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Function-Driven Metagenomics Jo Handelsman

Function-Driven Metagenomics Jo Handelsman. What is Function-Driven Metagenomics?. Clone DNA directly from environmental sample Screen clones for expressed functions Advantage: Not limited by sequence recognition. Points to Consider About Metagenomics.

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Function-Driven Metagenomics Jo Handelsman

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  1. Function-Driven MetagenomicsJo Handelsman

  2. What is Function-Driven Metagenomics? Clone DNA directly from environmental sample Screen clones for expressed functions Advantage: Not limited by sequence recognition

  3. Points to Consider About Metagenomics • Genes in environment have relevance to human health • Model systems necessary to develop ecological principles governing the human microbiome

  4. Function-Driven Metagenomics • Approach • Example #1: Antibiotic resistance genes in soil • Example #2: Insect gut microbiome

  5. Metagenomic library construction Collect soil Extract DNA Digest Ligate into vector Transform E. coli Screen transformants

  6. Example #1 Antibiotic Resistance Genes in Soil • Antibiotic resistance is a significant and growing problem • Where do resistance genes originate? Goal: Identify the source, diversity, and movement of antibiotic resistance genes in the environment

  7. Why Soil? • Most antibiotics originate from soil bacteria • Most antibiotic producers carry resistance genes • Other soil organisms must contend with antibiotics • Soil is the richest microbial environment we know of (5,000-10,000 species; enormous strain variation)

  8. Aminoglycoside modification sites 6’-N-acetyltranferase Adapted from Kotra et al. 2000. Antimicrobial Agents & Chemotherapy

  9. Phylogenetic analysis of aminoglycoside resistance genes (acetyltransferases) Nodes unmarked Probability >0.90 Probability between 0.80 and 0.90 Probability between 0.70 and 0.80 Probability between 0.50 and 0.70 Resistance genes from soil form a new clade of acetyltransferases.

  10. Penicillin core A. Penicillin G. B-E, semi-synthetic β-lactam antibiotics. B. Amoxicillin. C. Ampicillin. D. Carbenicillin. E. Piperacillin. Carbapenemcore Cephalosporin core B. R = imipenem. A1. Cephalexin. A2. Cefamandole. A3. Cefotaxime. b-Lactam antibiotics

  11. Enzymatic Inactivation of b-lactams Classes of b-lactamases - A, C, and D = serine β-lactamases - B = metallo-β-lactamases Cephalosporin core Penicillin core

  12. Examples of b-lactam Resistance in Soil Metagenome

  13. Metagenomics and Antibiotic Resistance • Functional screening identified resistance genes that would not have been recognized by sequence alone • Soil contains new clades of genes for resistance to aminoglycosides and β-lactams • Do these genes move from soil to clinical settings? • Do agricultural practices such as spraying apple trees with streptomycin affect the frequency of antibiotic resistance genes in soil?

  14. Example #2 Gypsy Moth

  15. Ecological Questions • What is the basis for robustness of a gut community? • How does interspecies microbial signaling affect robustness? Gypsy moth gut contains a simple model community with 5-10 species

  16. Metagenomic library construction Dissect larvae, separate bacteria Extract DNA Digest Ligate into vector Transform E. coli Screen transformants

  17. Search for Community Signals • Screen clones for ability to induce quorum sensing • Quorum sensing is bacterial communication • Usually mediated by acylated homoserine lactones

  18. Gypsy Moth Midgut Quorum Sensing Inducing Clone PUTATIVE TRANSMEMBRANE PROTEIN CONSERVED HYPOTHETICAL PROTEIN PROBABLE OXIDOREDUCTASE PROTEIN PUTATIVE OXYGENASE SUBUNIT PROTEIN CONSERVED HYPOTHETICAL PROTEIN Ralstonia solanacearum GMI1000 genome 2547-7634 bp, reversed 69.7% 78.0% Gypsy moth metagenomic clone pBSS3 500 bp Putative oxygenase and conserved hypothetical protein required for production of signal

  19. Scheme for Synthesis of Quorum Sensing Inducer O H O O N O Dioxindole N O2 O2 O O N O H O H N Isatin 2 - hydroxyindoxyl N Indoxyl

  20. Insect Gut Signal • Indoxyl respresents a new class of quorum sensing inducer • Genes would not have been recognized by sequence analysis alone • Signal affects community structure • Impact of signal on community robustness are in progress

  21. Thanks to…. Soil Metagenome Heather Allen Luke Moe Christian Reisenfeld Lynn Williamson Brad Borlee Changhui Guan Jitsupang (Koy) Rodbumrer NIH, NSF, and HHMI Insect Microbiome Nichole Broderick Changhui Guan Ben Shen/Jinhua Ju Ken Raffa

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