Microbes Can Grow On                 Anything• Energy    – Light    – Organic and inorganic chemicals• Carbon    – Organic...
QuickTime™ and aTIFF (Uncompressed) decompressor   are needed to see this picture.
ExtremophilyType        ConditionsTemperature Thermophiles,              PsychorphilepH          Alkaliphiles,            ...
QuickTime™ and aTIFF (Uncompressed) decompressor   are needed to see this picture.
How Survive at 100°C• Change amino acid composition of all  proteins• Change composition of membranes• Add enzymes to repa...
QuickTime™ and aTIFF (Uncompressed) decompressor   are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor   are needed to see this picture.
How Survive at High Salt• High salt will cause water to want to flow out of  cell• Compensate by increasing solute concent...
QuickTime™ and aTIFF (Uncompressed) decompressor   are needed to see this picture.
How Survive Desiccation?•   Spore formation•   Increase solute concentration•   Starvation tolerance•   Repair desiccation...
But ….
Great Plate Count Anomaly Culturing   Microscope  Count        Count
Great Plate Count Anomaly Culturing          Microscope  Count      <<<<     Count
Environmental Microbiology Era I:       Who is out There?
rRNA Revolution           • Morphology and             physiology evolve too             rapidly           • Molecular sys...
PCR Saves the Day
Solving the Plate Count Anomaly                    PCR    Culturing   Microscope     Count        Count
Compare PCR Amplified rRNA To Those of Cultured Species                                Eisen et                           ...
Majority of Microbes are “Uncultured”       Numbers and Diversity
Problems with rRNA PCR• Doesn’t predict biology of organisms well• Doesn’t work for viruses• Not very quantitative
Environmental Microbiology Era II:      What are they Doing?
Metagenomics by Large Inserts•   Isolate, by filtration, all microbes in a sample•   Extract total DNA in very large piece...
Phylogenetic Anchors                   Beja et al. 2000
Using a rRNA anchor                         allowed the                   identification of a new                    form ...
Beja O, et.al., Science 2000 289:1902-6, Nature (2001) 411: 786-789
Limits of Large Insert Approach• Large insert libraries less random and less  representative than small inserts• Lower thr...
Enviornmental Microbiology Era III:Environmental Shotgun Sequencing
Environmental Shotgun Sequencing                        shotgunWarner Brothers, Inc.                                  sequ...
Assemble Fragmentssequencer output                   assemble                   fragments                   Closure &     ...
Baumannia cicadellinicola genome project:1° symbionts of the Glassy-winged Sharpshooter                                • S...
Co-Symbiosis?
Sargasso Sea Shotgun Sequencing                    QuickTime™ and a               TIFF (LZW) decompressor                 ...
Can Learn By “Black Box”        Approach
Binning Much More Difficult in      Complex CommunitiesA                                TB                                ...
rRNA Phylotypes                  Venter et al., 2004
taxonomic content per SHOTGUN 16S100%90%80%70%60%50%40%30%20%10% 0%       G G G G G G G G G G G G G G G G G G G G G G G G ...
Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA)                           Venter et al., 2004
Other Markers Give Similar Phylotpyes                                           Sargasso Phylotypes        0.5       0.45 ...
Shotgun Sequencing Detects More  Diversity than PCR-methods
Biased Sampling of Genomes
Biased Sampling of Genomes
Proteobacteria TM6OS-K                        • At least 40AcidobacteriaTermite Group OP8                          phyla o...
Proteobacteria TM6OS-K                        • At least 40AcidobacteriaTermite Group OP8                          phyla o...
Proteobacteria TM6OS-K                        • At least 40AcidobacteriaTermite Group OP8                          phyla o...
Proteobacteria TM6OS-K                        • At least 40AcidobacteriaTermite Group OP8                          phyla o...
Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
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Microbes run the planet - Jonathan Eisen slides from #scifoo 2006

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Presentation by Jonathan Eisen from SciFoo meeting in 2006

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  • An example of the reasons predicting function is difficult comes from our work on Deincooccus radiodurans , the most radiation resistant organism known. When TIGR sequenced the genome of this species (just before I got to TIGR) I helped look at the predicted DNA repair genes in the genome in the hope that this analysis would tell us something about this species radiation resistance. I was very interested in this since my Ph.D was on comparative studies of DNA repair, especially in extremophiles.
  • Extension of rRNA analysis to uncultured organisms using PCR
  • Phylogenetic analysis of rRNAs led to the discovery of archaea
  • Functional prediction using a gene tree is just like predicting the biology of a species using a species tree
  • This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems.
  • Metagenomics involves cloning large DNA fragments from environmental samples and then selecting specific fragments for further sequencing. The selection of specific fragments can be based on mapping rRNA genes to them, mapping functional genes, or by first doing end-sequencing and then selecting fragments with interesting end-sequences.
  • This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems.
  • Metagenomic analysis led to the discovery of a new form of phototrophy in the ocean
  • PCR primers designed from the metagenomic sequences were then used to discover additional forms of the proteorhodopsin that is the basis for the new form of phototrophy
  • This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems. clone from the Sargasso Sea. This shows that this
  • Microbes run the planet - Jonathan Eisen slides from #scifoo 2006

    1. 1. Microbes Can Grow On Anything• Energy – Light – Organic and inorganic chemicals• Carbon – Organic degradation – Inorganic “fixation” • CO2, CO, CH4• Contol global cycling of most nutrients – N, S, P, – Can manipulate just about every form
    2. 2. QuickTime™ and aTIFF (Uncompressed) decompressor are needed to see this picture.
    3. 3. ExtremophilyType ConditionsTemperature Thermophiles, PsychorphilepH Alkaliphiles, AcidiphilesPressure BarophileSalt HalophilesRadiation Radiophiles
    4. 4. QuickTime™ and aTIFF (Uncompressed) decompressor are needed to see this picture.
    5. 5. How Survive at 100°C• Change amino acid composition of all proteins• Change composition of membranes• Add enzymes to repair heat specific damage (e.g., deamination of DNA)• Changing which metals are used as cofactors in biological processes• Cell wall coatings
    6. 6. QuickTime™ and aTIFF (Uncompressed) decompressor are needed to see this picture.
    7. 7. QuickTime™ and aTIFF (Uncompressed) decompressor are needed to see this picture.
    8. 8. How Survive at High Salt• High salt will cause water to want to flow out of cell• Compensate by increasing solute concentrations in cell• Many organisms use different solutes• Extreme halophiles fill up inside of cell with salts also• Enzymes from these organisms work well in industrial applications where salts are present
    9. 9. QuickTime™ and aTIFF (Uncompressed) decompressor are needed to see this picture.
    10. 10. How Survive Desiccation?• Spore formation• Increase solute concentration• Starvation tolerance• Repair desiccation damage
    11. 11. But ….
    12. 12. Great Plate Count Anomaly Culturing Microscope Count Count
    13. 13. Great Plate Count Anomaly Culturing Microscope Count <<<< Count
    14. 14. Environmental Microbiology Era I: Who is out There?
    15. 15. rRNA Revolution • Morphology and physiology evolve too rapidly • Molecular systematics is the only way • 16s rRNA is the choice • Three domains discovered
    16. 16. PCR Saves the Day
    17. 17. Solving the Plate Count Anomaly PCR Culturing Microscope Count Count
    18. 18. Compare PCR Amplified rRNA To Those of Cultured Species Eisen et al. 1992
    19. 19. Majority of Microbes are “Uncultured” Numbers and Diversity
    20. 20. Problems with rRNA PCR• Doesn’t predict biology of organisms well• Doesn’t work for viruses• Not very quantitative
    21. 21. Environmental Microbiology Era II: What are they Doing?
    22. 22. Metagenomics by Large Inserts• Isolate, by filtration, all microbes in a sample• Extract total DNA in very large pieces• Clone those pieces as BACs into E.coli to get enough.• ID BACs of interest (e.g., containing rRNA)• Sequence and analyze the BACs like a bacterial genomeSample Gene Filter Extract Clone Sequence DNA List concentrate Into BACs
    23. 23. Phylogenetic Anchors Beja et al. 2000
    24. 24. Using a rRNA anchor allowed the identification of a new form of phototrophy: ProteorhodopsinBeja et al. 2000
    25. 25. Beja O, et.al., Science 2000 289:1902-6, Nature (2001) 411: 786-789
    26. 26. Limits of Large Insert Approach• Large insert libraries less random and less representative than small inserts• Lower throughput• Requires some thinking
    27. 27. Enviornmental Microbiology Era III:Environmental Shotgun Sequencing
    28. 28. Environmental Shotgun Sequencing shotgunWarner Brothers, Inc. sequence
    29. 29. Assemble Fragmentssequencer output assemble fragments Closure & Annotation
    30. 30. Baumannia cicadellinicola genome project:1° symbionts of the Glassy-winged Sharpshooter • Sap feeding insects • Carriers of Xylella fastidiosa that causes Pierce’s disease of grapevines • There are >20000 sharpshooter species,Glassy-winged Sharpshooter within which intracellular symbiotic bacteria are wildspread
    31. 31. Co-Symbiosis?
    32. 32. Sargasso Sea Shotgun Sequencing QuickTime™ and a TIFF (LZW) decompressor shotgun are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. sequence Analysis led by Venter Institute. Eisen lab contributions by Dongying Wu, Martin Wu, Jonathan Badger
    33. 33. Can Learn By “Black Box” Approach
    34. 34. Binning Much More Difficult in Complex CommunitiesA TB UC VD WE XF YG Z
    35. 35. rRNA Phylotypes Venter et al., 2004
    36. 36. taxonomic content per SHOTGUN 16S100%90%80%70%60%50%40%30%20%10% 0% G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 36 Station
    37. 37. Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA) Venter et al., 2004
    38. 38. Other Markers Give Similar Phylotpyes Sargasso Phylotypes 0.5 0.45 0.4 0.35 EFG 0.3 EFTu HSP70 0.25 RecA 0.2 RpoB rRNA 0.15Weighted % of Clones 0.1 0.05 0 CFB Chlorobi Firmicutes Chloroflexi Fusobacteria Spirochaetes Cyanobacteria Actinobacteria Euryarchaeota Crenarchaeota BetaproteobacteriaDeltaproteobacteria Alphaproteobacteria Epsilonproteobacteria Gammaproteobacteria Deinococcus-Thermus Major Phylogenetic Group
    39. 39. Shotgun Sequencing Detects More Diversity than PCR-methods
    40. 40. Biased Sampling of Genomes
    41. 41. Biased Sampling of Genomes
    42. 42. Proteobacteria TM6OS-K • At least 40AcidobacteriaTermite Group OP8 phyla ofNitrospiraBacteroides bacteriaChlorobiFibrobacteresMarine GroupA WS3GemmimonasFirmicutesFusobacteriaActinobacteria OP9CyanobacteriaSynergistesDeferribacteresChrysiogenetesNKB19VerrucomicrobiaChlamydia OP3PlanctomycetesSpriochaetesCoprothmermobacterOP10ThermomicrobiaChloroflexi TM7Deinococcus-ThermusDictyoglomusAquificaeThermudesulfobacteriaThermotogae OP1OP11
    43. 43. Proteobacteria TM6OS-K • At least 40AcidobacteriaTermite Group OP8 phyla ofNitrospiraBacteroides bacteriaChlorobiFibrobacteresMarine GroupA • Genome WS3GemmimonasFirmicutes sequences areFusobacteriaActinobacteria mostly from OP9Cyanobacteria three phylaSynergistesDeferribacteresChrysiogenetesNKB19VerrucomicrobiaChlamydia OP3PlanctomycetesSpriochaetesCoprothmermobacterOP10ThermomicrobiaChloroflexi TM7Deinococcus-ThermusDictyoglomusAquificaeThermudesulfobacteriaThermotogae OP1OP11
    44. 44. Proteobacteria TM6OS-K • At least 40AcidobacteriaTermite Group OP8 phyla ofNitrospiraBacteroides bacteriaChlorobiFibrobacteresMarine GroupA • Genome WS3GemmimonasFirmicutes sequences areFusobacteriaActinobacteria mostly from OP9Cyanobacteria three phylaSynergistesDeferribacteresChrysiogenetes • Some otherNKB19VerrucomicrobiaChlamydia phyla are OP3PlanctomycetesSpriochaetes only sparselyCoprothmermobacterOP10 sampledThermomicrobiaChloroflexi TM7Deinococcus-ThermusDictyoglomusAquificaeThermudesulfobacteriaThermotogae OP1OP11
    45. 45. Proteobacteria TM6OS-K • At least 40AcidobacteriaTermite Group OP8 phyla ofNitrospiraBacteroides bacteriaChlorobiFibrobacteresMarine GroupA • Genome WS3Gemmimonas sequences areFirmicutesFusobacteria mostly fromActinobacteria OP9Cyanobacteria three phylaSynergistesDeferribacteresChrysiogenetes • Some otherNKB19VerrucomicrobiaChlamydia phyla are only OP3Planctomycetes sparselySpriochaetesCoprothmermobacterOP10 sampledThermomicrobiaChloroflexi TM7 • Solution:Deinococcus-ThermusDictyoglomusAquificae sequence moreThermudesulfobacteriaThermotogae phyla OP1OP11

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