Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

Brady, Sean F.

doi:10.1038/nprot.2007.195

Cited by 189 publications

(178 citation statements)

References 21 publications

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“…Demographic details for the ulcerative colitis patient (UC) (library 1), Crohn's patient (CD) (library 2), and healthy control (HC) (library 3) from whom stool was collected for this study are shown in SI Appendix. The construction of metagenomic libraries from patient stool samples proceeded using methods developed for cloning DNA directly from soil (36). In brief, high-molecular-weight environmental DNA (eDNA) was Step 1: The human reporter cell line and individual metagenomic bacterial clones are robotically arrayed in separate 384-well microplates.…”

Section: Resultsmentioning

confidence: 99%

“…8). We elected to specifically look at a tyrosine analog because long-chain N-acyl tyrosine producing clones are frequently found in soil eDNA libraries (15,20,21,36,56,60). All of the commendamide analogs tested, whether they contained changes in the head group or acyl chain, showed reduced GPR132/G2A activity.…”

Section: Comparative Phylogenetic and Functional Analysis Of Effectormentioning

confidence: 99%

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Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

Cohen

Kang

Chu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

151

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The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. Functional metagenomics provides a systematic means of surveying commensal DNA for genes that encode effector functions. Here, we examine 3,000 Mb of metagenomic DNA cloned from three phenotypically distinct patients for effectors that activate NF-κB, a transcription factor known to play a central role in mediating responses to environmental stimuli. This screen led to the identification of 26 unique commensal bacteria effector genes (Cbegs) that are predicted to encode proteins with diverse catabolic, anabolic, and ligand-binding functions and most frequently interact with either glycans or lipids. Detailed analysis of one effector gene family (Cbeg12) recovered from all three patient libraries found that it encodes for the production of N-acyl-3-hydroxypalmitoyl-glycine (commendamide). This metabolite was also found in culture broth from the commensal bacterium Bacteroides vulgatus, which harbors a gene highly similar to Cbeg12. Commendamide resembles long-chain N-acyl-amides that function as mammalian signaling molecules through activation of G-protein–coupled receptors (GPCRs), which led us to the observation that commendamide activates the GPCR G2A/GPR132. G2A has been implicated in disease models of autoimmunity and atherosclerosis. This study shows the utility of functional metagenomics for identifying potential mechanisms used by commensal bacteria for host interactions and outlines a functional metagenomics-based pipeline for the systematic identification of diverse commensal bacteria effectors that impact host cellular functions.

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Section: Resultsmentioning

confidence: 99%

Section: Comparative Phylogenetic and Functional Analysis Of Effectormentioning

confidence: 99%

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

Cohen

Kang

Chu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

151

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“…Total DNA was extracted from seawater using the MoBio power water DNA isolation kit. H. perlevis total DNA was extracted via a method adapted from Brady (2007). H. perlevis (4 g wet weight) was frozen in liquid nitrogen and ground to a fine powder in a sterile pestle and mortar.…”

Section: Methodsmentioning

confidence: 99%

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

et al. 2012

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Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis Catriona Harrington, 1,2,3 Antonio Del Casale, 3 Jonathan Kennedy, 1 Horst Neve, 4 Bernard E. Picton, 5 Marlies J. Mooij, 1,2,6 Fergal O'Gara, 1,2,6 Leonid A. Kulakov, 3 Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phagederived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction. INTRODUCTIONViruses are the most numerous and diverse biological entities in not only the marine environment, with 10 6 to 10 9 particles (ml seawater) 21 (Kristensen et al., 2010) and 10 30 unique viral genotypes (Suttle, 2007), but also on Earth (Angly et al., 2006). Viruses are extremely important life forms known to play a crucial role in microbial ecosystems. They are essential for maintaining and influencing the diversity of all microbial communities both by affecting them directly (by controlling numbers of bacterial cells) and by horizontally transferring genetic material to the host via the transduction process (Lohr et al., 2005). Phages can also affect microbial evolution as killing certain dominant bacteria may allow other related strains that are resistant to the phage to become dominant (Angly et al., 2006) in what is known as the 'kill the winner' hypothesis. This may help explain microbial diversity and the changes that can be observed within bacterial community composition ). However, this interaction between phages and their bacterial hosts has remained largely under studied, especially with respect to marine sponge ecosystems.Microbial communities of marine sponges are a major focus of current research, as sponge-associated microbes have been shown to produce a plethora of novel bioactive metabolites (Taylor et al., 2007). Due to the advent of high throughput sequencing, numerous metagenomic studies have facilitated the identification of sponge-associated bacterial groups, which have revealed remarkable levels of bacterial diversity, with 26 major phyla to date having been found to be present in close association with sponge species worldwide (Kennedy et al., 2008;Webster & Taylor 2012;Jackson et al., 2012). Some sponges have a low microbial abundance, with a microbial range similar to that of Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeat; OTU, operational taxonomic unit; TEM, transmission electron microscopy; TFF, tangential flow filtration.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences from viral DN...

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“…Crude eDNA samples were prepared from soil samples collected in Pennsylvania (samples A and B), New Jersey, Massachusetts (samples A and B), Utah, Oregon, North Carolina, Tanzania (samples A and B), and Costa Rica by using standard eDNA isolation methodology (27). In brief, a 1:1 mixture (wt/vol) of soil and lysis buffer (100 mM Tris⅐HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (wt/vol) CTAB, 2% (wt/vol) SDS, pH 8.0) was heated for 2 h at 70°C, and then, the soil was removed by centrifugation (4,000 ϫ g, 30 min).…”

Section: Methodsmentioning

confidence: 99%

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

Banik

Brady

2008

Proc. Natl. Acad. Sci. U.S.A.

103

100

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Glycopeptide antibiotics have long served as drugs of last resort for the treatment of antibiotic-resistant Gram-positive bacterial infections. Resistance to the clinically relevant glycopeptides, vancomycin and teicoplanin, threatens to undermine the usefulness of this important class of antibiotics. DNA extracted from a geographically diverse collection of soil samples was screened by PCR for the presence of sequences related to OxyC, an oxidative coupling enzyme found in glycopeptide biosynthetic gene clusters. Every soil sample examined contained at least 1 unique OxyC gene sequence. In an attempt to access the biosynthetic gene clusters associated with these OxyC sequences, a 10,000,000-membered environmental DNA (eDNA) megalibrary was created from a single soil sample. Two unique glycopeptide gene clusters were recovered from this eDNA megalibrary. Using the teicoplanin aglycone and the 3 sulfotransferases found in one of these gene clusters, mono-, di-, and trisulfated glycopeptide congeners were produced. The high frequency with which OxyC genes were found in environmental samples indicates that soil eDNA libraries are likely to be a rewarding source of glycopeptide gene clusters. Enzymes found in these gene clusters should be useful for generating new glycopeptides analogs. Environmental DNA megalibraries, like the one constructed for this study, can provide access to many of the natural product biosynthetic gene clusters that are predicted to be present in soil microbiomes.antibiotics ͉ eDNA ͉ metagenomics ͉ natural products ͉ uncultured bacteria

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Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

Cited by 189 publications

References 21 publications

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

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