Tracking the units of microbial communities with metagenomics

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1 Tracking the units of microbial communities with metagenomics Dr. Kostas Konstantinidis School of Civil and Environmental Engineering & School of Biology (Adjunct), Center for Bioinformatics and Computational Genomics Georgia Institute of Technology MBL STAMPS August 9 th, 2012

Overwhelming abundance & diversity 1 g of soil contains 1 million to 10 billion microbial cells representing about 4,000-10,000 species (Torsvik et

2 Overwhelming abundance & diversity 1 g of soil contains 1 million to 10 billion microbial cells representing about 4,000-10,000 species (Torsvik et al. 1990) Each species carries a few hundred unique genes of unknown function (Konstantinidis & Tiedje, PNAS 2005) Similar numbers in other habitats

3 What are the important units? (species?) [ ] we cannot understand functional diversity without first agreeing on what biological units to study (Alan Konopka, ASM Microbe, April 2006)

4 OTUs based on 16S rrna gene identity The Tree of Life based on 16S rrna gene phylogeny Grouping at 3% 16S rrna gene identity Pace, Science, 1997

5 How are species/units defined? The most popular species definition a genomically coherent (discreet) group of strains based on the hybridization of their purified DNA molecules Plus, a diagnostic phenotype Wayne et al. IJSB, 1987

6 The DNA-DNA hybridization method DDH general principle Isolate genomic DNA from strains A and B Random fragmentation Denature DNA Mix and let renature Quantify heteroduplex relative to homoduplex >70% => SAME species Good correspondence with phenotypically coherent clusters of strains in Enterobacteriaceae BUT + +? Difficult to do! Unclear how it relates to whole-genome relatedness. Need to have isolates available but only 1-2% of prokaryotic cells are cultivable! (the great plate count anomaly)

7 DDH vs. whole-genome sequence relatedness DDH vs. whole-genome Average Nucleotide Identity (ANI) 70% DDH <=> 95% ANI! (ANI) Goris, Konstantinidis, et al. IJSEM, 2007

8 ANI to measure relatedness Reference genome (CDS sequences) Tester genome (genomic sequence) Query Reciprocal (best match) BLASTP/N IF 30% Id. & 70% Len. of the query ORF (a.a. OR nt. level) Database Conserved Genes Conserved genes as percent of the total ORFs (normalize genome size effect) Average Nucleotide Identity of the conserved genes (measure evolutionary distance)

9 ANI vs 16S rrna gene identity >70% DDH => >95% ANI => >98.5% 16S rrna

10 Why the species definition does not work?

11 Genetic continuum OR discreet clusters? Whole-genome Max. Likelihood phylogeny (based on 2,000 core genes) E. coli Year Year Year Same Same picture 16S picture with with ANI identity ~98.3% ANI

12 Genetic continuum OR discreet clusters? Whole-genome Max. Likelihood phylogeny (based on 2,000 core genes) E. coli Year 2002

13 Genetic continuum OR discreet clusters? E. coli Year 2010 Alex, Bioinformatics Strains of Clades II-V come from the environment. (e.g., soil, freshwater beaches) Share same ecology/phenotype? Encode specific gene signatures?

14 Approach Illumina sequencing; 9 genomes in one sequencing run; $10K

15 Environmentals encode enterics core E. coli core genes conserved in environmental. Implications for coliform testing Coliform count test positive!

16 Gene signatures of clades Enteric genomes are enriched in functions selected in the human gut!

17 Quartet decomposition analysis Luo et al., PNAS 2011

18 Core gene exchange among clades Luo et al., PNAS 2011

19 Genetic continuum OR discreet clusters? E. coli Year S rrna gene identity >99% How to define species (e.g., where to draw the line)?

20 The new science of metagenomics

21 Metagenomics Very powerful methodology!

Metagenomic sampling of the Oceans Global Ocean Survey (GOS) sampling sites Hawaii Ocean Time Series ~200Mbp of shotgun library Deep, 4000m depth

22 Metagenomic sampling of the Oceans Global Ocean Survey (GOS) sampling sites Hawaii Ocean Time Series ~200Mbp of shotgun library Deep, 4000m depth (Konstantinidis & DeLong, ISME 2008) SAR3, SAR4, GOS18, GOS23, GOS34 ~ Mbp of shotgun library Surface, ~5m depth (Rusch et al, PLoS Biology 2007)

23 The approach From Konstantinidis, 2010 In: Handbook of Molecular Microbial Ecology. Volume I Metagenomics and Complementary Approaches. Editor: Frans J. de Bruijn See also: Caro-Quintero & Konstantinidis, Env. Microbiol. 2012

24 Clusters are ubiquitous in the Oceans! BAC clone from uncultured γ proteobacterium vs. Surface shotgun Based on a phylogenetic approach too! Coverage Sequence discontinuity Nucleotide Identity Synechococcus vs. Coastal Atlantic (mostly) sequencing errors Konstantinidis & DeLong, The ISME J 2008

25 What about populations from different environments or sites?

26 Hawaii Ocean Time- Series (HOT) Bermuda Atlan8c Time- Series (BATS) Coleman and Chisholm, PNAS, 2010

27 P-gene content differs in HOT vs BATS HOT BATS 20m 25m 50m 75m 100m 110m Sampling depths # of reads, BATS # of reads, HOT pho operon: alkaline phosphatase/regulator phn operon: phosphohydrolase Coleman and Chisholm, PNAS, 2010

28 Caveats? Has the collapsing of samples and ecotypes missed any important underlying patterns? From DeLong et al., Science, 2006

29 HOT 20m BATS 25m Our approach BATS HOT Community DNA Clone free (454) 50m 75m Reference sequence from BATS Data processing Blastn (fishing out) 100m 110m Sampling depths # of reads 80 Nucleo8de iden8ty Coverage plot (Konstantinidis & DeLong) 100

Surface populations indistinguishable Reference: HOT 25m contigs FracHon of reads mapped to conhgs Coleman and Chisholm, PNAS, 2010

30 Surface populations indistinguishable Reference: HOT 25m contigs FracHon of reads mapped to conhgs Coleman and Chisholm, PNAS, Nucleotide identity to reference At surface level, Prochlorococcus populations are indistinguishable between HOT and BATS.

Deeper populations differ Reference: BATS 100m contigs Fraction of reads mapped to contigs 85 90 95 100

31 Deeper populations differ Reference: BATS 100m contigs Fraction of reads mapped to contigs Nucleotide identity to reference At deeper depths, populations represent different ecotypes between HOT and BATS.

32 P-gene abundance with depth phn operon is differen8ally present only at deep phobr are equally present at HOT & BATS surface

33 Paired-ended read sequence analysis NCBI NR phobr phoa Prochlorococcus ref.? Expectation: both reads should have Prochlorococcus as best match

34 Most reads do not match Prochlorococcus

35 Bacterial species exist? Luo & Konstantinidis, PNAS 2011 Fraction of reads mapped to contigs Nucleotide identity to reference Bacterial species exist? Strong implications for population genetics studies 100

36 How populations cohere together??

37 The Baltic Sea A redox stratified environment rich in Shewanella North Sea Sweden Alejandro Caro-Quintero Stable stratification of water column from N N Gotland Deep Finland Finland 113 S. baltica strains were isolated in 1986 & 1987 In 1986, S. baltica strains accounted for 32-80% of total cultivable denitrifying bacteria

38 Water chemistry profile & isolation depth Depths where S. baltica strains isolated O 2 NO 3 OS155 (90m) OS185 (120m) & OS223 (120m) OS195 (140m) H 2 S Data from: Brettar, I. and Hofle, M Mar. Ecol. Prog. Ser. 94:

39 Recent exchange of core genes Clear evidence of homologous recombination between OS195 and OS185! % identity of orthologs across the genome Whole-genome Max Lik. tree (recombined parts excluded)

40 Genome-wide, unbiased recombination Spatial distribution of recombined fragments (OS195 & OS185) Recombination fragment length distribution No strong spatial or genefunction biases associated with the recombinant fragments Frequency OS195 Conservation in OS185 Recombinant fragments Length (in Kbp) Recombination to mutation ratio = 3/1 => Sexual speciation! (for OS195 vs. OS185) Caro-Quintero et al., The ISME Journal, 2011

41 Species-like populations!? Sequence-discrete populations Smaller intra-population gene-content differences compared to several named species (<5% vs 20-30%) Detectable intra-genomic homologous recombination; albeit lower levels compared to biofilm communities (e.g., AMD) Caro-Quintero et al., The ISME Journal, 2011 Genetic discontinuity 95% nt identity

42 Summary-Conclusions Translate old standards to portable sequence data Sequence-distinct populations 95% ANI level! Strains of same species can be very diverse Homologous recombination force of cohesion (?)

43 Environmental Microbial Genomics GaTech Alejandro Alex Bioinformatics Natasha Microbial Ecology Seung-Dae Env. Engineering Evolution/Bioinformatics Env. Engineering Luis Rachel Poretsky Mike Weigand Post-doc Despoina Post-doc Env. Engineering Miguel Bioinformatics kostas@ce.gatech.edu

Microbial & interdisciplinary research @ GaTech

Atlanta Interested? Email kostas@ce.gatech.

44 Microbial & interdisciplinary GaTech Environmental Engineering Building Downtown Atlanta Interested? kostas@ce.gatech.edu Natural & Engineered microbial systems (Bioremediation)

Acknowledgments People Kostas group @ Georgia Tech Alex Luo, Bioinformatics Alejandro Caro, Microbial Ecology/Bioinformatics Miguel Rodriguez, Bioinformatics Natasha DeLeong, Microbial Ecology

45 Acknowledgments People Kostas Georgia Tech Alex Luo, Bioinformatics Alejandro Caro, Microbial Ecology/Bioinformatics Miguel Rodriguez, Bioinformatics Natasha DeLeong, Microbial Ecology Seung-Dae Oh, Env. Engineering Despina Tsementzi, Env. Engineering Coto Orellana, Env. Engineering Rachel Poretsky, Post-Doc Mike Weigand, Post-Doc Support Genomes to Life Program Award #DE-FG02-07ER64389 & DE-SC National Science Foundation Prof. DeLong M.I.T. IOS & CBET Other collaborators Prof. Frank Loeffler (U of Tennessee), on bioremediation communities Prof. Spyros Pavlostathis (Gatech), antibiotic resistance Prof Jizhong Zhou (U of Oklahoma), climate change and soil communities Prof. James Tiedje (MSU), on the species concept Dr. Alban Ramette (Max Planck, Bremen), on biogeography

Day 3. Examine gels from PCR. Learn about more molecular methods in microbial ecology

Day 3. Examine gels from PCR. Learn about more molecular methods in microbial ecology Day 3 Examine gels from PCR Learn about more molecular methods in microbial ecology Genes We Targeted 1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control