1. Function-Driven Metagenomics Jo 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. Collect soil Extract DNA Digest Metagenomic library construction 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. 6’-N-acetyltranferase Aminoglycoside modification sites Adapted from Kotra et al. 2000. Antimicrobial Agents & Chemotherapy

9. Nodes Probability >0.90 Probability between 0.80 and 0.90 Probability between 0.70 and 0.80 Probability between 0.50 and 0.70 unmarked Phylogenetic analysis of aminoglycoside resistance genes (acetyltransferases) 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. Carbapenem core B. R = imipenem. Cephalosporin core A1. Cephalexin. A2. Cefamandole. A3. Cefotaxime.  -Lactam antibiotics

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

12. Examples of  -lactam Resistance in Soil Metagenome CloneInsert Size ResistanceResistance Gene Similarity βLR16.1ampicillin cephalothin 67% identity to class A serine β-lactamase Burkholderia βLR212.1amoxicillin 45% identity to metallo β-lactamase Janthinobacterium βLR310.1ceftazidime 48% identity to class B β-lactamase Chryseobacterium βLR168.0 carbenicillin (24 o C) 33% identity to membrane protein Magnetospirillum

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

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

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