Research Database (Denmark)

    Yang, Zhang; Drew, Damian Paul; Jørgensen, Bodil

    2011-01-01

    Human mucins are large heavily O-glycosylated glycoproteins (>200 kDa), which account for the majority of proteins in mucus layers that e.g. hydrate, lubricate and protect cells from proteases as well as from pathogens. O-linked mucin glycans are truncated in many cancers, yielding truncated cancer...... specific glyco-peptide epitopes, such as the Tn epitope (GalNAc sugar attached to either Serine or Threonine), which are antigenic to the immune system. In the present study, we have identified plant cells as the only eukaryotic cells without mammalian type O-glycosylation or competing (for sites) O...

  6. Aluminium, beta-amyloid and non-enzymatic glycosylation.

    Science.gov (United States)

    Exley, C; Schley, L; Murray, S; Hackney, C M; Birchall, J D

    1995-05-08

    The non-enzymatic glycosylation of beta-amyloid is implicated in the aetiology of Alzheimer's disease. However, controversy surrounds the nature of any involvement and a potential mechanism has not been fully elucidated. We present evidence of an aluminium-induced aggregation of the A beta P(25-35) peptide and speculate that the mechanism of formation of our ordered beta-amyloid aggregates might involve non-enzymatic glycosylation and/or site-specific crosslinking of beta-amyloid fibrils by atomic aluminium.

  7. Metagenomic Analysis of Koumiss in Kazakhstan

    Directory of Open Access Journals (Sweden)

    Samat Kozhakhmetov

    2014-12-01

    : Lactobacillus diolivorans, Lactobacillus acidophilus, L. casei, L. curvatus  yeast genus Torula (62.4% and Saccharomyces cerevisiae (37.6%.Conclusion. Thus, the first metagenomic research of koumiss, which was conducted in Kazakhstan, showed significant variations in microbial composition.

  8. Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea.

    Science.gov (United States)

    Abu-Qarn, Mehtap; Eichler, Jerry; Sharon, Nathan

    2008-10-01

    Of the many post-translational modifications proteins can undergo, glycosylation is the most prevalent and the most diverse. Today, it is clear that both N-glycosylation and O-glycosylation, once believed to be restricted to eukaryotes, also transpire in Bacteria and Archaea. Indeed, prokaryotic glycoproteins rely on a wider variety of monosaccharide constituents than do those of eukaryotes. In recent years, substantial progress in describing the enzymes involved in bacterial and archaeal glycosylation pathways has been made. It is becoming clear that enhanced knowledge of bacterial glycosylation enzymes may be of therapeutic value, while the demonstrated ability to introduce bacterial glycosylation genes into Escherichia coli represents a major step forward in glyco-engineering. A better understanding of archaeal protein glycosylation provides insight into this post-translational modification across evolution as well as protein processing under extreme conditions. Here, we discuss new structural and biosynthetic findings related to prokaryotic protein glycosylation, until recently a neglected topic.

  9. Effects of branched O-glycosylation on a semiflexible peptide linker.

    Science.gov (United States)

    Johnson, Quentin R; Lindsay, Richard J; Raval, Sherin R; Dobbs, Jeremy S; Nellas, Ricky B; Shen, Tongye

    2014-02-27

    Glycosylation is an essential modification of proteins and lipids by the addition of carbohydrate residues. These attached carbohydrates range from single monomers to elaborate branched glycans. Here, we examine how the level of glycosylation affects the conformation of a semiflexible peptide linker using the example of the hinge peptide from immunoglobulin A. Three sets of atomistic models of this hinge peptide with varying degrees of glycosylation are constructed to probe how glycosylation affects the physical properties of the linker. We found that glycosylation greatly altered the predominant conformations of the peptide, causing it to become elongated in reference to the unglycosylated form. Furthermore, glycosylation restricts the conformational exploration of the peptide. At the residue level, glycans are found to introduce a bias for the formation of more extended secondary structural elements for glycosylated serines. Additionally, the flexibility of this semiflexible proline-rich peptide is significantly reduced by glycosylation.

  10. Metagenome Skimming of Insect Specimen Pools: Potential for Comparative Genomics.

    Science.gov (United States)

    Linard, Benjamin; Crampton-Platt, Alex; Gillett, Conrad P D T; Timmermans, Martijn J T N; Vogler, Alfried P

    2015-05-14

    Metagenomic analyses are challenging in metazoans, but high-copy number and repeat regions can be assembled from low-coverage sequencing by "genome skimming," which is applied here as a new way of characterizing metagenomes obtained in an ecological or taxonomic context. Illumina shotgun sequencing on two pools of Coleoptera (beetles) of approximately 200 species each were assembled into tens of thousands of scaffolds. Repeated low-coverage sequencing recovered similar scaffold sets consistently, although approximately 70% of scaffolds could not be identified against existing genome databases. Identifiable scaffolds included mitochondrial DNA, conserved sequences with hits to expressed sequence tag and protein databases, and known repeat elements of high and low complexity, including numerous copies of rRNA and histone genes. Assemblies of histones captured a diversity of gene order and primary sequence in Coleoptera. Scaffolds with similarity to multiple sites in available coleopteran genome sequences for Dendroctonus and Tribolium revealed high specificity of scaffolds to either of these genomes, in particular for high-copy number repeats. Numerous "clusters" of scaffolds mapped to the same genomic site revealed intra- and/or intergenomic variation within a metagenome pool. In addition to effect of taxonomic composition of the metagenomes, the number of mapped scaffolds also revealed structural differences between the two reference genomes, although the significance of this striking finding remains unclear. Finally, apparently exogenous sequences were recovered, including potential food plants, fungal pathogens, and bacterial symbionts. The "metagenome skimming" approach is useful for capturing the genomic diversity of poorly studied, species-rich lineages and opens new prospects in environmental genomics.

  11. Remote homology and the functions of metagenomic dark matter

    Directory of Open Access Journals (Sweden)

    Briallen eLobb

    2015-07-01

    Full Text Available Predicted open reading frames (ORFs that lack detectable homology to known proteins are termed ORFans. Despite their prevalence in metagenomes, the extent to which ORFans encode real proteins, the degree to which they can be annotated, and their functional contributions, remain unclear. To gain insights into these questions, we applied sensitive remote-homology detection methods to functionally analyze ORFans from soil, marine, and human gut metagenome collections. ORFans were identified, clustered into sequence families, and annotated through profile-profile comparison to proteins of known structure.We found that a considerable number of metagenomic ORFans (73,896 of 484,121, 15.3% exhibit significant remote homology to structurally characterized proteins, providing a means for ORFan functional profiling. The extent of detected remote homology significantly exceeds that obtained for artificial protein families (1.4%. In addition, predicted ORFan functions show significant functional consistency with their gene neighbors (p < 0.001 as expected for real genes. Compared to genes annotated through standard homology searches, ORFans have intriguing functional differences such as an enrichment of virus-related functions and biological processes associated with extreme sequence diversity. Each environment also possesses many unique ORFan families that likely play important community roles such as identified ORFan polysaccharide degradation genes unique to the human gut metagenome. Lastly, ORFans are a valuable resource for finding novel enzymes of interest, as we demonstrate by identifying hundreds of ORFan metalloproteases that conserve a catalytic site despite a lack of overall sequence similarity to known proteins. Our ORFan functional predictions are a valuable resource for discovering novel protein families and exploring the boundaries of protein sequence space. Our resource of annotated metagenomic ORFans is available at http://doxey.uwaterloo.ca.

  12. Functional metagenomics to decipher food-microbe-host crosstalk.

    Science.gov (United States)

    Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

    2015-02-01

    The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk.

  13. Xenogenomics: Genomic Bioprospecting in Indigenous and Exotic Plants Through EST Discovery, cDNA Microarray-Based Expression Profiling and Functional Genomics

    Directory of Open Access Journals (Sweden)

    German C. Spangenberg

    2006-04-01

    Full Text Available To date, the overwhelming majority of genomics programs in plants have been directed at model or crop plant species, meaning that very little of the naturally occurring sequence diversity found in plants is available for characterization and exploitation. In contrast, ‘xenogenomics’ refers to the discovery and functional analysis of novel genes and alleles from indigenous and exotic species, permitting bioprospecting of biodiversity using high-throughput genomics experimental approaches. Such a program has been initiated to bioprospect for genetic determinants of abiotic stress tolerance in indigenous Australian flora and native Antarctic plants. Uniquely adapted Poaceae and Fabaceae species with enhanced tolerance to salt, drought, elevated soil aluminium concentration, and freezing stress have been identified, based primarily on their eco-physiology, and have been subjected to structural and functional genomics analyses. For each species, EST collections have been derived from plants subjected to appropriate abiotic stresses. Transcript profiling with spotted unigene cDNA micro-arrays has been used to identify genes that are transcriptionally modulated in response to abiotic stress. Candidate genes identified on the basis of sequence annotation or transcript profiling have been assayed in planta and other in vivo systems for their capacity to confer novel phenotypes. Comparative genomics analysis of novel genes and alleles identified in the xenogenomics target plant species has subsequently been undertaken with reference to key model and crop plants.

  14. Molecular cloning and heterologous expression of a true lipase in Pichia pastoris isolated via a metagenomic approach.

    Science.gov (United States)

    Zheng, Jianhua; Liu, Liguo; Liu, Cuina; Jin, Qi

    2012-01-01

    Lipases are important enzymes for various biotechnological applications. By using functional expression screening, lipZ03, a novel lipase gene, was isolated from a soil-derived metagenomic library. The gene was supposed to encode a protein of 617 amino acids with a C-terminal targeting signal region and four potential N-linked glycosylation sites. The protein sequence shared a conserved GXSXG motif (X represents any amino acid residue) with other microbial lipases. Gene lipZ03 was expressed in Pichia pastoris and the molecular weight was estimated to be approximately 65 kDa by electrophoresis. The optimum reaction temperature and pH value for LipZ03 was 50°C and 9.0, respectively. The enzyme was highly stable in the temperature range of 40-60°C and under alkaline conditions (pH 8-10). Lipolytic activity was significantly enhanced by Ca(2+) and Mg(2+) ions, but dramatically inhibited by Cu(2+), Ni(2+) and Hg(2+) ions and EDTA. The purified enzyme preferentially hydrolyzed relatively long-chain triacylglycerols and was a true lipase rather than an esterase. Using a multi-stepwise methanol supply, the purified LipZ03 achieved a conversion yield of biodiesel production up to 74% after 36 h. Some interesting characteristics described here showed that the recombinant lipase may have potential to be a useful enzyme in industrial applications.

  15. Characterization of a glycoside hydrolase family 1 β-galactosidase from hot spring metagenome with transglycosylation activity.

    Science.gov (United States)

    Gupta, Richa; Govil, Tanvi; Capalash, Neena; Sharma, Prince

    2012-11-01

    A novel, thermostable, alkalophilic β-D-galactosidase (Mbgl) was isolated from a metagenome of geothermal springs in northern Himalayan region of India. Mbgl was 447 amino acids in size and had conserved catalytic residues E170 and E358, indicating that it belonged to family 1 of glycosyl hydrolases showing maximum homology (89 %) with uncharacterized β-galactosidase of Eubacterium, Meiothermus ruber DSM1279. Temperature and pH optima of Mbgl were 65 °C and 8.0 respectively, and it retained 80 % activity even at pH 10.0. Mbgl was active as a homotetramer, recognized β-(1,4)-D-galactoside as the preferred glycosidic bond, and preferentially hydrolyzed pNPgal with K(m) 3.33 mM and k(cat) 2,000 s(-1). It displayed high transglycosylation activity with wide acceptor specificity including hexoses and pentoses leading to the formation of prebiotic galacto-oligosaccharides whereas its lactose hydrolysis potential was low.

  16. DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

    DEFF Research Database (Denmark)

    Petersen, Lars; Laursen, Jane B.; Larsen, K.;

    2003-01-01

    The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy ...

  17. Stabilization of exosome-targeting peptides via engineered glycosylation.

    Science.gov (United States)

    Hung, Michelle E; Leonard, Joshua N

    2015-03-27

    Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

  18. Deciphering a pathway of Halobacterium salinarum N-glycosylation.

    Science.gov (United States)

    Kandiba, Lina; Eichler, Jerry

    2015-02-01

    Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered.

  19. Insights into bacterial protein glycosylation in human microbiota.

    Science.gov (United States)

    Zhu, Fan; Wu, Hui

    2016-01-01

    The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.

  20. Congenital disorders of glycosylation with emphasis on cerebellar involvement.

    Science.gov (United States)

    Barone, Rita; Fiumara, Agata; Jaeken, Jaak

    2014-07-01

    Congenital disorders of glycosylation (CDG) are genetic diseases due to defective glycosylation of proteins and lipids. The authors present an update on these disorders affecting the central nervous system with a focus on cerebellar involvement. The rate of identification of novel CDG shows an exponential increase. Some 76 CDG are actually known, not taking into account the defects in glycan-modifying proteins. Neurologic involvement is present in the large majority of CDG. Screening methods are limited to serum transferrin isoelectrofocusing (for N-glycosylation disorders with sialic acid deficiency), and serum apolipoprotein C-III isoelectrofocusing (for core 1 mucin-type O-glycosylation disorders). Whole exome/genome sequencing is increasingly used in the diagnostic workup of patients with CDG-X. Treatment is greatly lagging behind because only one CDG is efficiently treatable (MPI-CDG). Cerebellar involvement is an important feature of PMM2-CDG, the congenital muscular dystrophies due to dystroglycanopathy, and SRD5A3-CDG. It has also been reported in some patients with ALG1-CDG, ALG3-CDG, ALG9-CDG, ALG6-CDG, ALG8-CDG, PIGA-CDG, DPM1-CDG, DPM2-CDG, B4GALT1-CDG, SLC35A2-CDG, COG1-CDG, COG5-CDG, COG7-CDG, and COG8-CDG.

  1. Species and strain glycosylation patterns of PrPSc.

    Directory of Open Access Journals (Sweden)

    Konstantinos Xanthopoulos

    Full Text Available BACKGROUND: A key event in transmissible spongiform encephalopathies (TSEs is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP(C to an abnormally structured, aggregated and partially protease-resistant isoform (PrP(Sc. Both PrP isoforms bear two potential glycosylation sites and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono- or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE strain and have been used to discriminate between them. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we used lectin-based western blotting to evaluate possible variations in composition within sugar chains carried by PrP(Sc purified from subjects affected with different TSEs. Our findings indicate that in addition to the already well-documented differences in electrophoretic mobility and amounts of the glycosylated PrP(Sc forms, TSE strains also vary in the abundance of specific N-linked sugars of the PrP(Sc protein. CONCLUSIONS/SIGNIFICANCE: These results imply that PrP glycosylation might fine-tune the conversion of PrP(C to PrP(Sc and could play an accessory role in the appearance of some of the characteristic features of TSE strains. The differences in sugar composition could also be used as an additional tool for discrimination between the various TSEs.

  2. Prognostic relevance of glycosylation-associated genes in breast cancer

    NARCIS (Netherlands)

    Milde-Langosch, Karin; Karn, Thomas; Schmidt, Marcus; Eulenburg, Christine Zu; Oliveira-Ferrer, Leticia; Wirtz, Ralph M.; Schumacher, Udo; Witzel, Isabell; Schuetze, Dina; Mueller, Volkmar

    2014-01-01

    Glycosylation of cellular proteins has important impact on their stability and functional properties, and glycan structures strongly influence cell adhesion. Many enzymes are involved in glycoconjugate synthesis and degradation, but there is only limited information about their role in breast cancer

  3. High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

    KAUST Repository

    Alam, Intikhab

    2016-01-26

    More and more genomes and metagenomes are being sequenced since the advent of Next Generation Sequencing Technologies (NGS). Many metagenomic samples are collected from a variety of environments, each exhibiting a different environmental profile, e.g. temperature, environmental chemistry, etc… These metagenomes can be profiled to unearth enzymes relevant to several industries based on specific enzyme properties such as ability to work on extreme conditions, such as extreme temperatures, salinity, anaerobically, etc.. In this work, we present the DMAP platform comprising of a high-throughput metagenomic annotation pipeline and a data-warehouse for comparisons and profiling across large number of metagenomes. We developed two reference databases for profiling of important genes, one containing enzymes related to different industries and the other containing genes with potential bioactivity roles. In this presentation we describe an example analysis of a large number of publicly available metagenomic sample from TARA oceans study (Science 2015) that covers significant part of world oceans.

  4. Fasting serum glucose and glycosylated hemoglobin level in obesity.

    Science.gov (United States)

    Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

    2014-04-01

    Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive correlation between BMI with fasting serum glucose and

  5. Identification of novel plasma glycosylation-associated markers of aging

    Science.gov (United States)

    Catera, Mariangela; Borelli, Vincenzo; Malagolini, Nadia; Chiricolo, Mariella; Venturi, Giulia; Reis, Celso A.; Osorio, Hugo; Abruzzo, Provvidenza M.; Capri, Miriam; Monti, Daniela; Ostan, Rita; Franceschi, Claudio; Dall'Olio, Fabio

    2016-01-01

    The pro- or anti-inflammatory activities of immunoglobulins G (IgGs) are controlled by the structure of the glycan N-linked to Asn297 of their heavy chain. The age-associated low grade inflammation (inflammaging) is associated with increased plasmatic levels of agalactosylated IgGs terminating with N-acetylglucosamine (IgG-G0) whose biogenesis has not been fully explained. Although the biosynthesis of glycans is in general mediated by glycosyltransferases associated with internal cell membranes, the extracellular glycosylation of circulating glycoproteins mediated by plasmatic glycosyltransferases has been recently demonstrated. In this study we have investigated the relationship between plasmatic glycosyltransferases, IgG glycosylation and inflammatory and aging markers. In cohorts of individuals ranging from infancy to centenarians we determined the activity of plasmatic β4 galactosyltransferase(s) (B4GALTs) and of α2,6-sialyltransferase ST6GAL1, the glycosylation of IgG, the GlycoAge test (a glycosylation-based marker of aging) and the plasma level of inflammatory and liver damage markers. Our results show that: 1) plasmatic B4GALTs activity is a new marker of aging, showing a linear increase throughout the whole age range. 2) plasmatic ST6GAL1 was high only in children and in people above 80, showing a quadratic relationship with age. 3) Neither plasmatic glycosyltransferase correlated with markers of liver damage. 4) plasmatic ST6GAL1 showed a positive association with acute phase proteins in offspring of short lived parents, but not in centenarians or in their offspring. 5) Although the glycosylation of IgGs was not correlated with the level of the two plasmatic glycosyltransferases, it showed progressive age-associated changes consistent with a shift toward a pro-inflammatory glycotype. PMID:26840264

  6. Metagenomes from two microbial consortia associated with Santa Barbara seep oil.

    Science.gov (United States)

    Hawley, Erik R; Malfatti, Stephanie A; Pagani, Ioanna; Huntemann, Marcel; Chen, Amy; Foster, Brian; Copeland, Alexander; del Rio, Tijana Glavina; Pati, Amrita; Jansson, Janet R; Gilbert, Jack A; Tringe, Susannah Green; Lorenson, Thomas D; Hess, Matthias

    2014-12-01

    The metagenomes from two microbial consortia associated with natural oils seeping into the Pacific Ocean offshore the coast of Santa Barbara (California, USA) were determined to complement already existing metagenomes generated from microbial communities associated with hydrocarbons that pollute the marine ecosystem. This genomics resource article is the first of two publications reporting a total of four new metagenomes from oils that seep into the Santa Barbara Channel.

  7. Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures

    Directory of Open Access Journals (Sweden)

    Pride David T

    2008-09-01

    Full Text Available Abstract Background Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC, where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. Results From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of

  8. MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies.

    Science.gov (United States)

    Norling, Martin; Karlsson-Lindsjö, Oskar E; Gourlé, Hadrien; Bongcam-Rudloff, Erik; Hayer, Juliette

    2016-01-01

    Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens' theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab.

  9. Metagenomic approaches to understanding phylogenetic diversity in quorum sensing.

    Science.gov (United States)

    Kimura, Nobutada

    2014-04-01

    Quorum sensing, a form of cell-cell communication among bacteria, allows bacteria to synchronize their behaviors at the population level in order to control behaviors such as luminescence, biofilm formation, signal turnover, pigment production, antibiotics production, swarming, and virulence. A better understanding of quorum-sensing systems will provide us with greater insight into the complex interaction mechanisms used widely in the Bacteria and even the Archaea domain in the environment. Metagenomics, the use of culture-independent sequencing to study the genomic material of microorganisms, has the potential to provide direct information about the quorum-sensing systems in uncultured bacteria. This article provides an overview of the current knowledge of quorum sensing focused on phylogenetic diversity, and presents examples of studies that have used metagenomic techniques. Future technologies potentially related to quorum-sensing systems are also discussed.

  10. Methylotrophs in natural habitats: current insights through metagenomics.

    Science.gov (United States)

    Chistoserdova, Ludmila

    2015-07-01

    The focus of this review is on the recent data from the omics approaches, measuring the presence of methylotrophs in natural environments. Both Bacteria and Archaea are considered. The data are discussed in the context of the current knowledge on the biochemistry of methylotrophy and the physiology of cultivated methylotrophs. One major issue discussed is the recent metagenomic data pointing toward the activity of "aerobic" methanotrophs, such as Methylobacter, in microoxic or hypoxic conditions. A related issue of the metabolic distinction between aerobic and "anaerobic" methylotrophy is addressed in the light of the genomic and metagenomic data for respective organisms. The role of communities, as opposed to single-organism activities in environmental cycling of single-carbon compounds, such as methane, is also discussed. In addition, the emerging issue of the role of non-traditional methylotrophs in global metabolism of single-carbon compounds and the role of methylotrophy pathways in non-methylotrophs is briefly mentioned.

  11. Gene and translation initiation site prediction in metagenomic sequences

    Energy Technology Data Exchange (ETDEWEB)

    Hyatt, Philip Douglas [ORNL; LoCascio, Philip F [ORNL; Hauser, Loren John [ORNL; Uberbacher, Edward C [ORNL

    2012-01-01

    Gene prediction in metagenomic sequences remains a difficult problem. Current sequencing technologies do not achieve sufficient coverage to assemble the individual genomes in a typical sample; consequently, sequencing runs produce a large number of short sequences whose exact origin is unknown. Since these sequences are usually smaller than the average length of a gene, algorithms must make predictions based on very little data. We present MetaProdigal, a metagenomic version of the gene prediction program Prodigal, that can identify genes in short, anonymous coding sequences with a high degree of accuracy. The novel value of the method consists of enhanced translation initiation site identification, ability to identify sequences that use alternate genetic codes and confidence values for each gene call. We compare the results of MetaProdigal with other methods and conclude with a discussion of future improvements.

  12. Metagenome of a Versatile Chemolithoautotroph from Expanding Oceanic Dead Zones

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, David A.; Zaikova, Elena; Howes, Charles L.; Song, Young; Wright, Jody; Tringe, Susannah G.; Tortell, Philippe D.; Hallam, Steven J.

    2009-07-15

    Oxygen minimum zones (OMZs), also known as oceanic"dead zones", are widespread oceanographic features currently expanding due to global warming and coastal eutrophication. Although inhospitable to metazoan life, OMZs support a thriving but cryptic microbiota whose combined metabolic activity is intimately connected to nutrient and trace gas cycling within the global ocean. Here we report time-resolved metagenomic analyses of a ubiquitous and abundant but uncultivated OMZ microbe (SUP05) closely related to chemoautotrophic gill symbionts of deep-sea clams and mussels. The SUP05 metagenome harbors a versatile repertoire of genes mediating autotrophic carbon assimilation, sulfur-oxidation and nitrate respiration responsive to a wide range of water column redox states. Thus, SUP05 plays integral roles in shaping nutrient and energy flow within oxygen-deficient oceanic waters via carbon sequestration, sulfide detoxification and biological nitrogen loss with important implications for marine productivity and atmospheric greenhouse control.

  13. The MG-RAST metagenomics database and portal in 2015.

    Science.gov (United States)

    Wilke, Andreas; Bischof, Jared; Gerlach, Wolfgang; Glass, Elizabeth; Harrison, Travis; Keegan, Kevin P; Paczian, Tobias; Trimble, William L; Bagchi, Saurabh; Grama, Ananth; Chaterji, Somali; Meyer, Folker

    2016-01-01

    MG-RAST (http://metagenomics.anl.gov) is an open-submission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. The system currently hosts over 200,000 datasets and is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. To show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.

  14. [Bacterial genomics and metagenomics: clinical applications and medical relevance].

    Science.gov (United States)

    Diene, S M; Bertelli, C; Pillonel, T; Schrenzel, J; Greub, G

    2014-11-12

    New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis.

  15. Metagenomic insights into the dynamics of microbial communities in food.

    Science.gov (United States)

    Kergourlay, Gilles; Taminiau, Bernard; Daube, Georges; Champomier Vergès, Marie-Christine

    2015-11-20

    Metagenomics has proven to be a powerful tool in exploring a large diversity of natural environments such as air, soil, water, and plants, as well as various human microbiota (e.g. digestive tract, lungs, skin). DNA sequencing techniques are becoming increasingly popular and less and less expensive. Given that high-throughput DNA sequencing approaches have only recently started to be used to decipher food microbial ecosystems, there is a significant growth potential for such technologies in the field of food microbiology. The aim of this review is to present a survey of recent food investigations via metagenomics and to illustrate how this approach can be a valuable tool in the better characterization of foods and their transformation, storage and safety. Traditional food in particular has been thoroughly explored by global approaches in order to provide information on multi-species and multi-organism communities.

  16. Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes.

    Science.gov (United States)

    Gonaus, Christoph; Maresch, Daniel; Schropp, Katharina; Ó Conghaile, Peter; Leech, Dónal; Gorton, Lo; Peterbauer, Clemens K

    2017-04-01

    Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N(75) and N(175), near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N(252) prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N(319), which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.

  17. Assessment of Common and Emerging Bioinformatics Pipelines for Targeted Metagenomics

    Science.gov (United States)

    Siegwald, Léa; Touzet, Hélène; Lemoine, Yves; Hot, David

    2017-01-01

    Targeted metagenomics, also known as metagenetics, is a high-throughput sequencing application focusing on a nucleotide target in a microbiome to describe its taxonomic content. A wide range of bioinformatics pipelines are available to analyze sequencing outputs, and the choice of an appropriate tool is crucial and not trivial. No standard evaluation method exists for estimating the accuracy of a pipeline for targeted metagenomics analyses. This article proposes an evaluation protocol containing real and simulated targeted metagenomics datasets, and adequate metrics allowing us to study the impact of different variables on the biological interpretation of results. This protocol was used to compare six different bioinformatics pipelines in the basic user context: Three common ones (mothur, QIIME and BMP) based on a clustering-first approach and three emerging ones (Kraken, CLARK and One Codex) using an assignment-first approach. This study surprisingly reveals that the effect of sequencing errors has a bigger impact on the results that choosing different amplified regions. Moreover, increasing sequencing throughput increases richness overestimation, even more so for microbiota of high complexity. Finally, the choice of the reference database has a bigger impact on richness estimation for clustering-first pipelines, and on correct taxa identification for assignment-first pipelines. Using emerging assignment-first pipelines is a valid approach for targeted metagenomics analyses, with a quality of results comparable to popular clustering-first pipelines, even with an error-prone sequencing technology like Ion Torrent. However, those pipelines are highly sensitive to the quality of databases and their annotations, which makes clustering-first pipelines still the only reliable approach for studying microbiomes that are not well described. PMID:28052134

  18. Recovery of a medieval Brucella melitensis genome using shotgun metagenomics.

    Science.gov (United States)

    Kay, Gemma L; Sergeant, Martin J; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara; Bianucci, Raffaella; Pallen, Mark J

    2014-07-15

    Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. Importance: Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries.

  19. Metagenomic Sequencing for Surveillance of Food- and Waterborne Viral Diseases

    Science.gov (United States)

    Nieuwenhuijse, David F.; Koopmans, Marion P. G.

    2017-01-01

    A plethora of viruses can be transmitted by the food- and waterborne route. However, their recognition is challenging because of the variety of viruses, heterogeneity of symptoms, the lack of awareness of clinicians, and limited surveillance efforts. Classical food- and waterborne viral disease outbreaks are mainly caused by caliciviruses, but the source of the virus is often not known and the foodborne mode of transmission is difficult to discriminate from human-to-human transmission. Atypical food- and waterborne viral disease can be caused by viruses such as hepatitis A and hepatitis E. In addition, a source of novel emerging viruses with a potential to spread via the food- and waterborne route is the repeated interaction of humans with wildlife. Wildlife-to-human adaptation may give rise to self- limiting outbreaks in some cases, but when fully adjusted to the human host can be devastating. Metagenomic sequencing has been investigated as a promising solution for surveillance purposes as it detects all viruses in a single protocol, delivers additional genomic information for outbreak tracing, and detects novel unknown viruses. Nevertheless, several issues must be addressed to apply metagenomic sequencing in surveillance. First, sample preparation is difficult since the genomic material of viruses is generally overshadowed by host- and bacterial genomes. Second, several data analysis issues hamper the efficient, robust, and automated processing of metagenomic data. Third, interpretation of metagenomic data is hard, because of the lack of general knowledge of the virome in the food chain and the environment. Further developments in virus-specific nucleic acid extraction methods, bioinformatic data processing applications, and unifying data visualization tools are needed to gain insightful surveillance knowledge from suspect food samples. PMID:28261185

  20. A retrospective metagenomics approach to studying Blastocystis

    DEFF Research Database (Denmark)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn

    2015-01-01

    a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals...... metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities....

  1. Biocatalysts and their small molecule products from metagenomic studies

    OpenAIRE

    2012-01-01

    The vast majority of bacteria present in environmental samples have never been cultured and therefore they have not been available to exploit their ability to produce useful biocatalysts or collections of biocatalysts that can biosynthesize interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent...

  2. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat.

    Directory of Open Access Journals (Sweden)

    Ana-Belen Martín-Cuadrado

    Full Text Available BACKGROUND: Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (approximately 14 degrees C than waters of similar depth in open oceans (approximately 2 degrees C. We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500-700 m deep in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source. CONCLUSIONS/SIGNIFICANCE: The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding

  3. Bioinformatic approaches reveal metagenomic characterization of soil microbial community.

    Directory of Open Access Journals (Sweden)

    Zhuofei Xu

    Full Text Available As is well known, soil is a complex ecosystem harboring the most prokaryotic biodiversity on the Earth. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated the progress of soil ecological studies. However, how to effectively understand the underlying biological features of large-scale sequencing data is a new challenge. In the present study, we used 33 publicly available metagenomes from diverse soil sites (i.e. grassland, forest soil, desert, Arctic soil, and mangrove sediment and integrated some state-of-the-art computational tools to explore the phylogenetic and functional characterizations of the microbial communities in soil. Microbial composition and metabolic potential in soils were comprehensively illustrated at the metagenomic level. A spectrum of metagenomic biomarkers containing 46 taxa and 33 metabolic modules were detected to be significantly differential that could be used as indicators to distinguish at least one of five soil communities. The co-occurrence associations between complex microbial compositions and functions were inferred by network-based approaches. Our results together with the established bioinformatic pipelines should provide a foundation for future research into the relation between soil biodiversity and ecosystem function.

  4. Culture-independent discovery of natural products from soil metagenomes.

    Science.gov (United States)

    Katz, Micah; Hover, Bradley M; Brady, Sean F

    2016-03-01

    Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.

  5. Functional metagenomic selection of RubisCOs from uncultivated bacteria

    Science.gov (United States)

    Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

    2016-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

  6. New viruses in veterinary medicine, detected by metagenomic approaches.

    Science.gov (United States)

    Belák, Sándor; Karlsson, Oskar E; Blomström, Anne-Lie; Berg, Mikael; Granberg, Fredrik

    2013-07-26

    In our world, which is faced today with exceptional environmental changes and dramatically intensifying globalisation, we are encountering challenges due to many new factors, including the emergence or re-emergence of novel, so far "unknown" infectious diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a limited group of infectious agents. This specificity complicates or even hinders the detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses or novel viral variants. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowledge of the infectious agent, thereby enabling a better diagnosis and disease control, in line with the "One World, One Health" principles (www.oneworldonehealth.org). Using metagenomic approaches, we have recently identified a broad variety of new viruses, such as novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in porcine disease syndromes, new virus variants in honeybee populations, as well as a range of other infectious agents in further host species. These findings indicate that the metagenomic detection of viral pathogens is becoming now a powerful, cultivation-independent, and useful novel diagnostic tool in veterinary diagnostic virology.

  7. PhyloSift: phylogenetic analysis of genomes and metagenomes

    Directory of Open Access Journals (Sweden)

    Aaron E. Darling

    2014-01-01

    Full Text Available Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection.In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata.These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454.

  8. Cyclodipeptides from metagenomic library of a japanese marine sponge

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui; Wang, Bochu; Zhub, Liancai, E-mail: wangbc2000@126.com [Bioengineering College, Chongqing University, Chongqing, (China); Wang, Manyuan [School of Traditional Chinese Medicine, Capital University of Medical Sciences, Beijing (China); Wakimoto, Toshiyuki; Abe, Ikuro, E-mail: abei@mol.f.u-tokyo.ac.jp [Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo (Japan)

    2013-12-01

    Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

  9. Challenges of the Unknown: Clinical Application of Microbial Metagenomics

    Directory of Open Access Journals (Sweden)

    Graham Rose

    2015-01-01

    Full Text Available Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

  10. Reconstruction of ribosomal RNA genes from metagenomic data.

    Directory of Open Access Journals (Sweden)

    Lu Fan

    Full Text Available Direct sequencing of environmental DNA (metagenomics has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.

  11. Patterns of glycemic control using glycosylated hemoglobin in diabetics

    Directory of Open Access Journals (Sweden)

    Arunpreet Singh Kahlon

    2011-01-01

    Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

  12. IMG/M: A data management and analysis system for metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    Markowitz, Victor M.; Ivanova, Natalia N.; Szeto, Ernest; Palaniappan, Krishna; Chu, Ken; Dalevi, Daniel; Chen, I-Min A.; Grechkin,Yuri; Dubchak,Inna; Anderson, Iain; Lykidis, Athanasios; Mavromatis,Konstantinos; Hug enholtz, Phil; Kyrpides, Nikos C.

    2007-08-01

    IMG/M is a data management and analysis system for microbial community genomes (metagenomes) hosted at the Joint Genome Institute (JGI). IMG/M consists of metagenome data integrated with isolate microbial genomes from the Integrated Microbial Genomes (IMG) system. IMG/M provides IMG's comparative data analysis tools extended to handle metagenome data, together with metagenome-specific analysis tools. IMG/M is available at http://img.jgi.doe.gov/m. Studies of the collective genomes (also known as metagenomes) of environmental microbial communities (also known as microbiomes) are expected to lead to advances in environmental cleanup, agriculture, industrial processes, alternative energy production, and human health (1). Metagenomes of specific microbiome samples are sequenced by organizations worldwide, such as the Department of Energy's (DOE) Joint Genome Institute (JGI), the Venter Institute and the Washington University in St. Louis using different sequencing strategies, technology platforms, and annotation procedures. According to the Genomes OnLine Database, about 28 metagenome studies have been published to date, with over 60 other projects ongoing and more in the process of being launched (2). The Department of Energy's (DOE) Joint Genome Institute (JGI) is one of the major contributors of metagenome sequence data, currently sequencing more than 50% of the reported metagenome projects worldwide. Due to the higher complexity, inherent incompleteness, and lower quality of metagenome sequence data, traditional assembly, gene prediction, and annotation methods do not perform on these datasets as well as they do on isolate microbial genome sequences (3, 4). In spite of these limitations, metagenome data are amenable to a variety of analyses, as illustrated by several recent studies (5-10). Metagenome data analysis is usually set up in the context of reference isolate genomes and considers the questions of composition and functional or metabolic

  13. NeSSM: a Next-generation Sequencing Simulator for Metagenomics.

    Directory of Open Access Journals (Sweden)

    Ben Jia

    Full Text Available BACKGROUND: Metagenomics can reveal the vast majority of microbes that have been missed by traditional cultivation-based methods. Due to its extremely wide range of application areas, fast metagenome sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of metagenomics analysis tools. RESULTS: We present here a customizable metagenome simulation system: NeSSM (Next-generation Sequencing Simulator for Metagenomics. Combining complete genomes currently available, a community composition table, and sequencing parameters, it can simulate metagenome sequencing better than existing systems. Sequencing error models based on the explicit distribution of errors at each base and sequencing coverage bias are incorporated in the simulation. In order to improve the fidelity of simulation, tools are provided by NeSSM to estimate the sequencing error models, sequencing coverage bias and the community composition directly from existing metagenome sequencing data. Currently, NeSSM supports single-end and pair-end sequencing for both 454 and Illumina platforms. In addition, a GPU (graphics processing units version of NeSSM is also developed to accelerate the simulation. By comparing the simulated sequencing data from NeSSM with experimental metagenome sequencing data, we have demonstrated that NeSSM performs better in many aspects than existing popular metagenome simulators, such as MetaSim, GemSIM and Grinder. The GPU version of NeSSM is more than one-order of magnitude faster than MetaSim. CONCLUSIONS: NeSSM is a fast simulation system for high-throughput metagenome sequencing. It can be helpful to develop tools and evaluate strategies for metagenomics analysis and it's freely available for academic users at http://cbb.sjtu.edu.cn/~ccwei/pub/software/NeSSM.php.

  14. Bridging the Gap between Glycosylation and Vesicle Traffic.

    Science.gov (United States)

    Fisher, Peter; Ungar, Daniel

    2016-01-01

    Glycosylation is recognized as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil tethers termed golgins and the multisubunit tethering complex known as the conserved oligomeric Golgi (COG) complex. In this review we discuss the mechanisms involved in vesicle tethering at the Golgi apparatus and highlight the importance of tethering in the context of glycan biosynthesis and a set of diseases known as congenital disorders of glycosylation.

  15. Bridging the gap between glycosylation and vesicle traffic

    Directory of Open Access Journals (Sweden)

    Daniel eUngar

    2016-03-01

    Full Text Available Glycosylation is recognised as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil tethers termed golgins and the multisubunit tethering complex known as the conserved oligomeric Golgi (COG complex. In this review we discuss the mechanisms involved in vesicle tethering at the Golgi apparatus and highlight the importance of tethering in the context of glycan biosynthesis and a set of diseases known as congenital disorders of glycosylation.

  16. Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae.

    Science.gov (United States)

    Han, Ah Reum; Park, Sung Ryeol; Park, Je Won; Lee, Eun Yeol; Kim, Dong-Myung; Kim, Byung-Gee; Yoon, Yeo Joon

    2011-06-01

    Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.

  17. Glycosylation status of vitamin D binding protein in cancer patients.

    Science.gov (United States)

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

  18. Cnidarians as a source of new marine bioactive compounds--an overview of the last decade and future steps for bioprospecting.

    Science.gov (United States)

    Rocha, Joana; Peixe, Luisa; Gomes, Newton C M; Calado, Ricardo

    2011-01-01

    Marine invertebrates are rich sources of bioactive compounds and their biotechnological potential attracts scientific and economic interest worldwide. Although sponges are the foremost providers of marine bioactive compounds, cnidarians are also being studied with promising results. This diverse group of marine invertebrates includes over 11,000 species, 7500 of them belonging to the class Anthozoa. We present an overview of some of the most promising marine bioactive compounds from a therapeutic point of view isolated from cnidarians in the first decade of the 21st century. Anthozoan orders Alcyonacea and Gorgonacea exhibit by far the highest number of species yielding promising compounds. Antitumor activity has been the major area of interest in the screening of cnidarian compounds, the most promising ones being terpenoids (monoterpenoids, diterpenoids, sesquiterpenoids). We also discuss the future of bioprospecting for new marine bioactive compounds produced by cnidarians.

  19. A cost-effective strategy for the bio-prospecting of mixed microalgae with high carbohydrate content: diversity fluctuations in different growth media.

    Science.gov (United States)

    Cea-Barcia, Glenda; Buitrón, Germán; Moreno, Gloria; Kumar, Gopalakrishnan

    2014-07-01

    In recent years, widespread efforts have been directed towards decreasing the costs associated with microalgae culture systems for the production of biofuels. In this study, a simple and inexpensive strategy to bio-prospect and cultivate mixed indigenous chlorophytes with a high carbohydrate content for biomethane and biohydrogen production was developed. Mixed microalgae were collected from four different water-bodies in Queretaro, Mexico, and were grown in Bold's basal mineral medium and secondary effluent from a wastewater treatment plant using inexpensive photo-bioreactors. The results showed large fluctuations in microalgal genera diversity based on different culture media and nitrogen sources. In secondary effluent, Golenkinia sp. and Scenedesmus sp. proliferated. The carbohydrate content, for secondary effluent, varied between 12% and 57%, and the highest volumetric and areal productivity were 61 mg L(-1)d(-1) and 4.6 g m(-2)d(-1), respectively. These results indicate that mixed microalgae are a good feedstock for biomethane and biohydrogen production.

  20. The Effect of Malnutrition on Protein Glycosylation in Children

    OpenAIRE

    2014-01-01

    Objective: The goal of this study was to evaluate the effect of protein energy malnutrition on protein glycosylation by investigating transferrin isoform pattern and its relationship to the degree of malnutrition and the biochemical markers of nutritional status in children. Methods: Forty one children with mild (n=23) and severely/moderately (n=18) acute malnutrition and 29 controls were enrolled in the study. Serum transferrin isoforms were determined by isoelectric focusing electrophoresis...

  1. Reduced apolipoprotein glycosylation in patients with the metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Olga V Savinova

    Full Text Available The purpose of this study was to compare the apolipoprotein composition of the three major lipoprotein classes in patients with metabolic syndrome to healthy controls.Very low density (VLDL, intermediate/low density (IDL/LDL, hereafter LDL, and high density lipoproteins (HDL fractions were isolated from plasma of 56 metabolic syndrome subjects and from 14 age-sex matched healthy volunteers. The apolipoprotein content of fractions was analyzed by one-dimensional (1D gel electrophoresis with confirmation by a combination of mass spectrometry and biochemical assays.Metabolic syndrome patients differed from healthy controls in the following ways: (1 total plasma--apoA1 was lower, whereas apoB, apoC2, apoC3, and apoE were higher; (2 VLDL--apoB, apoC3, and apoE were increased; (3 LDL--apoC3 was increased, (4 HDL--associated constitutive serum amyloid A protein (SAA4 was reduced (p<0.05 vs. controls for all. In patients with metabolic syndrome, the most extensively glycosylated (di-sialylated isoform of apoC3 was reduced in VLDL, LDL, and HDL fractions by 17%, 30%, and 25%, respectively (p<0.01 vs. controls for all. Similarly, the glycosylated isoform of apoE was reduced in VLDL, LDL, and HDL fractions by 15%, 26%, and 37% (p<0.01 vs. controls for all. Finally, glycosylated isoform of SAA4 in HDL fraction was 42% lower in patients with metabolic syndrome compared with controls (p<0.001.Patients with metabolic syndrome displayed several changes in plasma apolipoprotein composition consistent with hypertriglyceridemia and low HDL cholesterol levels. Reduced glycosylation of apoC3, apoE and SAA4 are novel findings, the pathophysiological consequences of which remain to be determined.

  2. Role of N-glycosylation in renal betaine transport.

    Science.gov (United States)

    Schweikhard, Eva S; Burckhardt, Birgitta C; Joos, Friedericke; Fenollar-Ferrer, Cristina; Forrest, Lucy R; Kempson, Stephen A; Ziegler, Christine

    2015-09-01

    The osmolyte and folding chaperone betaine is transported by the renal Na(+)-coupled GABA (γ-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn(171) and Asn(183)) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin-Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn(171) and Asn(183) contributed equally to protein activity and substrate affinity. Substitution of Asn(171) and Asn(183) by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.

  3. Bridging the Gap between Glycosylation and Vesicle Traffic

    OpenAIRE

    Fisher, Peter; Ungar, Daniel

    2016-01-01

    Glycosylation is recognized as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil te...

  4. Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

    DEFF Research Database (Denmark)

    Norén, K; Hansen, Gert Helge; Clausen, H;

    1997-01-01

    In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...

  5. Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

    Directory of Open Access Journals (Sweden)

    Anne Dell

    2010-01-01

    Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

  6. Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes.

    Science.gov (United States)

    Dell, Anne; Galadari, Alaa; Sastre, Federico; Hitchen, Paul

    2010-01-01

    Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i) oligosaccharyltransferase (OST)-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii) stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii) OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv) stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring "en bloc" to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

  7. A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

    Science.gov (United States)

    del Val, Ioscani Jimenez; Polizzi, Karen M.; Kontoravdi, Cleo

    2016-01-01

    Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation. PMID:27345611

  8. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

    Science.gov (United States)

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E

    2017-02-03

    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  9. Bioprospecting of microalgae: Proper extraction followed by high performance liquid chromatographic-high resolution mass spectrometric fingerprinting as key tools for successful metabolom characterization.

    Science.gov (United States)

    Stranska-Zachariasova, Milena; Kastanek, Petr; Dzuman, Zbynek; Rubert, Josep; Godula, Michal; Hajslova, Jana

    2016-03-15

    Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC-HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water-aqueous methanol-hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC-HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species

  10. Lipoprotein Modification by Advanced Glycosylation Endproducts (AGEs): Role in Atherosclerosis.

    Science.gov (United States)

    Bucala, R

    1997-02-01

    Recent progress in our understanding of advanced glycosylation reactions in vivo has affirmed the hypothesis that these products play an important role in the evolution of both diabetic and nondiabetic vascular disease. Utilizing newly developed advanced glycosylation end-products (AGE)-specific enzyme-linked immunosorbent assay (ELISA) techniques, AGEs have been identified to be present on a variety of vascular wall, lipoprotein, and lipid constituents. Vascular wall AGEs contribute to vascular pathology by increasing vascular permeability, enhancing subintimal protein and lipoprotein deposition, and inactivating nitric oxide. Lipid-linked AGEs present in low-density lipoprotein (LDL) also have been shown to initiate oxidative modification, promoting oxidation reactions that may proceed without the involvement of free metals or other radical generating systems. AGE-specific ELISA analysis has demonstrated a significantly increased level of AGE-modified LDL in the plasma of diabetic patients when compared to normal controls. AGE-modification impairs LDL-receptor-mediated clearance mechanisms in vivo and may contribute to elevated LDL levels in patients with diabetes. This concept has been substantiated further by the recent clinical observations that administration of the advanced glycosylation inhibitor aminoguanidine to diabetic patients significantly decreases circulating LDL levels. (Trends Cardiovasc Med 1997;7:39-47). © 1997, Elsevier Science Inc.

  11. Hormone induced changes in lactase glycosylation in developing rat intestine.

    Science.gov (United States)

    Chaudhry, Kamaljit Kaur; Mahmood, Safrun; Mahmood, Akhtar

    2008-11-01

    Lactase exists in both soluble and membrane-bound forms in suckling rat intestine. The distribution of lactase and its glycosylated isoforms in response to thyroxine or cortisone administration has been studied in suckling rats. 75% of lactase activity was detected, associated with brush borders, compared to 24% in the soluble fraction of 8-day-old rats. Thyroxine treatment enhanced soluble lactase activity to 34%, whereas particulate fraction was reduced to 67% compared to controls. Cortisone administration reduced soluble lactase activity from 24% in controls to 12% with a concomitant increase in membrane-bound activity to 89%. Western blot analysis revealed lactase signal, corresponding to 220 kDa in both the soluble and membrane fractions, which corroborated the enzyme activity data. The elution pattern of papain solubilized lactase from agarose-Wheat Germ agglutinin, or Concanavalin A or Jacalin agglutinin columns was different in the suckling and adult rat intestines. Also the elution profile of lactase activity from agarose-lectin columns was modulated in cortisone, thyroxine, and insulin injected pups, which suggests differences in glycosylated isoforms of lactase under these conditions. These findings suggest the role of these hormones in inducing changes in lactase glycosylation during postnatal development of intestine, which may contribute to adult-type hypolactasia in rats.

  12. BioCreative Workshops for DOE Genome Sciences: Text Mining for Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cathy H. [Univ. of Delaware, Newark, DE (United States). Center for Bioinformatics and Computational Biology; Hirschman, Lynette [The MITRE Corporation, Bedford, MA (United States)

    2016-10-29

    The objective of this project was to host BioCreative workshops to define and develop text mining tasks to meet the needs of the Genome Sciences community, focusing on metadata information extraction in metagenomics. Following the successful introduction of metagenomics at the BioCreative IV workshop, members of the metagenomics community and BioCreative communities continued discussion to identify candidate topics for a BioCreative metagenomics track for BioCreative V. Of particular interest was the capture of environmental and isolation source information from text. The outcome was to form a “community of interest” around work on the interactive EXTRACT system, which supported interactive tagging of environmental and species data. This experiment is included in the BioCreative V virtual issue of Database. In addition, there was broad participation by members of the metagenomics community in the panels held at BioCreative V, leading to valuable exchanges between the text mining developers and members of the metagenomics research community. These exchanges are reflected in a number of the overview and perspective pieces also being captured in the BioCreative V virtual issue. Overall, this conversation has exposed the metagenomics researchers to the possibilities of text mining, and educated the text mining developers to the specific needs of the metagenomics community.

  13. Taxonomic and functional assignment of cloned sequences from high Andean forest soil metagenome

    NARCIS (Netherlands)

    Montaña, José Salvador; Jiménez Avella, Diego; Angel, Tatiana; Hernández, Mónica; Baena, Sandra

    2012-01-01

    Total metagenomic DNA was isolated from high Andean forest soil and subjected to taxonomical and functional composition analyses by means of clone library generation and sequencing. The obtained yield of 1.7 μg of DNA/g of soil was used to construct a metagenomic library of approximately 20,000 clon

  14. Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening▿ †

    OpenAIRE

    2007-01-01

    A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in Escherichia coli. As proof of principle, several anti-Bacillus lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.

  15. Exploring Antibiotic Resistance Genes and Metal Resistance Genes in Plasmid Metagenomes from Wastewater Treatment Plants

    Directory of Open Access Journals (Sweden)

    An-Dong eLi

    2015-09-01

    Full Text Available Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer, they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge and digested sludge of two wastewater treatment plants. Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs database and a metal resistance genes (MRGs database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes and metal resistance genes (23 out of a total 23 types on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs than the activated sludge and the digested sludge metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in wastewater treatment plants could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  16. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

    Science.gov (United States)

    Li, An-Dong; Li, Li-Guan; Zhang, Tong

    2015-01-01

    Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  17. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    NARCIS (Netherlands)

    Nielsen, H.B.; Almeida, M.; Sierakowska Juncker, A.; Rasmussen, S.; Li, J.; Sunagawa, S.; Plichta, D.R.; Gautier, L.; Pedersen, A.G.; Chatelier, Le E.; Pelletier, E.; Bonde, I.; Nielsen, T.; Manichanh, C.; Arumugam, M.; Batto, J.M.; Quintanilha dos Santos, M.B.; Blom, N.; Borruel, N.; Burgdorf, K.S.; Boumezbeur, F.; Casellas, F.; Doré, J.; Dworzynski, P.; Guarner, F.; Hansen, T.; Hildebrand, F.; Kaas, R.S.; Kennedy, S.; Kristiansen, K.; Kultima, J.R.; Leonard, P.; Levenez, F.; Lund, O.; Moumen, B.; Paslier, Le D.; Pons, N.; Pedersen, O.; Prifti, E.; Qin, J.; Raes, J.; Sørensen, S.; Tap, J.; Tims, S.; Ussery, D.W.; Yamada, T.; Jamet, A.; Mérieux, A.; Cultrone, A.; Torrejon, A.; Quinquis, B.; Brechot, C.; Delorme, C.; M'Rini, C.; Vos, de W.M.; Maguin, E.; Varela, E.; Guedon, E.; Gwen, F.; Haimet, F.; Artiguenave, F.; Vandemeulebrouck, G.; Denariaz, G.; Khaci, G.; Blottière, H.; Knol, J.; Weissenbach, J.; Hylckama Vlieg, van J.E.; Torben, J.; Parkhil, J.; Turner, K.; Guchte, van de M.; Antolin, M.; Rescigno, M.; Kleerebezem, M.; Derrien, M.; Galleron, N.; Sanchez, N.; Grarup, N.; Veiga, P.; Oozeer, R.; Dervyn, R.; Layec, S.; Bruls, T.; Winogradski, Y.; Zoetendal, E.G.; Renault, D.; Sicheritz-Ponten,; Bork, P.; Wang, J.; Brunak, S.; Ehrlich, S.D.

    2014-01-01

    Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as part

  18. The great screen anomaly--a new frontier in product discovery through functional metagenomics.

    Science.gov (United States)

    Ekkers, David Matthias; Cretoiu, Mariana Silvia; Kielak, Anna Maria; Elsas, Jan Dirk van

    2012-02-01

    Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

  19. Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

    NARCIS (Netherlands)

    Hall, Richard J; Draper, Jenny L; Nielsen, Fiona G G; Dutilh, Bas E

    2015-01-01

    Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used

  20. Different glycosyltransferases are involved in lipid glycosylation and protein N-glycosylation in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Naparstek, Shai; Vinagradov, Evguenii; Eichler, Jerry

    2010-07-01

    Both the lipid and the protein components of biological membranes can be modified by the covalent addition of polysaccharides. Whereas eukaryal and bacterial pathways of lipid and protein glycosylation are relatively well defined, considerably less is known of the parallel processes in Archaea. Recent efforts have identified glycosyltransferases involved in N-glycosylation of the surface-layer glycoprotein of the halophilic archaeon Haloferax volcanii. In the present study, the involvement of these same glycosyltransferases in the biosynthesis of Hfx. volcanii glycolipids was considered by performing nuclear magnetic resonance analysis of the glycolipid fraction of Hfx. volcanii cells deleted of genes encoding those glycosyltransferases, as well as the oligosaccharyltransferase, AglB. The results reveal that different glycosyltransferases are involved in the biosynthesis of N-linked glycoproteins and glycolipids in Archaea.

  1. Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities

    DEFF Research Database (Denmark)

    Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

    Albertsen Keywords: Metagenomics; Accumulibacter; Micro-diversity; Enhanced Biological Phosphorus Removal Introduction Metagenomics, or environmental genomics, provides comprehensive information about the entire microbial community of a certain ecosystem, e.g. a wastewater treatment plant. So far......, metagenomic analyses have been hampered by high costs and high level of expertise needed to conduct the investigations, but it is changing now with development of new technologies allowing analyses of billions of DNA sequences (deep-sequencing) and user-friendly pipelines for analyses of the huge data sets...... in Albertsen et al., (2011). Results and Discussion We sequenced two metagenomes from Aalborg East and West EBPR wastewater treatment plants at a depth of 12 and 8 Gb using Illumina short read sequencing. The EBPR plants form a distinct group when compared to metagenomes from a wide range of environments, both...

  2. Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

    Science.gov (United States)

    Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

    2012-12-01

    Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

  3. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Science.gov (United States)

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  4. Soil-specific limitations for access and analysis of soil microbial communities by metagenomics.

    Science.gov (United States)

    Lombard, Nathalie; Prestat, Emmanuel; van Elsas, Jan Dirk; Simonet, Pascal

    2011-10-01

    Metagenomics approaches represent an important way to acquire information on the microbial communities present in complex environments like soil. However, to what extent do these approaches provide us with a true picture of soil microbial diversity? Soil is a challenging environment to work with. Its physicochemical properties affect microbial distributions inside the soil matrix, metagenome extraction and its subsequent analyses. To better understand the bias inherent to soil metagenome 'processing', we focus on soil physicochemical properties and their effects on the perceived bacterial distribution. In the light of this information, each step of soil metagenome processing is then discussed, with an emphasis on strategies for optimal soil sampling. Then, the interaction of cells and DNA with the soil matrix and the consequences for microbial DNA extraction are examined. Soil DNA extraction methods are compared and the veracity of the microbial profiles obtained is discussed. Finally, soil metagenomic sequence analysis and exploitation methods are reviewed.

  5. MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes.

    Science.gov (United States)

    Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2016-01-01

    The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a 'dark matter.' We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about the

  6. Reconstructing the genomic content of microbiome taxa through shotgun metagenomic deconvolution.

    Science.gov (United States)

    Carr, Rogan; Shen-Orr, Shai S; Borenstein, Elhanan

    2013-01-01

    Metagenomics has transformed our understanding of the microbial world, allowing researchers to bypass the need to isolate and culture individual taxa and to directly characterize both the taxonomic and gene compositions of environmental samples. However, associating the genes found in a metagenomic sample with the specific taxa of origin remains a critical challenge. Existing binning methods, based on nucleotide composition or alignment to reference genomes allow only a coarse-grained classification and rely heavily on the availability of sequenced genomes from closely related taxa. Here, we introduce a novel computational framework, integrating variation in gene abundances across multiple samples with taxonomic abundance data to deconvolve metagenomic samples into taxa-specific gene profiles and to reconstruct the genomic content of community members. This assembly-free method is not bounded by various factors limiting previously described methods of metagenomic binning or metagenomic assembly and represents a fundamentally different approach to metagenomic-based genome reconstruction. An implementation of this framework is available at http://elbo.gs.washington.edu/software.html. We first describe the mathematical foundations of our framework and discuss considerations for implementing its various components. We demonstrate the ability of this framework to accurately deconvolve a set of metagenomic samples and to recover the gene content of individual taxa using synthetic metagenomic samples. We specifically characterize determinants of prediction accuracy and examine the impact of annotation errors on the reconstructed genomes. We finally apply metagenomic deconvolution to samples from the Human Microbiome Project, successfully reconstructing genus-level genomic content of various microbial genera, based solely on variation in gene count. These reconstructed genera are shown to correctly capture genus-specific properties. With the accumulation of metagenomic

  7. MG-Digger: an automated pipeline to search for giant virus-related sequences in metagenomes

    Directory of Open Access Journals (Sweden)

    Jonathan eVerneau

    2016-03-01

    Full Text Available The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter’. We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase pairs were collected, processed and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and five virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate hundreds of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are

  8. Importance of N-Glycosylation on CD147 for Its Biological Functions

    Directory of Open Access Journals (Sweden)

    Yang Bai

    2014-04-01

    Full Text Available Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

  9. Importance of N-glycosylation on CD147 for its biological functions.

    Science.gov (United States)

    Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

    2014-04-15

    Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

  10. Interrelationship of steric stabilization and self-crowding of a glycosylated protein

    DEFF Research Database (Denmark)

    Høiberg-Nielsen, Rasmus; Westh, Peter; Skov, L.K.;

    2009-01-01

    In the eukaryotic cell, protein glycosylation takes place in the crowded environment of the endoplasmatic reticulum. With the purpose of elucidating the impact of high concentration on the interactions of glycoproteins, we have conducted a series of small-angle x-ray scattering experiments...... from the glycans contribute to steric stabilization of the protein, and that glycosylation helps to sustain repulsive electrostatic interactions under crowded conditions. In combination, this aids in stabilizing high concentrations of glycosylated proteins....

  11. Assembling the Marine Metagenome, One Cell at a Time

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Xie, Gary; Copeland, Alex; Gonzalez, Jose M.; Han, Cliff; Kiss, Hajnalka; Saw, Jimmy H.; Senin, Pavel; Yang, Chi; Chatterji, Sourav; Cheng, Jan-Fang; Eisen, Jonathan A.; Sieracki, Michael E.; Stepanauskas, Ramunas

    2010-06-24

    The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91percent and 78percent, respectively. Only 0.24percent of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured

  12. Preparation of metagenomic libraries from naturally occurring marine viruses.

    Science.gov (United States)

    Solonenko, Sergei A; Sullivan, Matthew B

    2013-01-01

    Microbes are now well recognized as major drivers of the biogeochemical cycling that fuels the Earth, and their viruses (phages) are known to be abundant and important in microbial mortality, horizontal gene transfer, and modulating microbial metabolic output. Investigation of environmental phages has been frustrated by an inability to culture the vast majority of naturally occurring diversity coupled with the lack of robust, quantitative, culture-independent methods for studying this uncultured majority. However, for double-stranded DNA phages, a quantitative viral metagenomic sample-to-sequence workflow now exists. Here, we review these advances with special emphasis on the technical details of preparing DNA sequencing libraries for metagenomic sequencing from environmentally relevant low-input DNA samples. Library preparation steps broadly involve manipulating the sample DNA by fragmentation, end repair and adaptor ligation, size fractionation, and amplification. One critical area of future research and development is parallel advances for alternate nucleic acid types such as single-stranded DNA and RNA viruses that are also abundant in nature. Combinations of recent advances in fragmentation (e.g., acoustic shearing and tagmentation), ligation reactions (adaptor-to-template ratio reference table availability), size fractionation (non-gel-sizing), and amplification (linear amplification for deep sequencing and linker amplification protocols) enhance our ability to generate quantitatively representative metagenomic datasets from low-input DNA samples. Such datasets are already providing new insights into the role of viruses in marine systems and will continue to do so as new environments are explored and synergies and paradigms emerge from large-scale comparative analyses.

  13. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Directory of Open Access Journals (Sweden)

    Laura Millares

    Full Text Available The course of chronic obstructive pulmonary disease (COPD is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST, and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3 vs 3.6 (3.3-6.9, p = 0.012; and 1.8 (0-3.3 vs 3.6 (1.8-5.1, p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5 vs 0 (0-0.5, p = 0.043; and 7 (6.4-9 vs 5.9 (6.3-6.1, p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  14. Diel Metagenomics and Metatranscriptomics of Elkhorn Slough Hypersaline Microbial Mat

    Science.gov (United States)

    Lee, J.; Detweiler, A. M.; Everroad, R. C.; Bebout, L. E.; Weber, P. K.; Pett-Ridge, J.; Bebout, B.

    2014-12-01

    To understand the variation in gene expression associated with the daytime oxygenic phototrophic and nighttime fermentation regimes seen in hypersaline microbial mats, a contiguous mat piece was subjected to sampling at regular intervals over a 24-hour diel period. Additionally, to understand the impact of sulfate reduction on biohydrogen consumption, molybdate was added to a parallel experiment in the same run. 4 metagenome and 12 metatranscriptome Illumina HiSeq lanes were completed over day / night, and control / molybdate experiments. Preliminary comparative examination of noon and midnight metatranscriptomic samples mapped using bowtie2 to reference genomes has revealed several notable results about the dominant mat-building cyanobacterium Microcoleus chthonoplastes PCC 7420. Dominant cyanobacterium M. chthonoplastes PCC 7420 shows expression in several pathways for nitrogen scavenging, including nitrogen fixation. Reads mapped to M. chthonoplastes PCC 7420 shows expression of two starch storage and utilization pathways, one as a starch-trehalose-maltose-glucose pathway, another through UDP-glucose-cellulose-β-1,4 glucan-glucose pathway. The overall trend of gene expression was primarily light driven up-regulation followed by down-regulation in dark, while much of the remaining expression profile appears to be constitutive. Co-assembly of quality-controlled reads from 4 metagenomes was performed using Ray Meta with progressively smaller K-mer sizes, with bins identified and filtered using principal component analysis of coverages from all libraries and a %GC filter, followed by reassembly of the remaining co-assembly reads and binned reads. Despite having relatively similar abundance profiles in each metagenome, this binning approach was able to distinctly resolve bins from dominant taxa, but also sulfate reducing bacteria that are desired for understanding molybdate inhibition. Bins generated from this iterative assembly process will be used for downstream

  15. Assembling The Marine Metagenome, One Cell At A Time

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Gang [Los Alamos National Laboratory; Han, Shunsheng [Los Alamos National Laboratory; Kiss, Hajnalka [Los Alamos National Laboratory; Saw, Jimmy [Los Alamos National Laboratory; Senin, Pavel [Los Alamos National Laboratory; Woyke, Tanja [DOE JOINT GENOME INAT.; Copeland, Alex [DOE JOINT GENSOME INST.; Gonzalez, Jose [UNIV OF LAGUNA, SPAIN; Chatterji, Sourav [DOE JOINT GENSOME INST.; Cheng, Jan - Fang [DOE JOINT GENSOME INST.; Eisen, Jonathan A [DOE JOINT GENOME INST.; Sieracki, Michael E [UNIV OF CA-DAVIS; Stepanauskas, Ramunas [BIGELOW LAB

    2008-01-01

    The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex

  16. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Science.gov (United States)

    Millares, Laura; Pérez-Brocal, Vicente; Ferrari, Rafaela; Gallego, Miguel; Pomares, Xavier; García-Núñez, Marian; Montón, Concepción; Capilla, Silvia; Monsó, Eduard; Moya, Andrés

    2015-01-01

    The course of chronic obstructive pulmonary disease (COPD) is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST), and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes) did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index) showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3) vs 3.6 (3.3-6.9), p = 0.012; and 1.8 (0-3.3) vs 3.6 (1.8-5.1), p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5) vs 0 (0-0.5), p = 0.043; and 7 (6.4-9) vs 5.9 (6.3-6.1), p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  17. Assembling the marine metagenome, one cell at a time.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    Full Text Available The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured

  18. Metagenomic Analysis of the Medicinal Leech Gut Microbiota

    Directory of Open Access Journals (Sweden)

    Michele A Maltz

    2014-04-01

    Full Text Available There are trillions of microbes found throughout the human body and they exceed the number of eukaryotic cells by ten-fold. Metagenomic studies have revealed that the majority of these microbes are found within the gut, playing an important role in the host’s digestion and nutrition. The complexity of the animal digestive tract, unculturable microbes and the lack of genetic tools for most culturable microbes make it challenging to explore the nature of theses microbial interactions within this niche. The medicinal leech, Hirudo verbana, has been shown to be a useful tool in overcoming these challenges, due to the simplicity of the microbiome and the availability of genetic tools for one of the two dominant gut symbionts, Aeromonas veronii. In this study, we utilize 16S rRNA gene pyrosequencing to further explore the microbial composition of the leech digestive tract, confirming the dominance of two taxa, the Rikenella-like bacterium and A. veronii. The deep sequencing approach revealed the presence of additional members of the microbial community that suggests the presence of a moderately complex microbial community with a richness of 36 taxa. The presence of a Proteus strain as a newly identified resident in the leech crop was confirmed using fluorescence in situ hybridization (FISH. The metagenome of this community was also pyrosequenced and the contigs were binned into the following taxonomic groups: Rikenella-like (3.1 MB, Aeromonas (4.5 MB, Proteus (2.9 MB, Clostridium (1.8 MB, Eryspelothrix (0.96 MB, Desulfovibrio (0.14 MB and Fusobacterium (0.27 MB. Functional analyses on the leech gut symbionts were explored using the metagenomic data and MG-RAST. A comparison of the COG and KEGG categories of the leech gut metagenome to that of other animal digestive-tract microbiomes revealed that the leech digestive-tract had a similar metabolic potential to the human digestive-tract, supporting the usefulness of this system as a model for studying

  19. Binning sequences using very sparse labels within a metagenome

    Directory of Open Access Journals (Sweden)

    Halgamuge Saman K

    2008-04-01

    Full Text Available Abstract Background In metagenomic studies, a process called binning is necessary to assign contigs that belong to multiple species to their respective phylogenetic groups. Most of the current methods of binning, such as BLAST, k-mer and PhyloPythia, involve assigning sequence fragments by comparing sequence similarity or sequence composition with already-sequenced genomes that are still far from comprehensive. We propose a semi-supervised seeding method for binning that does not depend on knowledge of completed genomes. Instead, it extracts the flanking sequences of highly conserved 16S rRNA from the metagenome and uses them as seeds (labels to assign other reads based on their compositional similarity. Results The proposed seeding method is implemented on an unsupervised Growing Self-Organising Map (GSOM, and called Seeded GSOM (S-GSOM. We compared it with four well-known semi-supervised learning methods in a preliminary test, separating random-length prokaryotic sequence fragments sampled from the NCBI genome database. We identified the flanking sequences of the highly conserved 16S rRNA as suitable seeds that could be used to group the sequence fragments according to their species. S-GSOM showed superior performance compared to the semi-supervised methods tested. Additionally, S-GSOM may also be used to visually identify some species that do not have seeds. The proposed method was then applied to simulated metagenomic datasets using two different confidence threshold settings and compared with PhyloPythia, k-mer and BLAST. At the reference taxonomic level Order, S-GSOM outperformed all k-mer and BLAST results and showed comparable results with PhyloPythia for each of the corresponding confidence settings, where S-GSOM performed better than PhyloPythia in the ≥ 10 reads datasets and comparable in the ≥ 8 kb benchmark tests. Conclusion In the task of binning using semi-supervised learning methods, results indicate S-GSOM to be the best of

  20. Metagenomics and development of the gut microbiota in infants

    DEFF Research Database (Denmark)

    Vallès, Y.; Gosalbes, M. J.; de Vries, Lisbeth Elvira

    2012-01-01

    Clin Microbiol Infect 2012; 18 (Suppl. 4): 21–26 The establishment of a balanced intestinal microbiota is essential for numerous aspects of human health, yet the microbial colonization of the gastrointestinal tract of infants is both complex and highly variable among individuals. In addition......, the gastrointestinal tract microbiota is often exposed to antibiotics, and may be an important reservoir of resistant strains and of transferable resistance genes from early infancy. We are investigating by means of diverse metagenomic approaches several areas of microbiota development in infants, including...

  1. Phylogeny, culturing, and metagenomics of the human gut microbiota.

    Science.gov (United States)

    Walker, Alan W; Duncan, Sylvia H; Louis, Petra; Flint, Harry J

    2014-05-01

    The human intestinal tract is colonised by a complex community of microbes, which can have major impacts on host health. Recent research on the gut microbiota has largely been driven by the advent of modern sequence-based techniques, such as metagenomics. Although these are powerful and valuable tools, they have limitations. Traditional culturing and phylogeny can mitigate some of these limitations, either by expanding reference databases or by assigning functionality to specific microbial lineages. As such, culture and phylogeny will continue to have crucially important roles in human microbiota research, and will be required for the development of novel therapeutics.

  2. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn;

    2010-01-01

    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence......, from faecal samples of 124 European individuals. The gene set, ,150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes...

  3. Separating metagenomic short reads into genomes via clustering

    Directory of Open Access Journals (Sweden)

    Tanaseichuk Olga

    2012-09-01

    Full Text Available Abstract Background The metagenomics approach allows the simultaneous sequencing of all genomes in an environmental sample. This results in high complexity datasets, where in addition to repeats and sequencing errors, the number of genomes and their abundance ratios are unknown. Recently developed next-generation sequencing (NGS technologies significantly improve the sequencing efficiency and cost. On the other hand, they result in shorter reads, which makes the separation of reads from different species harder. Among the existing computational tools for metagenomic analysis, there are similarity-based methods that use reference databases to align reads and composition-based methods that use composition patterns (i.e., frequencies of short words or l-mers to cluster reads. Similarity-based methods are unable to classify reads from unknown species without close references (which constitute the majority of reads. Since composition patterns are preserved only in significantly large fragments, composition-based tools cannot be used for very short reads, which becomes a significant limitation with the development of NGS. A recently proposed algorithm, AbundanceBin, introduced another method that bins reads based on predicted abundances of the genomes sequenced. However, it does not separate reads from genomes of similar abundance levels. Results In this work, we present a two-phase heuristic algorithm for separating short paired-end reads from different genomes in a metagenomic dataset. We use the observation that most of the l-mers belong to unique genomes when l is sufficiently large. The first phase of the algorithm results in clusters of l-mers each of which belongs to one genome. During the second phase, clusters are merged based on l-mer repeat information. These final clusters are used to assign reads. The algorithm could handle very short reads and sequencing errors. It is initially designed for genomes with similar abundance levels and then

  4. Synthesis of glycosyl-amino acids of biological interest; Sintese de glicoaminoacidos de interesse biologico

    Energy Technology Data Exchange (ETDEWEB)

    Campo, Vanessa Leiria; Carvalho, Ivone [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Ciencias Farmaceuticas]. E-mail: carronal@usp.br

    2008-07-01

    This work describes the synthesis of the glycosylated amino acids {alpha}GlcNAc-Thr, {beta}GlcNAc-Thr and {alpha}LacNAc-Thr by the glycosylation reaction of the amino acid threonine with the corresponding glycosyl donors {alpha}GlcNAcCl and {alpha}LacN3Cl. The glycosylated amino acids containing the sugar units {alpha}-D-GlcNAc and {alpha}-D-LacNAc O-linked to threonine amino acids are related to O-glycans found in mucins of the parasite Trypanosoma cruzi, while the corresponding {beta}-D-GlcNAc isomer is involved in cellular signaling events. (author)

  5. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing...... response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-¿ release, and antibody induction. Gal...

  6. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

    DEFF Research Database (Denmark)

    Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek;

    2004-01-01

    than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network...... could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

  7. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

    DEFF Research Database (Denmark)

    Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek;

    2005-01-01

    than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network...... could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

  8. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

    Institute of Scientific and Technical Information of China (English)

    LI Bi-feng; MAO Hua-ming; YANG Shu-Li; ZHU Ya-xin; GU Zhao-bing; CHEN Yuan; LENG Jing; GOU Xiao; FENG Li; LI Qing; XI Dong-mei

    2016-01-01

    Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as wel as reeds and other plant species, and grow to higher mature live weights than Yunnan Yelow cattle maintained in similar harsh environments. The aim of this study was to identify speciifc celulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. TheUmcel-1 gene was isolated from this library. Investigation of the celulase activity of 24 random clones led to the identiifcation of theUmcel-1 gene, which exhibited the most potent celulase activity. Sequencing theUmcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putativegene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the celulase (GenBank accession no. YP_004310852.1) fromClostridium lentocelum DSM 5427, with 44% identity and 62% similarity. TheUmcel-1 gene was heterologously expressed inEscherichia coli BL21, and recombinant Umcel-1 was puriifed. The activity of puriifed recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl celulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the ifrst evidence for a celulase produced by bacteria in gayal rumen.

  9. Optimization of a colorimetric assay for glycosylated human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Bohney, J.P.; Feldhoff, R.C.

    1986-05-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

  10. A Graph-Centric Approach for Metagenome-Guided Peptide and Protein Identification in Metaproteomics.

    Science.gov (United States)

    Tang, Haixu; Li, Sujun; Ye, Yuzhen

    2016-12-01

    Metaproteomic studies adopt the common bottom-up proteomics approach to investigate the protein composition and the dynamics of protein expression in microbial communities. When matched metagenomic and/or metatranscriptomic data of the microbial communities are available, metaproteomic data analyses often employ a metagenome-guided approach, in which complete or fragmental protein-coding genes are first directly predicted from metagenomic (and/or metatranscriptomic) sequences or from their assemblies, and the resulting protein sequences are then used as the reference database for peptide/protein identification from MS/MS spectra. This approach is often limited because protein coding genes predicted from metagenomes are incomplete and fragmental. In this paper, we present a graph-centric approach to improving metagenome-guided peptide and protein identification in metaproteomics. Our method exploits the de Bruijn graph structure reported by metagenome assembly algorithms to generate a comprehensive database of protein sequences encoded in the community. We tested our method using several public metaproteomic datasets with matched metagenomic and metatranscriptomic sequencing data acquired from complex microbial communities in a biological wastewater treatment plant. The results showed that many more peptides and proteins can be identified when assembly graphs were utilized, improving the characterization of the proteins expressed in the microbial communities. The additional proteins we identified contribute to the characterization of important pathways such as those involved in degradation of chemical hazards. Our tools are released as open-source software on github at https://github.com/COL-IU/Graph2Pro.

  11. Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial

    Directory of Open Access Journals (Sweden)

    Adina eHowe

    2015-07-01

    Full Text Available Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs, providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow.

  12. Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial).

    Science.gov (United States)

    Howe, Adina; Chain, Patrick S G

    2015-01-01

    Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs), providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow.

  13. WebMGA: a customizable web server for fast metagenomic sequence analysis

    Directory of Open Access Journals (Sweden)

    Niu Beifang

    2011-09-01

    Full Text Available Abstract Background The new field of metagenomics studies microorganism communities by culture-independent sequencing. With the advances in next-generation sequencing techniques, researchers are facing tremendous challenges in metagenomic data analysis due to huge quantity and high complexity of sequence data. Analyzing large datasets is extremely time-consuming; also metagenomic annotation involves a wide range of computational tools, which are difficult to be installed and maintained by common users. The tools provided by the few available web servers are also limited and have various constraints such as login requirement, long waiting time, inability to configure pipelines etc. Results We developed WebMGA, a customizable web server for fast metagenomic analysis. WebMGA includes over 20 commonly used tools such as ORF calling, sequence clustering, quality control of raw reads, removal of sequencing artifacts and contaminations, taxonomic analysis, functional annotation etc. WebMGA provides users with rapid metagenomic data analysis using fast and effective tools, which have been implemented to run in parallel on our local computer cluster. Users can access WebMGA through web browsers or programming scripts to perform individual analysis or to configure and run customized pipelines. WebMGA is freely available at http://weizhongli-lab.org/metagenomic-analysis. Conclusions WebMGA offers to researchers many fast and unique tools and great flexibility for complex metagenomic data analysis.

  14. MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

    Science.gov (United States)

    Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

    2012-12-07

    MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

  15. Use of whole genome shotgun metagenomics: a practical guide for the microbiome-minded physician scientist.

    Science.gov (United States)

    Ma, Jun; Prince, Amanda; Aagaard, Kjersti M

    2014-01-01

    Whole genome shotgun sequencing (WGS) has been increasingly recognized as the most comprehensive and robust approach for metagenomics research. When compared with 16S-based metagenomics, it offers the advantage of identification of species level taxonomy and the estimation of metabolic pathway activities from human and environmental samples. Several large-scale metagenomic projects have been recently conducted or are currently underway utilizing WGS. With the generation of vast amounts of data, the bioinformatics and computational analysis of WGS results become vital for the success of a metagenomics study. However, each step in the WGS data analysis, including metagenome assembly, gene prediction, taxonomy identification, function annotation, and pathway analysis, is complicated by the shear amount of data. Algorithms and tools have been developed specifically to handle WGS-generated metagenomics data with the hope of reducing the requirement on computational time and storage space. Here, we present an overview of the current state of metagenomics through WGS sequencing, challenges frequently encountered, and up-to-date solutions. Several applications that are uniquely applicable to microbiome studies in reproductive and perinatal medicine are also discussed.

  16. Orphan protein function and its relation to glycosylation

    DEFF Research Database (Denmark)

    Gupta, Ramneek; Jensen, Lars Juhl; Brunak, Søren

    2002-01-01

    , and inference of protein function by exploiting direct sequence similarity indeed goes a long way in describing a proteome’s functional capacity. However, at least 40% of the gene products in newly sequenced genomes typically remain uncharacterised. Proteins without an annotated function are also known...... as orphan proteins since they do not belong to a functionally characterised protein family. Many sequences must, therefore, be compared using their features rather than by direct comparison in the conventional sequence space. Here we focus on one such feature — glycosylation — that is common in eukaryotic...

  17. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  18. Protecting groups in carbohydrate chemistry: influence on stereoselectivity of glycosylations.

    Science.gov (United States)

    Guo, Jian; Ye, Xin-Shan

    2010-10-20

    Saccharides are polyhydroxy compounds, and their synthesis requires complex protecting group manipulations. Protecting groups are usually used to temporarily mask a functional group which may interfere with a certain reaction, but protecting groups in carbohydrate chemistry do more than protecting groups usually do. Particularly, protecting groups can participate in reactions directly or indirectly, thus affecting the stereochemical outcomes, which is important for synthesis of oligosaccharides. Herein we present an overview of recent advances in protecting groups influencing stereoselectivity in glycosylation reactions, including participating protecting groups, and conformation-constraining protecting groups in general.

  19. Gene prediction in metagenomic fragments: A large scale machine learning approach

    Directory of Open Access Journals (Sweden)

    Morgenstern Burkhard

    2008-04-01

    Full Text Available Abstract Background Metagenomics is an approach to the characterization of microbial genomes via the direct isolation of genomic sequences from the environment without prior cultivation. The amount of metagenomic sequence data is growing fast while computational methods for metagenome analysis are still in their infancy. In contrast to genomic sequences of single species, which can usually be assembled and analyzed by many available methods, a large proportion of metagenome data remains as unassembled anonymous sequencing reads. One of the aims of all metagenomic sequencing projects is the identification of novel genes. Short length, for example, Sanger sequencing yields on average 700 bp fragments, and unknown phylogenetic origin of most fragments require approaches to gene prediction that are different from the currently available methods for genomes of single species. In particular, the large size of metagenomic samples requires fast and accurate methods with small numbers of false positive predictions. Results We introduce a novel gene prediction algorithm for metagenomic fragments based on a two-stage machine learning approach. In the first stage, we use linear discriminants for monocodon usage, dicodon usage and translation initiation sites to extract features from DNA sequences. In the second stage, an artificial neural network combines these features with open reading frame length and fragment GC-content to compute the probability that this open reading frame encodes a protein. This probability is used for the classification and scoring of gene candidates. With large scale training, our method provides fast single fragment predictions with good sensitivity and specificity on artificially fragmented genomic DNA. Additionally, this method is able to predict translation initiation sites accurately and distinguishes complete from incomplete genes with high reliability. Conclusion Large scale machine learning methods are well-suited for gene

  20. Comparative metagenome of a stream impacted by the urbanization phenomenon

    Directory of Open Access Journals (Sweden)

    Julliane Dutra Medeiros

    Full Text Available Abstract Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

  1. Metagenomic characterization of viral communities in Goseong Bay, Korea

    Science.gov (United States)

    Hwang, Jinik; Park, So Yun; Park, Mirye; Lee, Sukchan; Jo, Yeonhwa; Cho, Won Kyong; Lee, Taek-Kyun

    2016-12-01

    In this study, seawater samples were collected from Goseong Bay, Korea in March 2014 and viral populations were examined by metagenomics assembly. Enrichment of marine viral particles using FeCl3 followed by next-generation sequencing produced numerous sequences. De novo assembly and BLAST search showed that most of the obtained contigs were unknown sequences and only 0.74% of sequences were associated with known viruses. As a result, 138 viruses, including bacteriophages (87%), viruses infecting algae and others (13%) were identified. The identified 138 viruses were divided into 11 orders, 14 families, 34 genera, and 133 species. The dominant viruses were Pelagibacter phage HTVC010P and Roseobacter phage SIO1. The viruses infecting algae, including the Ostreococcus species, accounted for 9.4% of total identified viruses. In addition, we identified pathogenic herpes viruses infecting fishes and giant viruses infecting parasitic acanthamoeba species. This is a comprehensive study to reveal the viral populations in the Goseong Bay using metagenomics. The information associated with the marine viral community in Goseong Bay, Korea will be useful for comparative analysis in other marine viral communities.

  2. Diversity and genome dynamics of marine cyanophages using metagenomic analyses.

    Science.gov (United States)

    Ma, Yingfei; Allen, Lisa Zeigler; Palenik, Brian

    2014-12-01

    Cyanophages are abundant in the oceanic environment and directly impact cyanobacterial distributions, physiological processes and evolution. Two samples collected from coastal Maine in July and September 2009 were enriched for Synechococcus cells using flow cytometry and examined through metagenomic sequencing. Homology-based sequence prediction indicated cyanophages, largely myoviruses, accounted for almost half the reads and provided insights into environmental infection events. T4-phage core-gene phylogenetic reconstruction revealed unique diversity among uncultured cyanophages and reference isolates resulting in identification of a new phylogenetic cluster. Genomic comparison of reference cyanophage strains S-SM2 and Syn1 with putative homologous contigs recovered from metagenomes provided evidence that gene insertion, deletion and recombination have occurred among, and are likely important for diversification of, natural populations. Identification of putative genetic exchange between cyanophage and non-cyanophage viruses, i.e. Micromonas virus and Pelagibacter phage, supports hypotheses related to a significant role for viruses in mediating transfer of genetic material between taxonomically diverse organisms with overlapping ecological niches.

  3. Metagenomics reveals flavour metabolic network of cereal vinegar microbiota.

    Science.gov (United States)

    Wu, Lin-Huan; Lu, Zhen-Ming; Zhang, Xiao-Juan; Wang, Zong-Min; Yu, Yong-Jian; Shi, Jin-Song; Xu, Zheng-Hong

    2017-04-01

    Multispecies microbial community formed through centuries of repeated batch acetic acid fermentation (AAF) is crucial for the flavour quality of traditional vinegar produced from cereals. However, the metabolism to generate and/or formulate the essential flavours by the multispecies microbial community is hardly understood. Here we used metagenomic approach to clarify in situ metabolic network of key microbes responsible for flavour synthesis of a typical cereal vinegar, Zhenjiang aromatic vinegar, produced by solid-state fermentation. First, we identified 3 organic acids, 7 amino acids, and 20 volatiles as dominant vinegar metabolites. Second, we revealed taxonomic and functional composition of the microbiota by metagenomic shotgun sequencing. A total of 86 201 predicted protein-coding genes from 35 phyla (951 genera) were involved in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Metabolism (42.3%), Genetic Information Processing (28.3%), and Environmental Information Processing (10.1%). Furthermore, a metabolic network for substrate breakdown and dominant flavour formation in vinegar microbiota was constructed, and microbial distribution discrepancy in different metabolic pathways was charted. This study helps elucidating different metabolic roles of microbes during flavour formation in vinegar microbiota.

  4. Metagenome fragment classification using N-mer frequency profiles.

    Science.gov (United States)

    Rosen, Gail; Garbarine, Elaine; Caseiro, Diamantino; Polikar, Robi; Sokhansanj, Bahrad

    2008-01-01

    A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique N-mer frequency profiles of 635 microbial genomes publicly available as of February 2008. These profiles are used to train a naive Bayes classifier (NBC) that can be used to identify the genome of any fragment. We show that our method is comparable to BLAST for small 25 bp fragments but does not have the ambiguity of BLAST's tied top scores. We demonstrate that this approach is scalable to identify any fragment from hundreds of genomes. It also performs quite well at the strain, species, and genera levels and achieves strain resolution despite classifying ubiquitous genomic fragments (gene and nongene regions). Cross-validation analysis demonstrates that species-accuracy achieves 90% for highly-represented species containing an average of 8 strains. We demonstrate that such a tool can be used on the Sargasso Sea dataset, and our analysis shows that NBC can be further enhanced.

  5. Tracking Strains in the Microbiome: Insights from Metagenomics and Models.

    Science.gov (United States)

    Brito, Ilana L; Alm, Eric J

    2016-01-01

    Transmission usually refers to the movement of pathogenic organisms. Yet, commensal microbes that inhabit the human body also move between individuals and environments. Surprisingly little is known about the transmission of these endogenous microbes, despite increasing realizations of their importance for human health. The health impacts arising from the transmission of commensal bacteria range widely, from the prevention of autoimmune disorders to the spread of antibiotic resistance genes. Despite this importance, there are outstanding basic questions: what is the fraction of the microbiome that is transmissible? What are the primary mechanisms of transmission? Which organisms are the most highly transmissible? Higher resolution genomic data is required to accurately link microbial sources (such as environmental reservoirs or other individuals) with sinks (such as a single person's microbiome). New computational advances enable strain-level resolution of organisms from shotgun metagenomic data, allowing the transmission of strains to be followed over time and after discrete exposure events. Here, we highlight the latest techniques that reveal strain-level resolution from raw metagenomic reads and new studies that are tracking strains across people and environments. We also propose how models of pathogenic transmission may be applied to study the movement of commensals between microbial communities.

  6. Metagenomes of the picoalga Bathycoccus from the Chile coastal upwelling.

    Directory of Open Access Journals (Sweden)

    Daniel Vaulot

    Full Text Available Among small photosynthetic eukaryotes that play a key role in oceanic food webs, picoplanktonic Mamiellophyceae such as Bathycoccus, Micromonas, and Ostreococcus are particularly important in coastal regions. By using a combination of cell sorting by flow cytometry, whole genome amplification (WGA, and 454 pyrosequencing, we obtained metagenomic data for two natural picophytoplankton populations from the coastal upwelling waters off central Chile. About 60% of the reads of each sample could be mapped to the genome of Bathycoccus strain from the Mediterranean Sea (RCC1105, representing a total of 9 Mbp (sample T142 and 13 Mbp (sample T149 of non-redundant Bathycoccus genome sequences. WGA did not amplify all regions uniformly, resulting in unequal coverage along a given chromosome and between chromosomes. The identity at the DNA level between the metagenomes and the cultured genome was very high (96.3% identical bases for the three larger chromosomes over a 360 kbp alignment. At least two to three different genotypes seemed to be present in each natural sample based on read mapping to Bathycoccus RCC1105 genome.

  7. [Metagenomics as a Tool for the Investigation of Uncultured Microorganisms].

    Science.gov (United States)

    Ravin, N V; Mardanova, A V; Skryabin, K G

    2015-05-01

    Uncultured microorganisms represent a significant part of the Earth's biodiversity. Natural ecosystems contain less than 0.1-1% of the microorganisms that can be cultured in the laboratory. Therefore, new methodological approaches are required for the identification and description of uncultured microorganisms, for studies of their genetic diversity and the structure of microbial associations, and for an understanding of their ecological importance in the biosphere. Metagenomics, a method of analyzing the collective genome.of a microbial community without cultivation, makes it possible to unravel fundamental matters of the microbiology and ecology of microorganisms. Another efficient method of analysis of uncultured forms of microorganisms is "single cell genomics," which involves the isolation of single cells from microbial communities and the sequencing of their genomes. Developed in the last decade, the high throughput technologies of next-generation sequencing provide important input into the investigation of genome reconstruction for all of the microorganisms residing and interacting within ecosystems. This review describes the major methodological approaches used in metagenomic analysis of microbial communities, as well as accomplishments in the search for new uncultured microorganism, the unraveling of their genomes, and an elucidation of their role in ecosystems.

  8. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    Science.gov (United States)

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  9. Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

    Science.gov (United States)

    Martínez, Asunción; Osburne, Marcia S

    2013-01-01

    One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.

  10. Glycosylation of resveratrol protects it from enzymic oxidation.

    Science.gov (United States)

    Regev-Shoshani, Gilly; Shoseyov, Oded; Bilkis, Itzhak; Kerem, Zohar

    2003-01-01

    Plant polyphenols, including dietary polyphenols such as resveratrol, are important components in the plant antioxidant and defence systems. They are also known to exert beneficial effects on human health through diet. As they are produced, these polyphenols may be subjected to deleterious enzymic oxidation by the plant polyphenol oxidases. They are generally synthesized as glycosides like 5,4'-dihydroxystilbene-3-O-beta-D-glucopyranoside, the 3-glucoside of resveratrol. The effects of the glycosylation and methylation of the parent resveratrol on its enzymic oxidation were studied. Methyl and glucosyl derivatives were synthesized using simple one-step methodologies. The kinetics of their enzymic oxidation by tyrosinases were defined. Substitution at the p-hydroxy group, by either glucose or methyl, abolished enzymic oxidation by both mushroom and grape tyrosinases. Substitution at the m-hydroxy group with methyl had a small effect, but substitution with glucose resulted in a much lower affinity of the enzymes for the glycoside. We suggest that glycosylation of polyphenols in nature helps to protect these vital molecules from enzymic oxidation, extending their half-life in the cell and maintaining their beneficial antioxidant capacity and biological properties. PMID:12697026

  11. Heterogeneity of IgG glycosylation in adult periodontal disease.

    Science.gov (United States)

    Novak, J; Tomana, M; Shah, G R; Brown, R; Mestecky, J

    2005-10-01

    Periodontal disease is a chronic inflammatory disease of bacterial etiology. In many other chronic inflammatory diseases, IgG glycans are galactose-deficient and thus capable of complement activation through the lectin pathway. In this study, we examined whether IgG in serum and gingival crevicular fluid, and IgG locally produced by plasma cells in gingiva of periodontal disease patients, display altered glycosylation. We developed a lectin-ELISA to measure levels of galactose-deficient IgG in the fluids and immunofluorescence staining to detect galactose-deficient IgG-producing cells in gingiva. Our results indicated higher levels of galactose-deficient IgG in sera and gingival crevicular fluid from periodontal disease patients, compared with levels in healthy controls. Furthermore, gingivae from periodontal disease patients exhibited infiltration of IgG-producing plasma cells; many of them contained galactose-deficient IgG in the cytoplasm. Analysis of our data suggests that IgG secreted by B-cells was aberrantly glycosylated, which resulted in the production of pro-inflammatory galactose-deficient IgG.

  12. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy.

    Science.gov (United States)

    Solá, Ricardo J; Griebenow, Kai

    2010-02-01

    During their development and administration, protein-based drugs routinely display suboptimal therapeutic efficacies due to their poor physicochemical and pharmacological properties. These innate liabilities have driven the development of molecular strategies to improve the therapeutic behavior of protein drugs. Among the currently developed approaches, glycoengineering is one of the most promising, because it has been shown to simultaneously afford improvements in most of the parameters necessary for optimization of in vivo efficacy while allowing for targeting to the desired site of action. These include increased in vitro and in vivo molecular stability (due to reduced oxidation, cross-linking, pH-, chemical-, heating-, and freezing-induced unfolding/denaturation, precipitation, kinetic inactivation, and aggregation), as well as modulated pharmacodynamic responses (due to altered potencies from diminished in vitro enzymatic activities and altered receptor binding affinities) and improved pharmacokinetic profiles (due to altered absorption and distribution behaviors, longer circulation lifetimes, and decreased clearance rates). This article provides an account of the effects that glycosylation has on the therapeutic efficacy of protein drugs and describes the current understanding of the mechanisms by which glycosylation leads to such effects.

  13. Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Syakhovich, Vitaly E. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Saraswathi, N. T.; Ruff, Marc, E-mail: ruff@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Bokut, Sergey B. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Moras, Dino [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus)

    2006-02-01

    Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A{sub 1C} is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA{sub 1C} were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M}) of 9.70 Å{sup 3} Da{sup −1} and a solvent content of 49%.

  14. Defective glycosylation of α-dystroglycan contributes to podocyte flattening.

    Science.gov (United States)

    Kojima, Kenichiro; Nosaka, Hitonari; Kishimoto, Yuki; Nishiyama, Yuri; Fukuda, Seiichi; Shimada, Masaru; Kodaka, Kenzo; Saito, Fumiaki; Matsumura, Kiichiro; Shimizu, Teruo; Toda, Tatsushi; Takeda, Satoshi; Kawachi, Hiroshi; Uchida, Shunya

    2011-02-01

    In addition to skeletal muscle and the nervous system, α-dystroglycan is found in the podocyte basal membrane, stabilizing these cells on the glomerular basement membrane. Fukutin, named after the gene responsible for Fukuyama-type congenital muscular dystrophy, is a putative glycosyltransferase required for the post-translational modification of α-dystroglycan. Chimeric mice targeted for both alleles of fukutin develop severe muscular dystrophy; however, these mice do not have proteinuria. Despite the lack of a functional renal defect, we evaluated glomerular structure and found minor abnormalities in the chimeric mice by light microscopy. Electron microscopy revealed flattening of podocyte foot processes, the number of which was significantly lower in the chimeric compared to wild-type mice. A monoclonal antibody against the laminin-binding carbohydrate residues of α-dystroglycan did not detect α-dystroglycan glycosylation in the glomeruli by immunoblotting or immunohistochemistry. In contrast, expression of the core α-dystroglycan protein was preserved. There was no statistical difference in dystroglycan mRNA expression or in the amount of nephrin and α3-integrin protein in the chimeric compared to the wild-type mice as judged by immunohistochemistry and real-time RT-PCR. Thus, our results indicate that appropriate glycosylation of α-dystroglycan has an important role in the maintenance of podocyte architecture.

  15. Parallel-META 2.0: enhanced metagenomic data analysis with functional annotation, high performance computing and advanced visualization.

    Directory of Open Access Journals (Sweden)

    Xiaoquan Su

    Full Text Available The metagenomic method directly sequences and analyses genome information from microbial communities. The main computational tasks for metagenomic analyses include taxonomical and functional structure analysis for all genomes in a microbial community (also referred to as a metagenomic sample. With the advancement of Next Generation Sequencing (NGS techniques, the number of metagenomic samples and the data size for each sample are increasing rapidly. Current metagenomic analysis is both data- and computation- intensive, especially when there are many species in a metagenomic sample, and each has a large number of sequences. As such, metagenomic analyses require extensive computational power. The increasing analytical requirements further augment the challenges for computation analysis. In this work, we have proposed Parallel-META 2.0, a metagenomic analysis software package, to cope with such needs for efficient and fast analyses of taxonomical and functional structures for microbial communities. Parallel-META 2.0 is an extended and improved version of Parallel-META 1.0, which enhances the taxonomical analysis using multiple databases, improves computation efficiency by optimized parallel computing, and supports interactive visualization of results in multiple views. Furthermore, it enables functional analysis for metagenomic samples including short-reads assembly, gene prediction and functional annotation. Therefore, it could provide accurate taxonomical and functional analyses of the metagenomic samples in high-throughput manner and on large scale.

  16. Parallel-META 2.0: enhanced metagenomic data analysis with functional annotation, high performance computing and advanced visualization.

    Science.gov (United States)

    Su, Xiaoquan; Pan, Weihua; Song, Baoxing; Xu, Jian; Ning, Kang

    2014-01-01

    The metagenomic method directly sequences and analyses genome information from microbial communities. The main computational tasks for metagenomic analyses include taxonomical and functional structure analysis for all genomes in a microbial community (also referred to as a metagenomic sample). With the advancement of Next Generation Sequencing (NGS) techniques, the number of metagenomic samples and the data size for each sample are increasing rapidly. Current metagenomic analysis is both data- and computation- intensive, especially when there are many species in a metagenomic sample, and each has a large number of sequences. As such, metagenomic analyses require extensive computational power. The increasing analytical requirements further augment the challenges for computation analysis. In this work, we have proposed Parallel-META 2.0, a metagenomic analysis software package, to cope with such needs for efficient and fast analyses of taxonomical and functional structures for microbial communities. Parallel-META 2.0 is an extended and improved version of Parallel-META 1.0, which enhances the taxonomical analysis using multiple databases, improves computation efficiency by optimized parallel computing, and supports interactive visualization of results in multiple views. Furthermore, it enables functional analysis for metagenomic samples including short-reads assembly, gene prediction and functional annotation. Therefore, it could provide accurate taxonomical and functional analyses of the metagenomic samples in high-throughput manner and on large scale.

  17. O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

    DEFF Research Database (Denmark)

    Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof;

    1999-01-01

    O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

  18. Stannylene‐Mediated Regioselective 6‐O‐Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides

    DEFF Research Database (Denmark)

    Maggi, Agnese; Madsen, Robert

    2013-01-01

    A straightforward procedure is described for the synthesis of (1→6)‐linked saccharides by regioselective glycosylation of unprotected glycosyl acceptors. Phenyl 1‐thioglycopyranosides derived from D‐glucose, D‐galactose and D‐mannose were treated with dibutyltin oxide to introduce a stannylene...

  19. Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

    DEFF Research Database (Denmark)

    Sherlock, O.; Dobrindt, U.; Jensen, J.B.;

    2006-01-01

    Glycosylation is a common modulation of protein function in eukaryotes and is biologically important. However, in bacteria protein glycosylation is rare, and relatively few bacterial glycoproteins are known. In Escherichia coli only two glycoproteins have been described to date. Here we introduce...

  20. RNA-Seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney

    Science.gov (United States)

    The UT-A1 urea transporter is crucial to the kidney’s ability to generate the concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, protein abundance, particularly the 117 kD isoform, is si...