Microbial Genomics Workshop Elizabeth Dinsdale Rio October - 2012

1. Marine Microbial communities Microbial Genomics Workshop Elizabeth Dinsdale Rio October - 2012

2. Oceans • 71 % of planet is covered by ocean • Mean depth 3800 m – deepest 10, 800m • Mean height of Earth 840 m, highest 8936 • Temperature, pressure • Circulation – old and young water • High and low nutrient areas • Upwellings 1% area – most productivity

3. Biogeochemistry - carbon

4. What factors affect carbon uptake? • Human activity – more CO2 in atmosphere • More dissolving into ocean • Movement of current • Sinking of heavy CO2 cold water at poles • Abiotic processes a small fraction of the removal/use of carbon dioxide • Biological pump removes most CO2

5. Sequester of carbon – microbially driven Chisholm Nature407, 685-687

6. Recognition of the abundance of microbes in the oceans • 1970- 1980 DNA staining methods that identified the numbers of microbes in ocean • Local, regional, bulk estimates of numbers and consumption • Large effect of microbes on biogeochemical production • Problem could not identify individual species, like eukaryotes

7. DNA sequencing • Culturing - description of 1 % of all microbes • 16S rDNA analysis 1980 - Pace • Defined taxonomy of microbes • Reduced bias • Identified new organisms • More diversity • Targeted culturing – ID new species and ecotypes

8. Prochlorococcus - ecotypes • Highly related, but physiologically and genetically distinct • Investigated distribution in ocean • Different physiological conditions – high light – gene that repairs photo-damage - use reduced forms of nitrogen • Low light – no repair gene - use nitrite and ammonium

9. Prochlorococcus Cruise Southern hemisphere Nitrite depth profile Mixed layer Bouman et al Science 312:918 2006

10. Prochlorococcus abundance • DivinylChla • Cell #

11. Regional variation in Prochlrococcus strains • Low numbers – competition Synecococcus • Low nitrates – shallow mixed layer HL adapted

12. Metagenomes: genetic analysis of all the microbes from an environment www.evolutionpages.com www.cebitec.uni-bielefeld.de

13. Filter Capture organisms Extract DNA Microbial genomes Metagenomics Sample the environment Taxonomic Functional Description Sequence Comparison to database

14. Metagenomics – BAC libraries • Photorhodopsin genes – Beja et al 2000 • light driven proton pump, chloride ion pump or photosensory receptor • First found in a Halophilic Archaea • y- proteobacterium from the ocean, first time seen in a Bacterium non-extreme environment • SAR86 – common in the ocean • Seen deep water suggesting new functionality

15. Metagenomes- new genes • Archaeal ammonia oxidation • ammonia mooxygenasae gene • Crenarchaeota • Ammonium as sole energy source • Archaea important in nitrogen cycle, not just Bacteria as thought Beja et al 2002

16. Sargasso Sea • Sargasso Sea – Venter 2004 • Random sample of the entire microbial community • 6 sites of surface water • Suggested could assemble microbial genomes • Estimated 1800 species • New genes • 748 new photorhodopsin genes

17. Global Ocean survey • 6.12 million predicted proteins from 7.7 million reads • All known microbial proteins PLUS • 1,700 new ones – each with 20 representatives • Rate of new proteins identified, with more than 2 representatives, was linear with each sequence

18. Metagenomic momentum • Black Bac libraries, Pink – Fosmid libraries,Green - 454 Hughenholt and Tyson 2008

19. Functional profile of microbial community • Sequences from a large range of microbes in a community • Microbes from the same environment have similar functions • Functional or metabolic potential

20. Metagenomic sample location

21. Microbial metagenomes group by environment

22. Identify the metabolism that drive differences between environments Cell wall Membrane transport Virulence Protein Sulfur Stress Signaling Motility Respiration Dinsdale et al 2008 Nature 452:629-632

23. Targeted metagenomic Dinsdale and Rohwer 2008 review on Kalyuzhnaya et al 2008

24. Nitrogen fixing cyanobacteria (UCYN-A) flow sorted metagenomes • Flow sorting • PCR to confirm Sorting • 15% of community • 5000 cells • Amplified • Sequenced • Zehr et al 2008 Science 322:1110-1112

25. UCYN-A metagenome components from 16S

26. NIF genes all present • Analysis of NIF gene • Grey areas of matches, white noncoding

27. Photosystem genes identified in flow sorted metagenomes • No carbon fixing • What is PS1 doing? • Not producing oxygen • Is it symbiotic? • No poisoning of nitrogen fixing by oxygen

28. Two major organisms in biofilm air-liquid interface Microbial Evolution • Leptospirillum group II – iron oxidation, does not fix nitrogen • Ferroplasma type II – pyrite dilution and H+ production • Denef and Banfield, Science 2012

29. Filter Capture organisms Extract DNA Microbial genomes Metagenomics Sample the environment Taxonomic Functional Description Sequence Comparison to database Kmer, sequencing depth, GC content Bin sequences – compiled genomes

30. Location and samples over nine year period Leptospirillum group II type 1- 6

31. Genome type III, combination of type 1 (red) and VI (blue)

34. Genome evolution

35. RNA analysis

37. Transcriptome vs metagenome • Metagenome provide a functional potential • Transcriptome provides an analysis of the RNA’s that are being produced • Transcriptome difficult to obtain, small quantities, mostly rRNA not mRNA • Proteomics – proteins that are being produced

38. Marine microbiology • Extensive progress over last 40 year • High diversity • Novel genes • Novel activity • Clarified some biogeochemical pathways • Evolution of microbial communities • Do we know how much carbon sequestering will occur in the oceans?

39. Delong 2006 Nature 459 Metagenomic data adding to the description of the food web

40. Samba • Thursday 10:30pm • Lapa 40O – Diogo Nogueira • Rua Riachuelo, 97 – Lapa • Print the flyer you get a discount ~ R5 – logon to website • $R40 for Men! • $R25 for Women!! • BUT YOU have to be here on Friday morning