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Janelle R. Thompson Massachusetts Institute of Technology

From populations to genomes: Extensive genotypic diversity in a natural bacterioplankton population. Janelle R. Thompson Massachusetts Institute of Technology. Outline. Introduction Microbial diversity and organization “ Vibrio spendidus” model system

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Janelle R. Thompson Massachusetts Institute of Technology

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  1. From populations to genomes: Extensive genotypic diversity in a natural bacterioplankton population Janelle R. Thompson Massachusetts Institute of Technology

  2. Outline Introduction Microbial diversity and organization “Vibrio spendidus” model system Plum Island Sound, MA and Barnegat Bay NJ Population dynamics Genomic Diversity Ecological and evolutionary considerations

  3. We live on a microbial world Microbes are the most abundant living organisms on the Earth Estimates 4.15 - 6.40 x1030 organisms 353 - 546 Pg carbon 70% Ocean!! Credit M.Polz (Whitman et al. 1998) http://visibleearth.nasa.gov/

  4. We live on a microbial world Microbes are the most abundant living organisms on the Earth • Diverse metabolism • Global processes Estimates 4.15 - 6.40 x1030 organisms 353 - 546 Pg carbon 70% Ocean!! Credit M.Polz (Whitman et al. 1998) http://visibleearth.nasa.gov/

  5. Nutrient cycling A B Microbial Assemblages A. SEM B. Light microscopy with fluorescent-stain Complex Microbial Communities DeLong & Karl 1 µm System response P. Franks Harmful algal blooms Structure and Function Relationship? Sanitation

  6. Evolutionary Relationships A B Marine Assemblages A. SEM B. Light microscopy with fluorescent-stain Community Diversity Accessible only by molecular methods DNA/RNA ~100% community ? Isolates Biomarker genes e.g. 16S ribosomal RNA (ribotypes) <0.1 to 10% community

  7. Community Organization? Individuals ? Populations Genetic diversity (e.g. 16S rRNA ribotypes) How do they work together to mediate activities in the environment? Community

  8. The challenge: identifying functional units in natural communities Population theory informs search… Community Biomarker Tree Co-existing variation fitness advantage Selective Sweep Diversification sequence cluster Based on work by F. Cohan

  9. 0.05 subst/site 0.01 subst/site 0.1 subst/site Organization of a bacterioplankton community Plum Island Sound, MA 16SrRNA clone library Most of the ribotype diversity partitioned into sequence clusters with >99% identity =functional units? Acinas & Klepac-Ceraj, et al, Nature, 2004

  10. Motivating Questions: I) Can we identify populations in microbial communities? II) How diverse are the individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

  11. I) Can we identify populations in microbial communities? A Test: Do sequence clusters have coherent environmental dynamics?

  12. Quantification and Identification of Vibrios Barnegat Bay, NJ 4˚ to 27.5 ˚C mapquest.com * Thompson et al., 2002, 2004

  13. 16S rRNA V. coralilyticus Survey of Vibrio Diversity Barnegat Bay, NJ V. shiloi V. parahaemolyticus V. alginolyticus V. carchariea/harveyi V. pectenicida Culture-independent survey matched collections of cultured strains. V. splendidus V. anguillarum V.wodanis V.logei/fischeri 0.05 subst/site Thompson, et al, 2004

  14. 16S rRNA V. coralilyticus Survey of Vibrio Diversity Barnegat Bay, NJ V. shiloi V. parahaemolyticus V. alginolyticus V. carchariea/harveyi >99% Clusters Aug 01 Dec 01 Feb 02 Aug 02 V. pectenicida V. splendidus V. anguillarum Sequence clusters change with season and recur in summer V.wodanis V.logei/fischeri 0.05 subst/site Thompson, et al, 2004

  15. Clone Libraries Vibrio Dynamics Year-Round Vibrios (QPCR) CV ~10 to 25% cells/ml V. pectenicida-like V. splendidus-1,2 V. logei/wodanis

  16. Clone Libraries Vibrio Dynamics Coral pathogen Late-Summer Vibrios (QPCR) V. spp. CV ~10 to 25% cells/ml V. parahaemolyticus V. pectenicida-like V. splendidus-1,2 V. logei/wodanis [Thompson, et al, AEM 2004]

  17. Temperature relationship: summer and year-round Vibrios cells/ml Degrees C Persistence Currents Barnegat Bay, NJ Gulf Stream TEMPERATURE [Thompson, et al, AEM 2004]

  18. I) Can we identify populations in microbial communities? Diversity and dynamics of Vibrio ribotype clusters √Year-round and warm-water Vibrio ribotype clusters suggest differentiation with respect to seasonal parameters (e.g. temperature). √Coherent environmental dynamics in V. parahaemolyticus-like cluster (>98% rRNA identity) V. splendidus-like cluster (>98% rRNA identity) --> Closer examination of V. splendidus

  19. I) Can we identify populations in microbial communities? II) How diverse are the individuals in microbial populations? -1˚ to 16 ˚C • V. splendidus (>99% 16S rRNA) • - one of 500+ microdiverse clusters [Acinas and Klepac-Ceraj, et al 2004] • seasonal population dynamics? • genomic diversity? • Do some genotypes have different dynamics? Plum Island Sound Ipswich, MA

  20. Isolate and analyze strains in ribotype cluster Vibrio selective media Identification: clusters Internal standard Higher resolution gene Genome Fingerprint * Thompson et al., AEM 2004 DNA Digest Gel

  21. Red = V. splendidus strains V. splendidus dynamics Plum Island Sound, MA QPCR Isolation • V.splendidus ribotypes • Detected year round • Dominant isolate in summer • dynamics of genotypes (PFGE and Hsp60)? [Thompson, et al, Science 2005]

  22. V. splendidus diversity 16S rRNA ribotypes - 333 strains isolated - 20 taxa of Vibrio and Photobacterium - 232 Vibrio splendidus Hsp60 sequences 60% unique N=232 AMOVA: Random distribution of sequence-types 99% 87% unique N=206 Genome-typing (PFGE)

  23. What is the estimated diversity of “V. splendidus” genomes in the samples? Number of Hsp60 alleles: 141 (of 232 strains)Number of PFGE genotypes: 180 (of 206 strains) Chao-1 estimator: 100 to 300 Hsp60 alleles per month 500 to 900 PFGE genotypes per month at least 1,300 genomes overall

  24. What is the estimated diversity of “V. splendidus” genomes in the samples? QPCR estimation of “V. splendidus” population size: - summer months is 640 to 1,890 cells/ml • Genome concentration* = (population size/diversity) • - each month 2 to 15 cells/ml share identical Hsp60 alleles • - on average <1 cell/ml identical genome* *Conservative estimate: - Chao-1 predicts minimum diversity (richness) - “culture biases” would underestimate diversity (richness)

  25. What is the estimated diversity of “V. splendidus” genomes in the samples? Abundance: 103 cells/ml Diversity: 103 genome types/ml 1 ml 1 16S rRNA cluster 100 Hsp60 types* 1000 Genomes* * Based on the Chao-1

  26. Size variation among “V. splendidus” genomes Relationships of Hsp60 sequences How are genomes differentiated? Isolates paired by identical Hsp60 sequences spectrum of observed diversity Genome sizes (4.5 to 5.6 Mb) ~1000 genes Some diversification is due to large-scale genome changes

  27. What drives genome diversification? GENOME Elements Out Elements In • Duplication • Horizontal gene transfer • -homologous recombination • -mobile genetic elements: • phage-related gene clusters • >1% ORFs are integrases or transposases in strain 12B01 • Gene Loss Dynamic genome size & content

  28. gene transfer among closely related strains What drives population cohesion? Fraser, Science 2007 (A) (B) Considerations: (A) Frequency of recombination increases with sequence similarity “Microdiverse” organisms may have access a shared genetic pool. Are microbial populations genetically cohesive via biospecies-like evolution?

  29. Mechanisms for co-existing diversity • Genomic variants represent ecologically distinct populations consistent with niche theory. • Genome variation does not confer a time-averaged fitness advantage in a stochastic environment. • Variation is neutral • Variation is contextually-neutral i.e. it may be under selection in alternate unknown environments • Variation affects fitness; is maintained by balancing selection (e.g. kill the winner, environmental heterogeneity) ---> no single genotype may gain a lasting growth advantage.

  30. Conclusions I) Can we identify populations in microbial communities? II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

  31. Conclusions Microdiverse ribotype clusters Coherent environmental dynamics I) Can we identify populations in microbial communities? II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

  32. Conclusions Microdiverse ribotype clusters Coherent environmental dynamics I) Can we identify populations in microbial communities? V. splendidus 103 genome types/103 cells ml-1 heterogeneity up to ~1000 genes II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

  33. Conclusions Microdiverse ribotype clusters Coherent environmental dynamics I) Can we identify populations in microbial communities? V. splendidus 103 genome types/103 cells ml-1 heterogeneity up to ~1000 genes II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations? • Diversity within a population: balance of HGT & cohesion by recombination. • - Vast genomic variation may be contextually neutral or adaptive

  34. Acknowledgements Funding National Science Foundation Seagrant Department of Energy Joint Genome Institute Collaborators Dr. Martin Polz Polz Lab: Sarah Pacocha Vanja Klepac-Ceraj Chanathip Pharino Dana Hunt Jennifer Benoit Ramahi Sarma-Rupavtarm Dr. Luisa Marcelino Dr. Aoy Tomita-Mitchell Dr. Ee Lin Lim (Temple University) Dr. Daniel Distel (Ocean Genome Legacy, New England Biolabs) Dr. William Thilly (MIT)

  35. Questions?

  36. How diverse are natural microbial communities?

  37. Units of Biology Biological species Evolutionary species Reproductive Isolation Single lineage Species pluralism (Multiple definitions relevant!) Ecological species Ecological niche

  38. Are co-occurring genomes with same “ribotype” ecologically-equivalent? Three E. coli strains share <40% of total protein genes in genomes • Core genome: • shared by all • (e.g., housekeeping) • Flexible genome: • strain specific • (e.g., pathogenicity islands, integrons) Welch et al. (2002) • strains from different environments Would strains co-exist in nature outside a human host?

  39. Challenge: To identify ecologically-differentiated populations Correlation of ribotype to genome similarity 99% 16S rRNA 16S rRNA similarity 70% DNA-DNA hybridization Genome similarity Stackebrandt and Goebel, 1994

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