DNA & MicrobesDNA and the Hidden World of Microbes          CLIMB Symposium          September 12, 2011           Jonathan...
Robin in London Examples
MICROBES
Microbes are smallSize* They are small, by definition* Were not really known untilmicroscope invention
But there are LOTS of themNumbers* 100 million in gram of soil* More cells on Earth than starsin universe* More biomass th...
Diversity I: Form
Diversity II: Function
Function 1: The Bad
Function 2: The Good Nitrogen Fixation   Animal NutritionCarbon Fixation
Function 3: The Unknown
Function 4: The UnusualH2S, pH 0, 95°C   CO, 80°C    High salt                  low pH           CO2 4°C105°CCH3
Function 5: Food, Fuel, etc                   Feed                   microbes a                   little carbon           ...
Function 6: Running the Planet Carbon cycle         Nitrogen cycle
Studying microbial diversity• Two main questions  • Who is out there?  • What are they doing?
Sequencing and Microbes• Sequencing is useful as a tool in studies  of microbial diversity for many reasons• It is complim...
Era I: rRNA Tree of Life
Era I: rRNA Tree of Life  Bacteria                                             • Appearance of                            ...
Diversity III: Phylogenetic• Three main kinds of organisms  • Bacteria  • Archaea  • Eukaryotes• Viruses not alive, but so...
The Tree of Life                                        2006adapted from Baldauf, et al., in Assembling the Tree of Life, ...
The Tree of Life                                        2006adapted from Baldauf, et al., in Assembling the Tree of Life, ...
Era II: rRNA in environment
Culturing Microbes
Great Plate Count AnomalyCulturing    Microscope Count        Count
Great Plate Count AnomalyCulturing      Microscope Count      <<<< Count
Great Plate Count Anomaly                         DNACulturing      Microscope Count      <<<< Count
Culturing Microbes
Culturing Microbes
Plant/Animal Field Studies
Plant/Animal Field Studies
Plant/Animal Field Studies
Plant/Animal Field Studies
Plant/Animal Field Studies
Plant/Animal Field Studies
Plant/Animal Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
Microbial Field Studies
CSI Microbiology  Collect from  environment
CSI Microbiology  Collect from  environment
rRNA PCR              DNA              extraction                             PCR                                         ...
Comparing Sequences• Analogous to comparing bones
1992                            rRNA PCR and me ....                                                                      ...
Major phyla of bacteria & archaea                         No cultures                         Some cultures
The Hidden Majority            Richness estimates             Hugenholtz 2002         Bohannan and Hughes 2003
CensoredCensored
Era III: Genome Sequencing
1st Genome Sequence                  Fleischmann et al.                  1995
Genomes Revolutionized Microbiology• Predictions of metabolic processes• Better vaccine and drug design• New insights into...
Metabolic Predictions
Lateral Gene TransferPerna et al. 2003
Network of LifeBacteria                                       Archaea Eukaryotes    Figure from Barton, Eisen et al.      ...
Using the Core
WhWhole genome treebuilt usingAMPHORAby Martin Wu andDongying Wu
Microbial genomes     From http://genomesonline.org
Phylogenetic Diversity• Phylogenetic  diversity poorly  sampled• GEBA project at DOE-  JGI correcting this
Era IV: Genomes in Environment
Novel Form of Phototrophy                        Beja et al. 2000
Era IV: Genomes in Environment                 shotgun                      sequenceMetagenomics
Metagenomics Challenge
Binning challenge
Weighted % of Clones                                                                                           0          ...
Metagenomics & Ecology
Field Diversity
Sequencing Technology
Generation I: Manual Sanger
Generation II: Automation
Generation III: No clones
Generation IV: ????
Acknowledgements• $$$  •   DOE  •   NSF  •   GBMF  •   Sloan  •   DARPA• People, places  • DOE JGI: Eddy Rubin, Phil Hugen...
DNA and the hidden world of microbes
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DNA and the hidden world of microbes

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Talk by Jonathan Eisen for CLIMB2011 Symposium at UC Davis.

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  • Small organisms - too small to see without help\n giant microbes\n \n Mostly live as single cells\n \n Many different kinds (more on this in a bit)\n \n VIruses not included by some, but I think they count\n \n (Show flu bug slide)\n \n
  • Small organisms - too small to see without help\n giant microbes\n \n Mostly live as single cells\n \n Many different kinds (more on this in a bit)\n \n VIruses not included by some, but I think they count\n \n (Show flu bug slide)\n \n
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  • Do lots of nasty things\n Get all the good names by the way b/c mostly known through their diseses\n Yersinia pestis\n Vibrio cholerae\n Bacillus anthracis\n Smallpox - ok not all\n Mycobacterium tuberculosis\n Mycobacterium leprae\n Clostridium tetanus\n Clostridium botulinum\n
  • Mutualists (though names not so good)\n N2 fixation\n C fixation - Chloroplasts inside plants are actually symbiotic bacteria\n Digestion - ruminants and all cellulose\n\n
  • Cloud of microbes living in / on organisms\n More microbial cells than human\n
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  • Microbes run the planet\n All photosynthesis\n Number of microbial cells\n
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  • Composite tree of life based on diverse data\nmain point - euks monophyletic\n\nSignif LGT in euks, but mostly bacterial genes\nso bulk of euk genome is vertically transmitted\nand we can use these gene to reconstruct their history\n
  • Composite tree of life based on diverse data\nmain point - euks monophyletic\n\nSignif LGT in euks, but mostly bacterial genes\nso bulk of euk genome is vertically transmitted\nand we can use these gene to reconstruct their history\n
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  • Many prokaryotes cannot be grown and cannot get them from harsh envts, or replicate the envt (i.e volcanic)\nHow do we approach this problem?\n\nResearcher go to various areas to collect samples from envt\n\nGet samples, put into tube\n
  • Send it out for sequencing, do an alignment with your gene and blast it (search for other organisms) with a similar sequence\n
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  • Functional prediction using a gene tree is just like predicting the biology of a species using a species tree\n
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  • 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.\n
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  • DNA and the hidden world of microbes

    1. DNA & MicrobesDNA and the Hidden World of Microbes CLIMB Symposium September 12, 2011 Jonathan A. Eisen University of California, Davis
    2. Robin in London Examples
    3. MICROBES
    4. Microbes are smallSize* They are small, by definition* Were not really known untilmicroscope invention
    5. But there are LOTS of themNumbers* 100 million in gram of soil* More cells on Earth than starsin universe* More biomass than plants,animals* 10x cells on humans thanhuman cells* 50x10^6 viruses/ml sea water
    6. Diversity I: Form
    7. Diversity II: Function
    8. Function 1: The Bad
    9. Function 2: The Good Nitrogen Fixation Animal NutritionCarbon Fixation
    10. Function 3: The Unknown
    11. Function 4: The UnusualH2S, pH 0, 95°C CO, 80°C High salt low pH CO2 4°C105°CCH3
    12. Function 5: Food, Fuel, etc Feed microbes a little carbon and they can make some nice things
    13. Function 6: Running the Planet Carbon cycle Nitrogen cycle
    14. Studying microbial diversity• Two main questions • Who is out there? • What are they doing?
    15. Sequencing and Microbes• Sequencing is useful as a tool in studies of microbial diversity for many reasons• It is complimentary to other means of study• Four major “ERAs” in use of sequencing for microbial diversity studies
    16. Era I: rRNA Tree of Life
    17. Era I: rRNA Tree of Life Bacteria • Appearance of microbes not informative (enough) • rRNA Tree of Life Archaea identified two major groups of organisms w/o nuclei • rRNA powerful for many reasons, though not perfect EukaryotesBarton, Eisen et al. “Evolution”, CSHL Press. 2007.Based on tree from Pace 1997 Science 276:734-740
    18. Diversity III: Phylogenetic• Three main kinds of organisms • Bacteria • Archaea • Eukaryotes• Viruses not alive, but some call them microbes• Many misclassifications occurred before the use of molecular methods
    19. The Tree of Life 2006adapted from Baldauf, et al., in Assembling the Tree of Life, 2004
    20. The Tree of Life 2006adapted from Baldauf, et al., in Assembling the Tree of Life, 2004
    21. Era II: rRNA in environment
    22. Culturing Microbes
    23. Great Plate Count AnomalyCulturing Microscope Count Count
    24. Great Plate Count AnomalyCulturing Microscope Count <<<< Count
    25. Great Plate Count Anomaly DNACulturing Microscope Count <<<< Count
    26. Culturing Microbes
    27. Culturing Microbes
    28. Plant/Animal Field Studies
    29. Plant/Animal Field Studies
    30. Plant/Animal Field Studies
    31. Plant/Animal Field Studies
    32. Plant/Animal Field Studies
    33. Plant/Animal Field Studies
    34. Plant/Animal Field Studies
    35. Microbial Field Studies
    36. Microbial Field Studies
    37. Microbial Field Studies
    38. Microbial Field Studies
    39. Microbial Field Studies
    40. Microbial Field Studies
    41. Microbial Field Studies
    42. Microbial Field Studies
    43. CSI Microbiology Collect from environment
    44. CSI Microbiology Collect from environment
    45. rRNA PCR DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’...ACACACATAGGTGGAGC TAGCGATCGATCGA... 3’ Phylogenetic tree Sequence alignment = Data matrix rRNA2 rRNA1 rRNA2 rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCT rRNA4 AGCGACGATCGA... 3’rRNA3 rRNA2 T A C A G T rRNA3 rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGA E. coli Humans rRNA4 C A C A G T TTCTAGCGATATAGA... 3’ Yeast E. coli A G A C A G rRNA4 5’...ACGGCCCGATAGGTGG Humans T A T A G T ATTCTAGCGCCATAGA... 3’ Yeast T A C A G T
    46. Comparing Sequences• Analogous to comparing bones
    47. 1992 rRNA PCR and me .... NOTES 3419 A. pisum P A. piswn S Tx. nivea L awaaa sym L equizenata syr L Cud orbgcdar s,ym rs. gesgosterorn - / I -- V I N. gonorrhoeae B. Uhar.opkiuns sym 5% C. magncisca sym Tns. sp. L-12 A. tnefaciens JOURNAL OF BACTERIOLOGY, May 1992, p. 3416-3421 0021-9193/92/103416-06$02.00/0 Vol. 174, No. 10 Copyright © 1992, American Society for Microbiology Phylogenetic Relationships of Chemoautotrophic Bacterial R. ricketsil Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Received 4 November 1991/Accepted 9 March 1992 The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain rRNA regions and hybridization of an Escherichia coli gene probe to S. velum
    48. Major phyla of bacteria & archaea No cultures Some cultures
    49. The Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003
    50. CensoredCensored
    51. Era III: Genome Sequencing
    52. 1st Genome Sequence Fleischmann et al. 1995
    53. Genomes Revolutionized Microbiology• Predictions of metabolic processes• Better vaccine and drug design• New insights into mechanisms of evolution• Genomes serve as template for functional studies• New enzymes and materials for engineering and synthetic biology
    54. Metabolic Predictions
    55. Lateral Gene TransferPerna et al. 2003
    56. Network of LifeBacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.
    57. Using the Core
    58. WhWhole genome treebuilt usingAMPHORAby Martin Wu andDongying Wu
    59. Microbial genomes From http://genomesonline.org
    60. Phylogenetic Diversity• Phylogenetic diversity poorly sampled• GEBA project at DOE- JGI correcting this
    61. Era IV: Genomes in Environment
    62. Novel Form of Phototrophy Beja et al. 2000
    63. Era IV: Genomes in Environment shotgun sequenceMetagenomics
    64. Metagenomics Challenge
    65. Binning challenge
    66. Weighted % of Clones 0 0.1250 0.2500 0.3750 0.5000 Al ph a Be pro ta teo G p b am rot ac m eo te ba ria Ep ap ct si ro lo t e np eob ria D el rot ac ta e t pr ob eria ot ac C eo te ya b rEFG no ac iaEFTurRNARecARpoB b teHSP70 Fi act ria rm e Ac ic ria tin ut es ob a C cte hl r or ia ob C i FB C hl o Major Phylogenetic Group Sp rof Sargasso Phylotypes iro lex i Fu cha D304: 66. 2004 Metagenomic Phylotyping ei so et no ba es co ct cc er Euus ia ry -T a hVenter et al., Science C rcherm re na aeous rc t ha a eo ta
    67. Metagenomics & Ecology
    68. Field Diversity
    69. Sequencing Technology
    70. Generation I: Manual Sanger
    71. Generation II: Automation
    72. Generation III: No clones
    73. Generation IV: ????
    74. Acknowledgements• $$$ • DOE • NSF • GBMF • Sloan • DARPA• People, places • DOE JGI: Eddy Rubin, Phil Hugenholtz et al. • UC Davis: Aaron Darling, Dongying Wu • Other: Jessica Green, Katie Pollard, Martin Wu, Tom Slezak, Jack Gilbert
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