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Sequence-Based Approaches for Uncultivated Microbes

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Sequence-Based Approaches for Uncultivated Microbes Sequence-based approaches to uncultivated microbes Susannah Green Tringe DOE Joint Genome Institute Metagenome Program Lead Department of Energy: Mission areas Bioenergy Carbon Cycle DOE Joint Genome Institute Walnut Creek, CA since 1999 Mission: To advance genomics in support of the DOE mission Biogeochemistry Uncultivated microbial communities Wetland carbon cycling Root-associated communities Uncultivated microbial communities Wetland carbon cycling Root-associated communities JGI Programs & Infrastructure Bioenergy Carbon Cycling Biogeochemistry Metagenomes Plants Fungi Microbes Synthesis DNA Genomic Computational Synthesis Sequencing Technologies Analysis What is metagenomics? Isolate (pure culture) Microbial community Genomics Metagenomics tame wild ≠ Most bacteria don’t grow on plates: “the great plate count anomaly” 16S ribosomal RNA as a phylogenetic marker 21 proteins 16S rRNA 30S 70S Ribosome subunits 50S 5S rRNA 34 proteins 23S rRNA Escherichia coli 16S rRNA Primary and Secondary Structure Falk Warnecke 16S-based phylogeny Carl Woese 1928-2012 Woese Microbiol Rev 1987 Norm Pace 16S rRNA in environmental microbiology Falk Warnecke Bacterial phylogenetic tree expansion cultured uncultured Modified from Baker et al 2013 (Microbe) The situation is similar for archaea Baker et al 2013 (Microbe) Why metagenomics? Industrial enzymes Most microbes are uncultured! Antibiotics Greenhouse gas cycling Suizenbacher et al 1997; www.chm.bris.ac.uk; Functional metagenomics Gillespie 2002 Turbomycin synthesis genes isolated from a soil metagenome library Schloss & Handelsman 2003 Discovery of proteorhodopsin de la Torre, J. R. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 12830-12835 Rhodopsin-like gene – never before seen in bacteria! Bacterial rRNA operon Copyright ©2003 by the National Academy of Sciences Shotgun metagenomics Shotgun sequencing: Could it be done? Schloss & Handelsman 2003 Metagenome assembly - like putting together several jigsaw puzzles Falk Warnecke . with some pieces missing Falk Warnecke Can we still reconstruct? Falk Warnecke Can we still reconstruct? Falk Warnecke One approach: a simple community sedimentation anaerobic aerobic tank influent effluent return sludge waste sludge EUB PAO Enhanced Biological Phosphate Removal (EBPR) reactors are dominated by Candidatus Accumulibacter phosphatis (CAP) but it cannot be grown in pure culture Crocetti et al., 2001 Proposed biological model for polyP- accumulating organisms (PAOs) Anaerobic zone Aerobic zone Cell PHA Cell PHA NAD NADH Glycogen Acetyl-CoA Glycogen TCA cycle CO2 ADP ADP PolyP PolyP ATP ATP acetate PO4 PO4 CAP metabolic reconstruction Anaerobic Phase Aerobic Phase Garcia Martin et al. 2006 What about more complex communities? EBPR sludge Sargasso Sea Soil 1 10 100 1000 10000 Species complexity ? A Adaptive gene for habitat A Environmental Gene Tags Adaptive gene for habitat B Essential gene (EGTs) B Comparative metagenomics COG5524: Bacteriorhodopsin COG1292: Choline- glycine betaine transporter COG3459: Cellobiose phosphorylase Tringe et al 2005 Metagenome sequence output 100 Tb 100 90 JGI Sequenced Bases 80 Metagenome bases ) 70 (Tb 60 50 40 30 Tb 30 20 40 Gb Sequence output output Sequence 10 0 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 Fiscal Year meta-GENOMEs Hess 2012: 15 rumen genomes Wrighton 2012: 3 uncultivated phyla 49 genomes Iverson 2012: Marine Group A Annotation of metagenomes Shotgun data Singlet fasta ORF prediction Contig fasta Assembly RNA prediction Contig stats Contig Depth G+C Jill Banfield, UCB Contig1 10.1 .49 Chongle Pan, ORNL Contig2 5.6 .35 Contig3 19.8 .38 Genome binning, extraction, improvement ORF prediction RNA prediction The single-cell approach: how it works isolation lysis + + dNTPs + polymerase random hexamers template DNA MDA Tanja Woyke Single cell genomics: key challenges CHALLENGE isolation Sample contamination (‘hitchhiker’ DNA) No universal lysis for all taxa lysis Chimerism MDA Reagent contamination MDA bias Tanja Woyke JGI single-cell sequencing pipeline Whole genome Single cell Sample Community amplification isolation profiling 16S rRNA gene Draft genome identification Genome Assembly Data QC Annotation Data curation sequencing Tanja Woyke Current limitations: 100 cells ≠ 100 SAGs Whole genome Single cell Sample Community amplification isolation profiling ??? 16S rRNA gene Not every cell can be isolated Draft genome identification o o Not every cell can be lysed and WGA’d o Not every cell can be 16S ID’d Genome Assembly Data QC Annotation Data curation sequencing Tanja Woyke Recovered diversity: 16S tags vs SAGs Tanja Woyke Modified from Clingenpeel et al 2014 (Frontiers in Microbiol) marker genes culture single cell target cell enrichment metagenomics draft/ complete partial draft genomes, draft genomes, unassembled data, genomes complete genomes complete genomes genome bins, (rarely) complete genomes Tanja Woyke culture single cell target cell enrichment metagenomics Tanja Woyke metagenomic approach single-cell approach Tanja Woyke marker genes Uncultivated microbial communities Wetland carbon cycling Root-associated communities Wetland restoration Carbon stored by Global wetlands Wetlands Global carbon (35% of all (9%) land area terrestrial carbon) CH4 CO2 Wetland restoration What determines if a wetland serves as a greenhouse gas source or a greenhouse gas sink? CH4 CO2 San Francisco Bay wetlands Farmland PAST PRESENT Wetland Salt pond www.wrmp.org Subsidence and carbon loss 1850s Elevation loss Levee failure Mount and Twiss 2005 Subsidence and carbon loss 1850s Elevation loss Levee failure Mount and Twiss 2005 Twitchell Island restored wetland - Est. 1997 - Peat accretion: ~4 cm/year - Net GHG budget: -494 g CO eq/m2 2 Twitchell wetland Shaomei He Mark Waldrop Lisamarie Windham-Myers Sampling site gradients Site A B C/L Methane flux Water inlet Do the microbial communities reflect these changes in geochemistry? Peat Water accretion outflow Oxygen, Nitrate, Sulfate What controls methane? CHCH4 Abundance Activity Species composition Relative Gene Family Abundances Samples with more methanogenesis genes have fewer genes in denitrification, dissimilatory sulfate reduction, and metal reduction. Methane oxidation genes were more abundant in rhizomes. Methanogen abundance DNA abundance Methanogen marker genes from RNA abundance metagenome (÷2) CH4 - Methane emissions correlated to methanogen CH4 ABUNDANCE and ACTIVITY Methanogen diversity Hydrogenotrophic Methanogenesis: CO2 + 4 H2 → CH4 + 2 H2O Acetoclastic Methanogenesis: CH3COOH → CH4 + CO2 Hydrogenotrophic Aug_C Methanomicrobiales (order) [H] Methanosaetaceae;Methanosaeta [A] Methanobacteriaceae;Methanobacterium [H] Feb_L Methanobacteriales (order) [H] Methanosarcinaceae;Methanosarcina [A] Aug_B Methanocellaceae;Methanocella [H] Methanomicrobiaceae;Methanofollis [H] Feb_B MSBL1;SAGMEG-1 [H] Methanospirillaceae [H] Methanococcales (order) [H] Aug_A Methanosarcinaceae;Methanomethylovorans [A] Methanobacteriaceae;Methanobrevibacter [H] Feb_A Methanobacteriaceae;Methanosphaera [H] pMC1;pMC1FA [H] 0% 50% 100% Acetoclastic Bay / Delta Salinity Gradient Historic and restored wetlands sampled along a salinity gradient Methane flux varies with salinity and wetland age Salinity (ppt) Average seawater Susie Theroux Uncultivated microbial communities Wetland carbon cycling Root-associated communities Rhizosphere Grand Challenge 1) Rhizosphere / endophyte microbes -provide nutrients -protect from pathogens and stress -influence growth -sequester carbon 2) Challenges -soil microbial communities are notoriously complex -plant genomes are complex -crosses multiple disciplines and programs -statistical rigor requires high sample numbers 52 Arabidopsis rhizosphere project Rhizosphere Endosphere Soil Are there unique communities in each compartment? Does the plant control access? 53 Rhizosphere Grand Challenge Identifying the major determinants of microbial community assembly Host factors Root-associated microbial communities Variables investigating: Soil type – Mason Farm vs. Clayton Full factorial design Sample fraction – Bulk soil vs. rhizosphere vs. endophyte 1117 samples Plant age – bolting (young) vs. senescent (old) 16S pyrotag profiles Genotype – 8 ecotypes 54 Individual – Aim for 10 individuals per condition Jeff Dangl The Arabidopsis microbiome The endophyte community is unique and reproducible and similar across soil types OTUs Lundberg Nature 2012 Rhizosphere/ Soil 1 Rhizosphere/ Soil 2 Sample type Endophyte The Arabidopsis Microbiome Cultured isolates Single cells “Plate scrape” metagenomes Flow-sorted “mini-metagenomes” Sphingobacteriales OTU 2324 Streptomyces Pseudonocardiaciae OTU 14834 OTU 13797 Dangl lab, UNC Woyke lab, JGI An endophyte genome catalog Actinobacteria 99 isolates and 130 SAGs fully sequenced >50% of target OTUs Alphaproteobacteria Firmicutes Cyanobacteria Single Cells Isolates Bacteroidetes Chloroflexi Betaproteobacteria S. Clingenpeel Recolonization and RNA-seq Full phosphate No bacteria full P + Bacteria full P Low phosphate Inoculation with No bacteria low P + Bacteria low P root-associated microbes reverses low P phenotype Sterile Colonized Conclusions • Most microorganisms cannot be readily grown in the lab • Nucleic acid sequencing provides a means to study uncultivated organisms via their genomes • Next-gen sequencing is opening up new opportunities in metagenomics and single cell genomics • These techniques are enabling advances in diverse areas of science Acknowledgments • EBPR sludge project –Trina McMahon,
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