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

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 Nature 407,

6. Recognition of the abundance of microbes in the oceans
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 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:

10. Prochlorococcus abundance
Divinyl Chla
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

13. Metagenomics Sample the environment Filter Capture organisms
Extract DNA
Microbial genomes
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 Estimated 1800 species
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
Culturing tests the function of the microbes, eg ph, salinity, temperature, heavy metals etc

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
Similar environment across a Wide geographic area

21. Microbial metagenomes group by environment
Metagenomes grouped
2 dimensional space – of where the metagenomes site in the metabolic space
80% of the variance explained!!! If you do this with 16s there is about 12 % of the variance explained
Each point is one metagenome
Lots of variance explained by the analysis

22. Identify the metabolism that drive differences between environments
Cell wall
Membrane transport
Virulence
Protein
Sulfur
Stress
Signaling
Motility
Lozupone 2007 pnas comparisons of 16S genes
Which metabolic process driving the variation
One metagenome is not sitting with its sister group
Respiration
Dinsdale et al 2008 Nature 452:

23. Targeted metagenomic
Dinsdale and Rohwer 2008 review on Kalyuzhnaya et al 2008
Culturing tests the function of the microbes, eg ph, salinity, temperature, heavy metals etc

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:
Culturing tests the function of the microbes, eg ph, salinity, temperature, heavy metals etc

25. UCYN-A metagenome components from 16S
Culturing tests the function of the microbes, eg ph, salinity, temperature, heavy metals etc

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
Culturing tests the function of the microbes, eg ph, salinity, temperature, heavy metals etc

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

29. Metagenomics Sample the environment Filter Capture organisms
Extract DNA
Microbial genomes
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)
They aregue that the alignment homologous recombination points of the peptide data and the sequence data suggests that the recombination occurred in single cell and its desendents rose to fixation in the population?

32. Most recent to oldest, shaded areas not considered for snp analysis – because of recombination
Disproportional changes in transduction genes and transcriptional regulation genes

33. Most recent to oldest, shaded areas not considered for snp analysis – because of recombination
Disproportional changes in transduction genes and transcriptional regulation genes

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

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