Beiko ANL Soil Metagenomics presentation
- 1. Soil, lateral gene transfer, and hybrid genomes Robert Beiko 20 October 2015
- 2. A. microbe Lateral gene transfer http://genome.cbs.dtu.dk/staff/dave/MScourse/Lekt_11Feb2003c.html
- 3. In the gut Butyrate synthesis in Lachnospiraceae and other organisms Meehan and Beiko (2014) Genome Biol Evol Lachnospiraceae
- 4. LGT across habitats Smillie et al. (2011) Nature Within-site transfer rates are highest in host-associated (i.e., human) habitats AR genes are frequently transferred BETWEEN habitats
- 5. Villegas-Torres et al. (2011) International Biodeterioration & Biodegradation Forsberg et al. (2012) Science Sorangium cellulosum So157-2 14.8 Mbp; 11,599 coding sequences >1200 putative LGT acquisitions Han et al. (2013) Sci Rep
- 6. An ecological view of genomes Genes as individuals, Genomes as communities Key concept mappings: • Diversity: counts of genes / distribution across functional categories • Community: set of genes and their interactions • Migration: lateral gene transfer
- 7. Metacommunity Leibold et al., Ecol Lett, 2004 A set of local communities that are linked by dispersal Species B Species A Habitat Habitat Habitat Pattern and intensity of migration for species A
- 8. Genome Metacommunity Hypothesis • Since… • Genes are agents whose trajectories are not bound to their host organisms • Genes can evolve and take on new functional roles in concert with other genes • A genome can be viewed as a community of genes • Related sets of genomes comprise a metacommunity of genes
- 9. Genome Metacommunities Boon et al., Fems Microbiol Rev, 2014 A set of genomes that are linked by LGT Gene B Gene A Genome Genome Genome Pattern and intensity of LGT for gene A
- 10. Genome Metacommunities Boon et al., Fems Microbiol Rev, 2014 Related to the pan-genome, but not restricted to specific taxonomic groups Why is a given gene present in a given genome at a given time? How are functional roles partitioned across a community?
- 11. Soil thinking How important is LGT in soil communities? Does it make sense to think of gene metacommunities in the soil context? Lots of LGT YES Minimal LGT NO
- 12. The procedure • In the absence of a coherent set of known genomes from a given habitat… 1. Identify an interesting sample 2. Select genomes with very high marker-gene (i.e., 16S) similarity to sequences in the sample (gOTUs) 3. Mine genomes for evidence of LGT, examine patterns of connectivity
- 13. Conclusions • Positive relationship between abundance, diversity and pH • Specific relationships between different bacterial (notably Acidobacteria) and fungal groups vs. pH • Fungal OTUs appear to tolerate wider pH ranges (1) Chosen sample: http://metagenomics.anl.gov/?page=MetagenomeOverview&metagenome=4455674.3#org_ref (pH = 4.1)
- 14. Meet the Sample (MG-RAST) 1277 rRNA gene sequences
- 15. Meet the Sample (Matching genomes) 99% 16S identity (e-value < 1e-20): 1211 – No match Bradyrhizobiaceae: 61 Pseudomonas: 2 Nocardioides: 1 Acidithiobacillus: 1 Cyanobium: 1 Total: 18 genomes covering 8 genera 97% 16S identity: 1100 – No match Bradyrhizobiaceae: 77 Pseudoxanthomonas / Cycloclasticus: 25 Acidobacteria: 20 Other Proteobacteria: 48 Other: 10 Total: 114 genomes covering 74 genera 1277 rRNA gene sequences (1)
- 16. gOTUs 16S sequence from sample Rhodopseudomonas palustris TIE 1 Rhodopseudomonas palustris DX 1 Rhodopseudomonas palustris CGA009 Bradyrhizobium japonicum USDA 6 99% 16S identity 97% 16S identity 141824_31298 Rhodopseudomonas palustris HaA2 Rhodopseudomonas palustris BisA53 Bradyrhizobium BTAi1Nitrobacter winogradskyi Oligotropha carboxidovorans Agromonas oligotrophica Bradyrhizobium ORS278
- 17. Weird gOTUs 141824_229613 Bordetella pertussis Bordetella bronchiseptica Bordetella parapertussis Gross et al., 2008
- 18. Homology search • Compare proxy genomes against nr database • Identify interesting patterns: • Unusual best matches (e.g., best nonself match is to a completely different group) • Patchy distributions, phylogenetic trees • Linked sets of genes: co-transfer? • Implicated biological processes?
- 20. Beiko (2011) Biol Direct 504 gene trees in which A. ferrooxidans has a unique genus as partner Not shown: 795 genes w/multiple partners Also not shown: 333 other trees with less frequent, unique partner genera Split by 16S; reunited by genome sequencing?
- 21. Genome 1 – Acidithiobacillus ferrivorans (renaming of A. ferrooxidans) 3093 predicted proteins / 3035 with homology matches Observed / Predicted capabilities: • Facultatively anaerobic • Psychrotolerant • Optimal pH = 2.5 • Oxidation of iron and inorganic sulfur • Carbon fixation, nitrate reduction • Trehalose synthesis • “Bioleaching” Liljeqvist et al. (2011) J Bacteriol
- 22. Genome 1 – Acidithiobacillus ferrivorans Best nonself match is to… (273 non-Acidithiobacillus)
- 23. Mobile element signatures dominate • 14 x restriction system-associated • 8 x transposase • 8 x transcriptional regulators (incl CopG, TetR) • Other resistance (LacZ, bleomycin, …) • Integrase, reverse transcriptase, toxin/antitoxin, bacteriocin, … • Nitrate reductase & related • >90 unknown
- 24. 1877 found in other Acidithiobacillus + other genera Best non-Acidithiobacillus match is to… (only 11 Acidobacteria!)
- 26. Acidobacterial connections • short-chain dehydrogenase/reductase SDR • HNH endonuclease • Glycoside hydrolase family 8 (x3) • RES domain protein • Transposase x 5
- 27. Phylogenetic profiles # of similar genes (evalue < 10-50) Min 30 connections Proteobacteria Actinobacteria Cyanobacteria Planctomycetes Acidobacteria Bacteroidetes Acidithiobacillus Key observations • Connections to many other groups, mostly Proteobacteria (not surprising) • No between-group connections outside Proteobacteria at this threshold • Acidithiobacillus as hub rather than part of gene-exchange community? 65
- 28. Mutual information-based network (do groups co-occur > random?) Acidithiobacillus Gammaproteobacteria Alpha/Betaproteobacteria Key observations • Connections mostly predictable by phylogeny • Again, no interesting partners outside of Proteobacteria • However, many connections between Alpha/Betaproteobacteria
- 29. Phosphate ABC transporters gi 343775109 periplasmic (eval < 10-100) gi 343775110 inner membrane subunit PstC (eval < 10-50) gi 343775111 inner membrane subunit PstA (eval < 10-50) Distribution: • Acidithiobacillus • Acidobacterium • Alpha/Beta/Gamma • Actinobacteria • Firmicutes Recurrent grouping of Acidithiobacillus (Gamma) Acidobacterium Thiobacillus (Beta) Defluviimonas (Alpha) Salinisphaera (Gamma)
- 30. Genome 2 – Terriglobus roseus Eichorst et al (2007) IJSEM • “Group 1” acidobacterium • Preferred pH: ~6 • Aerobic • Catalase, carotenoids for defense against reactive oxygen • Oligotrophic; can grow on a wide range of carbon sources • 4245 protein-coding genes (2735 with nr matches, 558 species-specific) Rousk et al. Eichorst et al.
- 31. Best nonself matches (183 non-Acidobacteria) Multidrug resistance / cation efflux / prophage
- 32. Best matches outside Terriglobus
- 33. Phylogenetic profiles Profiles are wider and more diverse for Terriglobus than for Acidithiobacillus
- 34. LPS O-antigen biosynthesis gi 390412425 CDP-glucose 4,6-dehydratase gi 390412426 glucose-1-phosphate cytidylyltransferase gi 390412427 “LPS biosynthesis protein” Distribution: • Acidithiobacillus • Acidobacteria • Other proteobacteria • Other Flavobacteria Spirochaetes Spirochaetes Cyanobacteria
- 35. Contrasting Acidithiobacillus vs Terriglobus relationships: Same partners, different dance Compare profiles vs Streptomycetaceae (five strains found in sample gOTU) Acidithiobacillus Common Terriglobus Polyphosphate kinase glucose-6- phosphate 1- dehydrogenase Carbon monoxide dehydrogenase More glycolytic enzymes Heavy-metal resistance / export Multidrug resistance Ammonium transporter Catalase / peroxidase Exopolysaccaride
- 36. Conclusions • Different layers of LGT: • Very recent: mostly mobile elements (proxies unsuitable) • Less recent (outside species / genus) (proxies potentially more justifiable) • Taxonomy is a pain • What’s the story with gene metacommunities? • Lots of LGT! • Recurrent patterns of sharing among groups not evident • Metacommunities at the pan-genome level? • Need many isolate genomes from single samples
- 37. Technical impacts of LGT and gene metacommunities Metagenomic read assignment • Recently acquired genes will still look like they belong in the donor • These are some of the most interesting genes!! Functional prediction (e.g., PICRUSt) • Phylogeny will fail to accurately predict the distribution of these genes. Be very careful with extreme or poorly characterized samples! Phylogenetic beta diversity may be misleading
- 38. Key questions in LGT and gene metacommunities • Are gene-sharing networks: • Random? • Driven by shared location / habitat? • Constrained by phylogenetic relatedness? • Are shared genes: • Neutral or adaptive? • Driven by specific types of mobile element?