DNA Sequencing                              and the                      Modern Revolution in                   Studies of...
Monday, November 26, 12
Social Networking in ScienceMonday, November 26, 12
Bacterial evolveMonday, November 26, 12
Outline        • Introduction: Diversity of microbes        • I: The Tree of Life        • II: Genome Sequencing        • ...
Introduction                          Diversity of Microbes                                                  6Monday, Nove...
D. Diversity of form                          Diversity of function                                                  7Mond...
Many major pathogens are bacteriaMonday, November 26, 12
Bacteria and archaea are key commensals of many eukaryotesMonday, November 26, 12
Extreme conditions are dominated by bacteria and archaeaMonday, November 26, 12
Microbes run global cyclesMonday, November 26, 12
Photosynthetic Organisms Changed Earth’s Atmosphere                      The first photosynthetic cells were similar to cy...
6.15 Metabolic Pathways                          13Monday, November 26, 12
D. Diversity of form                    Diversity of form: prokaryotes                                                    ...
More shape diversity                          15Monday, November 26, 12
16Monday, November 26, 12
Diversity of form II: complexity and size                                            17Monday, November 26, 12
Fruiting bodiesPhoto 26.24 Fruiting body of gliding bacterium Stigmatella aurantiaca. SEM.   18Monday, November 26, 12
Diversity of form III: biofilms                                                                    Attraction of     Free-...
D. Diversity of form                           Diversity of form:                          microbial eukaryotes           ...
Part I:                          The Tree of Life                                             21Monday, November 26, 12
Darwin and a Single Tree of Life      George Richmond. Darwin Heirlooms Trust    Darwin Origin of Species 1859    Set stag...
Ernst Haeckel 1866        Plantae        Protista        Animalia                                          23             ...
Whittaker – Five Kingdoms 1969       Monera       Protista       Plantae        Fungi       Animalia                      ...
The Microbe Problem         Most trees of life did not deal with microbes very         well         Trees were not based o...
Carl Woesehttp://mcb.illinois.edu/faculty/profile/1204                                               26Monday, November 26...
12.3 From Gene to Protein                            27Monday, November 26, 12
The Ribosome                          28Monday, November 26, 12
rRNA Systematics        • All cellular organisms have ribosomes        • All have homologous subunits of the ribosomes inc...
Alignments  • Method of assigning    homology to    individual residues    in different    sequences  • Allows one to have...
Alignments        • Similar in          concept to lining          up bones from          different          species      ...
Woese 1987 - rRNA                          Microbiological Reviews 51:221                                                 ...
4.7 Eukaryotic Cells (Part 1)                                33Monday, November 26, 12
4.4 A Prokaryotic Cell                          34Monday, November 26, 12
26.23 Some Would Call It Hell; These Archaea Call It Home                                                            35Mon...
The Tree of Life                          2006                                                                            ...
The Tree of Life                          2006                                                                            ...
Why tree useful?        • Reclassification of many organisms, including diversity of          pathogens           Changes...
Part II:                          Genome Sequencing                                              39Monday, November 26, 12
Fleischmann et al.                          1995Monday, November 26, 12
Whole Genome Shotgun SequencingMonday, November 26, 12
Whole Genome Shotgun SequencingMonday, November 26, 12
Whole Genome Shotgun Sequencing Warner Brothers, Inc.Monday, November 26, 12
Whole Genome Shotgun Sequencing                               shotgun Warner Brothers, Inc.Monday, November 26, 12
Whole Genome Shotgun Sequencing                               shotgun Warner Brothers, Inc.Monday, November 26, 12
Whole Genome Shotgun Sequencing                               shotgun Warner Brothers, Inc.                               ...
Whole Genome Shotgun Sequencing                               shotgun Warner Brothers, Inc.                               ...
Assemble FragmentsMonday, November 26, 12
Assemble Fragments                          sequencer outputMonday, November 26, 12
Assemble Fragments                          sequencer outputMonday, November 26, 12
Assemble Fragments                          sequencer output                                             assemble         ...
Assemble Fragments                          sequencer output                                             assemble         ...
Microbial genomes                               From http://genomesonline.orgMonday, November 26, 12
General Steps in Analysis of                             Complete Genomes          • Identification/prediction of genes    ...
Vibrio cholerae MetabolismMonday, November 26, 12
Genome Sequences Have                     Revolutionized Microbiology            • Predictions of metabolic processes     ...
Genome SizeMonday, November 26, 12
Genome              Structure:                More               Variable              than OnceMonday, November 26, 12
Monday, November 26, 12
Figure 7.6 - Gene contentMonday, November 26, 12
Figure 7.7 - Gene content E. coliMonday, November 26, 12
Figure 7.10 - K12 vs O157H7Monday, November 26, 12
Lateral Transfer                                             from Doolittle, 1999Monday, November 26, 12
from Lerat et alMonday, November 26, 12
A. Studying microbes                                Part III:                          Microbes in the field              ...
How to study microbes        • Key questions about microbes in environment:           Who are they? (i.e., what kinds of ...
57Monday, November 26, 12
Figure 26.24 Extreme Halophiles                                  58Monday, November 26, 12
Deep Sea Ecosystems                          59Monday, November 26, 12
• For any particular environment, there are many different          ways one could go about characterizing the microbes th...
A. Method 1                             Method 1:                          Observe in the field                           ...
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       62Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       63Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
Field Observations an Important Tool                                       64Monday, November 26, 12
B. Method 2                          Method 2:                          Culturing                                      65M...
Method 2: Culturing                          66Monday, November 26, 12
Examples of Benefits of Culturing:        • Allows one to connect processes and properties to single          types of org...
Optimal salt concentration for different species                                                   68Monday, November 26, 12
Halophile adaptations        • Some stresses of high salt           Osmotic pressure on cells   H20           Desiccatio...
Halophile adaptations        • Some stresses of high salt           Osmotic pressure on cells          H20           Des...
Halophile adaptations        • Some stresses of high salt           Osmotic pressure on cells           Desiccation     ...
Halophile adaptations        • Some stresses of high salt           Osmotic pressure on cells           Desiccation     ...
Extreme halophiles are a monophyletic group                                              73Monday, November 26, 12
Uses of extremophiles        Type of           Examples           Example of        Practical Uses        environment     ...
Method III:                          CSI Microbiology                                             75Monday, November 26, 12
Great Plate Count Anomaly                          Culturing   Microscopy                           Count       Count     ...
Great Plate Count Anomaly                          Culturing          Microscopy                           Count      <<<<...
Great Plate Count Anomaly    Problem because     appearance not   effective for “who    is out there?” or     “what are th...
Great Plate Count Anomaly                                                      Solution?    Problem because     appearance...
Great Plate Count Anomaly                                                      Solution?    Problem because     appearance...
Analysis of uncultured microbes                          Collect from                          environment                ...
Analysis of uncultured microbes                          Collect from                          environment                ...
Polymerase Chain Reaction- PCR                                                   82Monday, November 26, 12
PCR and phylogenetic analysis of rRNA genes                          DNA                          extraction              ...
Deep Sea Ecosystems                          84Monday, November 26, 12
ChemosymbiontsMonday, November 26, 12
Analysis of uncultured microbes1992                                                                                       ...
Analysis of uncultured microbes                          Collect from                          environment                ...
Analysis of uncultured microbes                          Collect from                          environment                ...
PCR and phylogenetic analysis of rRNA genes                          DNA                          extraction              ...
PCR and phylogenetic analysis of rRNA genes                           DNA                           extraction            ...
Major phyla of bacteria and archaea (as of 2002)                                                   No cultures            ...
Uses of rDNA PCR                                            Bohannan and                                            Hughes...
92Monday, November 26, 12
Censored                          Censored                                     93Monday, November 26, 12
94Monday, November 26, 12
Part IV:                          Metagenomics                                         95Monday, November 26, 12
4.                          Microbes in the world I:                               rRNA PCR                            Per...
Metagenomics                                  shotgun                                            cloneMonday, November 26,...
Novel Form of Phototrophy                                                      Beja et al. 2000Monday, November 26, 12
Monday, November 26, 12
clone assembled with the appropriate orientation and separation, as                        unpublished data), and local ge...
Monday, November 26, 12
Metagenomics ChallengeMonday, November 26, 12
Metagenomics Challenge                           Who is out there?                          What are they doing?Monday, No...
Glassy Winged Sharpshooter                                           • Feeds on xylem sap                                 ...
Wu et al. 2006 PLoS Biology 4: e188.Monday, November 26, 12
Sharpshooter Shotgun Sequencing                                                      shotgun                          Coll...
Monday, November 26, 12
Binning challenge        A                                                        T        B                              ...
CFB PhylaMonday, November 26, 12
Sulcia makes vitamins and cofactors    Baumannia makes amino acids                                  Wu et al. 2006 PLoS Bi...
Part V:                  Knowing What We Don’t Know                                               111Monday, November 26, 12
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DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity

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Talk by Jonathan Eisen at the California Academy of Sciences December 16, 2010

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Transcript of "DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity"

  1. 1. DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity Jonathan A. Eisen UC Davis Talk at Calacademy December 17, 2010 1Monday, November 26, 12
  2. 2. Monday, November 26, 12
  3. 3. Social Networking in ScienceMonday, November 26, 12
  4. 4. Bacterial evolveMonday, November 26, 12
  5. 5. Outline • Introduction: Diversity of microbes • I: The Tree of Life • II: Genome Sequencing • III: Microbes in the Field • IV: Metagenomics 5Monday, November 26, 12
  6. 6. Introduction Diversity of Microbes 6Monday, November 26, 12
  7. 7. D. Diversity of form Diversity of function 7Monday, November 26, 12
  8. 8. Many major pathogens are bacteriaMonday, November 26, 12
  9. 9. Bacteria and archaea are key commensals of many eukaryotesMonday, November 26, 12
  10. 10. Extreme conditions are dominated by bacteria and archaeaMonday, November 26, 12
  11. 11. Microbes run global cyclesMonday, November 26, 12
  12. 12. Photosynthetic Organisms Changed Earth’s Atmosphere The first photosynthetic cells were similar to cyanobacteria.Monday, November 26, 12
  13. 13. 6.15 Metabolic Pathways 13Monday, November 26, 12
  14. 14. D. Diversity of form Diversity of form: prokaryotes 14Monday, November 26, 12
  15. 15. More shape diversity 15Monday, November 26, 12
  16. 16. 16Monday, November 26, 12
  17. 17. Diversity of form II: complexity and size 17Monday, November 26, 12
  18. 18. Fruiting bodiesPhoto 26.24 Fruiting body of gliding bacterium Stigmatella aurantiaca. SEM. 18Monday, November 26, 12
  19. 19. Diversity of form III: biofilms Attraction of Free-swimming Signal other organisms prokaryotes molecules Binding to surface Matrix Signal molecules Single-species biofilm Irreversible attachment Growth and division, formation of matrix Mature biofilm 19Monday, November 26, 12
  20. 20. D. Diversity of form Diversity of form: microbial eukaryotes 20Monday, November 26, 12
  21. 21. Part I: The Tree of Life 21Monday, November 26, 12
  22. 22. Darwin and a Single Tree of Life George Richmond. Darwin Heirlooms Trust Darwin Origin of Species 1859 Set stage for “tree thinking” 22Monday, November 26, 12
  23. 23. Ernst Haeckel 1866 Plantae Protista Animalia 23 www.mblwhoilibrary.orgMonday, November 26, 12
  24. 24. Whittaker – Five Kingdoms 1969 Monera Protista Plantae Fungi Animalia 24Monday, November 26, 12
  25. 25. The Microbe Problem Most trees of life did not deal with microbes very well Trees were not based on comparing homologous traits between all organismsMonday, November 26, 12
  26. 26. Carl Woesehttp://mcb.illinois.edu/faculty/profile/1204 26Monday, November 26, 12
  27. 27. 12.3 From Gene to Protein 27Monday, November 26, 12
  28. 28. The Ribosome 28Monday, November 26, 12
  29. 29. rRNA Systematics • All cellular organisms have ribosomes • All have homologous subunits of the ribosomes including specific ribosomal proteins and ribosomal RNAs (i.e., these are universally homologous genes) • Woese determined the sequences of ribosomal RNAs from different species • The sequences are highly similar but have some variation • Each position in a rRNA can be considered a distinct character trait • Each position has multiple possible character states (A, C, U, G) 29Monday, November 26, 12
  30. 30. Alignments • Method of assigning homology to individual residues in different sequences • Allows one to have multiple traits within individual genes • Each column in alignment = a different character • Each residue (ACTG) = state 30Monday, November 26, 12
  31. 31. Alignments • Similar in concept to lining up bones from different species 31Monday, November 26, 12
  32. 32. Woese 1987 - rRNA Microbiological Reviews 51:221 32Monday, November 26, 12
  33. 33. 4.7 Eukaryotic Cells (Part 1) 33Monday, November 26, 12
  34. 34. 4.4 A Prokaryotic Cell 34Monday, November 26, 12
  35. 35. 26.23 Some Would Call It Hell; These Archaea Call It Home 35Monday, November 26, 12
  36. 36. The Tree of Life 2006 36 adapted from Baldauf, et al., in Assembling the Tree of Life, 2004Monday, November 26, 12
  37. 37. The Tree of Life 2006 37 adapted from Baldauf, et al., in Assembling the Tree of Life, 2004Monday, November 26, 12
  38. 38. Why tree useful? • Reclassification of many organisms, including diversity of pathogens Changes how to design treatments • Interpret comparative data Convergence vs. homology 38Monday, November 26, 12
  39. 39. Part II: Genome Sequencing 39Monday, November 26, 12
  40. 40. Fleischmann et al. 1995Monday, November 26, 12
  41. 41. Whole Genome Shotgun SequencingMonday, November 26, 12
  42. 42. Whole Genome Shotgun SequencingMonday, November 26, 12
  43. 43. Whole Genome Shotgun Sequencing Warner Brothers, Inc.Monday, November 26, 12
  44. 44. Whole Genome Shotgun Sequencing shotgun Warner Brothers, Inc.Monday, November 26, 12
  45. 45. Whole Genome Shotgun Sequencing shotgun Warner Brothers, Inc.Monday, November 26, 12
  46. 46. Whole Genome Shotgun Sequencing shotgun Warner Brothers, Inc. sequenceMonday, November 26, 12
  47. 47. Whole Genome Shotgun Sequencing shotgun Warner Brothers, Inc. sequenceMonday, November 26, 12
  48. 48. Assemble FragmentsMonday, November 26, 12
  49. 49. Assemble Fragments sequencer outputMonday, November 26, 12
  50. 50. Assemble Fragments sequencer outputMonday, November 26, 12
  51. 51. Assemble Fragments sequencer output assemble fragmentsMonday, November 26, 12
  52. 52. Assemble Fragments sequencer output assemble fragments Closure & AnnotationMonday, November 26, 12
  53. 53. Microbial genomes From http://genomesonline.orgMonday, November 26, 12
  54. 54. General Steps in Analysis of Complete Genomes • Identification/prediction of genes • Characterization of gene features • Characterization of genome features • Prediction of gene function • Prediction of pathways • Integration with known biological data • Comparative genomics 44Monday, November 26, 12
  55. 55. Vibrio cholerae MetabolismMonday, November 26, 12
  56. 56. Genome Sequences Have 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 biologyMonday, November 26, 12
  57. 57. Genome SizeMonday, November 26, 12
  58. 58. Genome Structure: More Variable than OnceMonday, November 26, 12
  59. 59. Monday, November 26, 12
  60. 60. Figure 7.6 - Gene contentMonday, November 26, 12
  61. 61. Figure 7.7 - Gene content E. coliMonday, November 26, 12
  62. 62. Figure 7.10 - K12 vs O157H7Monday, November 26, 12
  63. 63. Lateral Transfer from Doolittle, 1999Monday, November 26, 12
  64. 64. from Lerat et alMonday, November 26, 12
  65. 65. A. Studying microbes Part III: Microbes in the field 55Monday, November 26, 12
  66. 66. How to study microbes • Key questions about microbes in environment: Who are they? (i.e., what kinds of microbes are they) What are they doing? (i.e., what functions and processes do they possess) 56Monday, November 26, 12
  67. 67. 57Monday, November 26, 12
  68. 68. Figure 26.24 Extreme Halophiles 58Monday, November 26, 12
  69. 69. Deep Sea Ecosystems 59Monday, November 26, 12
  70. 70. • For any particular environment, there are many different ways one could go about characterizing the microbes there • 1. Observe directly in the field • 2. Grow in the laboratory • 3. CSI Microbiology (collect & analyze DNA from field) 60Monday, November 26, 12
  71. 71. A. Method 1 Method 1: Observe in the field 61Monday, November 26, 12
  72. 72. Field Observations an Important Tool 62Monday, November 26, 12
  73. 73. Field Observations an Important Tool 62Monday, November 26, 12
  74. 74. Field Observations an Important Tool 62Monday, November 26, 12
  75. 75. Field Observations an Important Tool 62Monday, November 26, 12
  76. 76. Field Observations an Important Tool 62Monday, November 26, 12
  77. 77. Field Observations an Important Tool 62Monday, November 26, 12
  78. 78. Field Observations an Important Tool 62Monday, November 26, 12
  79. 79. Field Observations an Important Tool 63Monday, November 26, 12
  80. 80. Field Observations an Important Tool 63Monday, November 26, 12
  81. 81. Field Observations an Important Tool 63Monday, November 26, 12
  82. 82. Field Observations an Important Tool 63Monday, November 26, 12
  83. 83. Field Observations an Important Tool 63Monday, November 26, 12
  84. 84. Field Observations an Important Tool 63Monday, November 26, 12
  85. 85. Field Observations an Important Tool 63Monday, November 26, 12
  86. 86. Field Observations an Important Tool 64Monday, November 26, 12
  87. 87. Field Observations an Important Tool 64Monday, November 26, 12
  88. 88. Field Observations an Important Tool 64Monday, November 26, 12
  89. 89. Field Observations an Important Tool 64Monday, November 26, 12
  90. 90. Field Observations an Important Tool 64Monday, November 26, 12
  91. 91. Field Observations an Important Tool 64Monday, November 26, 12
  92. 92. Field Observations an Important Tool 64Monday, November 26, 12
  93. 93. Field Observations an Important Tool 64Monday, November 26, 12
  94. 94. B. Method 2 Method 2: Culturing 65Monday, November 26, 12
  95. 95. Method 2: Culturing 66Monday, November 26, 12
  96. 96. Examples of Benefits of Culturing: • Allows one to connect processes and properties to single types of organisms • Enhances ability to do experiments from genetics, to physiology to genomics • Provides possibility of large volumes of uniform material for study • Can supplement appearance based classification with other types of data. Many types are useful, though the standard is analysis of rRNA sequences. 67Monday, November 26, 12
  97. 97. Optimal salt concentration for different species 68Monday, November 26, 12
  98. 98. Halophile adaptations • Some stresses of high salt Osmotic pressure on cells H20 Desiccation 69Monday, November 26, 12
  99. 99. Halophile adaptations • Some stresses of high salt Osmotic pressure on cells H20 Desiccation • Halophile adaptations Increased osmolarity inside cell Proteins H20 Carbohydrates Salts Membrane pumps Desiccation resistance 70Monday, November 26, 12
  100. 100. Halophile adaptations • Some stresses of high salt Osmotic pressure on cells Desiccation • Halophile adaptations Increased osmolarity inside cell Proteins Carbohydrates Salts - only done in extremely halophilic archaea Membrane pumps Desiccation resistance 71Monday, November 26, 12
  101. 101. Halophile adaptations • Some stresses of high salt Osmotic pressure on cells Desiccation • Halophile adaptations Increased osmolarity inside cell Proteins Carbohydrates Salts - only done in extremely halophilic archaea Membrane pumps Desiccation resistance High internal salt requires ALL cellular components to be adapted to salt, charge. For example, all proteins must change surface charge and other properties. 72Monday, November 26, 12
  102. 102. Extreme halophiles are a monophyletic group 73Monday, November 26, 12
  103. 103. Uses of extremophiles Type of Examples Example of Practical Uses environment mechanism of survival High temp Deep sea vents, Amino acid Heat stable enzymes (thermophiles) hotsprings changes Low temp Antarctic ocean, Antifreeze Enhancing cold (psychrophile) glaciers proteins tolerance of crops High pressure Deep sea vents, Solute changes Industrial processes (barophile) hotsprings High salt Evaporating pools Incr. internal Soy sauce production (halophiles osmolarity High pH Soda lakes Transporters Detergents (alkaliphiles) Low pH Mine tailings Transporters Bioremediation (acidophiles) Desiccation Deserts Spore formation Freeze-drying (xerophiles) additives High radiation Nuclear reactor Absorption, repair Bioremediation, (radiophiles) waste sites damage space travel 74Monday, November 26, 12
  104. 104. Method III: CSI Microbiology 75Monday, November 26, 12
  105. 105. Great Plate Count Anomaly Culturing Microscopy Count Count 76Monday, November 26, 12
  106. 106. Great Plate Count Anomaly Culturing Microscopy Count <<<< Count 77Monday, November 26, 12
  107. 107. Great Plate Count Anomaly Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Microscopy Count <<<< Count 78Monday, November 26, 12
  108. 108. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Microscopy Count <<<< Count 79Monday, November 26, 12
  109. 109. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or DNA “what are they doing?” Culturing Microscopy Count <<<< Count 80Monday, November 26, 12
  110. 110. Analysis of uncultured microbes Collect from environment 81Monday, November 26, 12
  111. 111. Analysis of uncultured microbes Collect from environment 81Monday, November 26, 12
  112. 112. Polymerase Chain Reaction- PCR 82Monday, November 26, 12
  113. 113. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots of Sequence PCR copies of the rRNA genes rRNA genes in sample rRNA1 5’ ...TACAGTATAGGTGG Phylogenetic tree Sequence alignment = Data matrix AGCTAGCGATCGATC GA... 3’ rRNA1 Yeast rRNA1 A C A C A C Yeast T A C A G T E. coli A G A C A G E. coli Humans Humans T A T A G T 83Monday, November 26, 12
  114. 114. Deep Sea Ecosystems 84Monday, November 26, 12
  115. 115. ChemosymbiontsMonday, November 26, 12
  116. 116. Analysis of uncultured microbes1992 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 Vol. 174, No. 10 0021-9193/92/103416-06$02.00/0 Copyright © 1992, American Society for Microbiology R. ricketsil Phylogenetic Relationships of Chemoautotrophic Bacterial 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 86Unrooted phylogenetic tree showing the position of the S. velum symbionts in relation to that of other Proteobacteria species on intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum only genomic DNA restriction fragments. The symbionts appeared to have one copy of the 16S rRNA gene. TheMonday, November 26, 12 evolutionary distances in Table 1. Members of the alpha and beta variability lack of in the 16S sequence and hybridization patterns within and between individual S. velum
  117. 117. Analysis of uncultured microbes Collect from environment 87Monday, November 26, 12
  118. 118. Analysis of uncultured microbes Collect from environment 87Monday, November 26, 12
  119. 119. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots of Sequence PCR copies of the rRNA genes rRNA genes in sample rRNA1 5’ ...ACACACATAGGTG Phylogenetic tree Sequence alignment = Data matrix GAGCTAGCGATCGAT CGA... 3’ rRNA1 rRNA2 rRNA1 A C A C A C rRNA2 T A C A G T rRNA2 5’ E. coli A G A C A G ...TACAGTATAGGTGG E. coli Humans Humans T A T A G T AGCTAGCGATCGATC GA... 3’ Yeast Yeast T A C A G T 88Monday, November 26, 12
  120. 120. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots of Sequence PCR copies of the rRNA genes rRNA genes in sample rRNA1 5’...ACACACATAGGTGGAGCTA GCGATCGATCGA... 3’ Phylogenetic tree Sequence alignment = Data matrix rRNA2 rRNA1 rRNA2 rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCTAG CGACGATCGA... 3’ rRNA4rRNA3 rRNA2 T A C A G T rRNA3 rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGATT E. coli Humans rRNA4 C A C A G T CTAGCGATATAGA... 3’ Yeast E. coli A G A C A G rRNA4 5’...ACGGCCCGATAGGTGGATT Humans T A T A G T CTAGCGCCATAGA... 3’ Yeast T A C A G T 89Monday, November 26, 12
  121. 121. Major phyla of bacteria and archaea (as of 2002) No cultures Some cultures 90Monday, November 26, 12
  122. 122. Uses of rDNA PCR Bohannan and Hughes 2003 Hugenholtz 2002 91Monday, November 26, 12
  123. 123. 92Monday, November 26, 12
  124. 124. Censored Censored 93Monday, November 26, 12
  125. 125. 94Monday, November 26, 12
  126. 126. Part IV: Metagenomics 95Monday, November 26, 12
  127. 127. 4. Microbes in the world I: rRNA PCR Perna et al. 2003Monday, November 26, 12
  128. 128. Metagenomics shotgun cloneMonday, November 26, 12
  129. 129. Novel Form of Phototrophy Beja et al. 2000Monday, November 26, 12
  130. 130. Monday, November 26, 12
  131. 131. clone assembled with the appropriate orientation and separation, as unpublished data), and local gene orde expected for a low rate of mispairing error (tracking and chimaeric conserved (Supplementary Fig. S7). The clones). represent a nearly complete genome of Acid Mine Drainage 2004 The first step in assignment of scaffolds to organism types was to uncultured Ferroplasma species distinct this as Ferroplasma type II. The dominan was unexpected before the genomic analy We assigned the roughly 3£ coverage Leptospirillum group III on the basis of rRN up to 31 kb, totalling 2.66 Mb). Comparis those assigned to Leptospirillum group sequence divergence and only locally co firming that the scaffolds belong to a rel Leptospirillum group II. A partial 16S rR Sulfobacillus thermosulfidooxidans was assembled reads, suggesting very low cov any Sulfobacillus scaffolds .2 kb were a grouped with the Leptospirillum group II We compared the 3£ coverage, low Gþ 4.12 Mb) to the fer1 genome in order to types (Supplementary Fig. S6). Scaffold identity to fer1 were assigned to an enviro I genome (170 scaffolds up to 47 kb in 1.48 Mb of sequence). The remaining scaffolds are tentatively assigned to G-pla in this bin (62 kb) contains the G-plasma scaffolds assigned to G-plasma comprise partial 16S rRNA gene sequence from A-pl unassembled reads, suggesting low covera scaffolds from A-plasma .2 kb would be bin. Although eukaryotes are present in th in low abundance in the biofilm studied. eukaryotes have been detected. As independent evidence that the Lep Ferroplasma type II genomes are nearly co complement of transfer RNA synthetases Figure 1 The pink biofilm. a, Photograph of the biofilm in the Richmond mine (hand An almost complete set of these genes included for scale). b, FISH image of a. Probes targeting bacteria (EUBmix; fluorescein Leptospirillum group III. The G-plasma bin isothiocyanate (green)) and archaea (ARC915; Cy5 (blue)) were used in combination with a set of tRNA synthetases, consistent with in probe targeting the Leptospirillum genus (LF655; Cy3 (red)). Overlap of red and green scaffolds. In addition, we established (yellow) indicates Leptospirillum cells and shows the dominance of Leptospirillum. group II, Leptospirillum group III, Ferrop c, Relative microbial abundances determined using quantitative FISH counts. type II and G-plasma bins contained onlMonday, November 26, 12 2 ©2004 Nature Publishing Group NATURE | doi:10.1038/n
  132. 132. Monday, November 26, 12
  133. 133. Metagenomics ChallengeMonday, November 26, 12
  134. 134. Metagenomics Challenge Who is out there? What are they doing?Monday, November 26, 12
  135. 135. Glassy Winged Sharpshooter • Feeds on xylem sap • Vector for Pierce’s Disease • Potential bioterror agent • Collaboration with Nancy Moran to sequence symbiont genomes • Funded by NSF • Published in PLOS Biology 2006Monday, November 26, 12
  136. 136. Wu et al. 2006 PLoS Biology 4: e188.Monday, November 26, 12
  137. 137. Sharpshooter Shotgun Sequencing shotgun Collaboration with Nancy Moran’s Wu et al. 2006 PLoS Biology 4: e188. labMonday, November 26, 12
  138. 138. Monday, November 26, 12
  139. 139. Binning challenge A T B U C V D W E X F Y G No reference genome? What do you do? Z Phylogeny ....Monday, November 26, 12
  140. 140. CFB PhylaMonday, November 26, 12
  141. 141. Sulcia makes vitamins and cofactors Baumannia makes amino acids Wu et al. 2006 PLoS Biology 4: e188. 110Monday, November 26, 12
  142. 142. Part V: Knowing What We Don’t Know 111Monday, November 26, 12
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