1. The Evolution of Microbes and Their Genomes: A Phylogenomic Perspective Jonathan A. Eisen October 3, 2009

2. “Nothing in biology makes sense except in the light of evolution.” T. Dobzhansky (1973)

3. Origin of New Functions and Processes Species Evolution Genome Dynamics Eisen Lab - Phylogenomics of Novelty New genes Changes in old genes Changes in pathways Phylogenetic history Vertical vs. horizontal descent Needed to track gain/loss of processes, infer convergence Evolvability Repair and recombination processes Intragenomic variation

4. Phylogenomic Analysis Evolutionary reconstructions greatly improve genome analyses Genome analysis greatly improves evolutionary reconstructions There is a feedback loop such that these should be integrated

5. 56.1 The Global Ecosystem’s Compartments are Connected by the Flow of Elements

6. 26.23 Some Would Call It Hell; These Archaea Call It Home

8. Human commensals

9. Deep Sea Ecosystems

10. Fleischmann et al. 1995

11. Whole Genome Shotgun Sequencingshotgun sequence Warner Brothers, Inc.

12. Assemble Fragments sequencer output assemble fragments Closure & Annotation

13. From http://genomesonline.org

14. Major Microbial Sequencing Efforts Coordinated, top-down efforts –Fungal Genome Initiative (Broad/Whitehead)Fungal Genome Initiative –Gordon and Betty Moore Foundation Marine Microbial Genome Sequencing ProjectGordon and Betty Moore Foundation Marine Microbial Genome Sequencing Project –Sanger Center Pathogen Sequencing UnitSanger Center Pathogen Sequencing Unit –NHGRI Human Gut Microbiome ProjectNHGRI Human Gut Microbiome Project –NIH Human Microbiome Program White paper or grant systems –NIAID Microbial Sequencing CentersNIAID Microbial Sequencing Centers –DOE/JGI Community Sequencing ProgramDOE/JGI Community Sequencing Program –DOE/JGI BER Sequencing ProgramDOE/JGI BER Sequencing Program –NSF/USDA Microbial Genome SequencingNSF/USDA Microbial Genome Sequencing Covers lots of ground and biological diversity

15. 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 biology

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19. rRNA Tree of Life

20. The Tree is not Happy

21. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria As of 2002 Based on Hugenholtz, 2002

22. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla As of 2002 Based on Hugenholtz, 2002

23. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Some other phyla are only sparsely sampled As of 2002 Based on Hugenholtz, 2002

24. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Some other phyla are only sparsely sampled Same trend in Archaea As of 2002 Based on Hugenholtz, 2002

25. Need for Tree Guidance Well Established Common approach within some eukaryotic groups Many small projects funded to fill in some bacterial or archaeal gaps Phylogenetic gaps in bacterial and archaeal projects commonly lamented in literature

26. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Some other phyla are only sparsely sampled Solution I: sequence more phyla NSF-funded Tree of Life Project A genome from each of eight phyla Eisen, Ward, Badger, Wu, Wu, et al.

27. The Tree of Life is Still Angry

28. Major Lineages of Actinobacteria

29. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 100 phyla of bacteria Genome sequences are mostly from three phyla Most phyla with cultured species are sparsely sampled Lineages with no cultured taxa even more poorly sampled Solution - use tree to really fill gaps Well sampled phyla

30. http://www.jgi.doe.gov/programs/GEBA/pilot.html

31. GEBA Pilot Project Overview Identify major branches in rRNA tree for which no genomes are available Identify a cultured representative for each group Grow > 200 of these and prep. DNA Sequence and finish 100 Annotate, analyze, release data Assess benefits of tree guided sequencing

33. Some Lessons From GEBA

34. GEBA Lesson 1 rRNA Tree of Life is a Useful Guide and Genomes Improve Resolution

35. rRNA Tree of Life

38. GEBA Lesson 2 Phylogenetically Guided Selection Can Help Annotate Other Genomes

39. Predicting Function Identification of motifs –Small sequence elements that code for some general activity (e.g., ATPase) Homology/similarity based methods –Identify longer stretches of similarity to genes with known function Evolutionary reconstructions

40. Evolutionary Functional Prediction Based on Eisen, 1998 Genome Res 8: 163-167.Genome Res 8: 163-167.

41. Recent Functional Changes Phylogenomic functional prediction may not work well for very newly evolved functions Screen genomes for genes that have changed recently Examples: –Acquisition (e.g., LGT) –Unusual dS/dN ratios –Rapid evolutionary rates –Duplication and divergence E.coli gi1787690 B.subtilis gi2633766 Synechocystis sp. gi1001299 Synechocystis sp. gi1001300 Synechocystis sp. gi1652276 Synechocystis sp. gi1652103 H.pylori gi2313716 H.pylori99 gi4155097 C.jejuni Cj1190c C.jejuni Cj1110c A.fulgidus gi2649560 A.fulgidus gi2649548 B.subtilis gi2634254 B.subtilis gi2632630 B.subtilis gi2635607 B.subtilis gi2635608 B.subtilis gi2635609 B.subtilis gi2635610 B.subtilis gi2635882 E.coli gi1788195 E.coli gi2367378 E.coli gi1788194 E.coli gi1789453 C.jejuni Cj0144 C.jejuni Cj0262c H.pylori gi2313186 H.pylori99 gi4154603 C.jejuni Cj1564 C.jejuni Cj1506c H.pylori gi2313163 H.pylori99 gi4154575 H.pylori gi2313179 H.pylori99 gi4154599 C.jejuni Cj0019c C.jejuni Cj0951c C.jejuni Cj0246c B.subtilis gi2633374 T.maritima TM0014 T.pallidum gi3322777 T.pallidum gi3322939 T.pallidum gi3322938 B.burgdorferi gi2688522 T.pallidum gi3322296 B.burgdorferi gi2688521 T.maritima TM0429 T.maritima TM0918 T.maritima TM0023 T.maritima TM1428 T.maritima TM1143 T.maritima TM1146 P.abyssi PAB1308 P.horikoshii gi3256846 P.abyssi PAB1336 P.horikoshii gi3256896 P.abyssi PAB2066 P.horikoshii gi3258290 P.abyssi PAB1026 P.horikoshii gi3256884 D.radiodurans DRA00354 D.radiodurans DRA0353 D.radiodurans DRA0352 P.abyssi PAB1189 P.horikoshii gi3258414 B.burgdorferi gi2688621 M.tuberculosis gi1666149 V.cholerae VC0512 V.cholerae VCA1034 V.cholerae VCA0974 V.cholerae VCA0068 V.cholerae VC0825 V.cholerae VC0282 V.cholerae VCA0906 V.cholerae VCA0979 V.cholerae VCA1056 V.cholerae VC1643 V.cholerae VC2161 V.cholerae VCA0923 V.cholerae VC0514 V.cholerae VC1868 V.cholerae VCA0773 V.cholerae VC1313 V.cholerae VC1859 V.cholerae VC1413 V.cholerae VCA0268 V.cholerae VCA0658 V.cholerae VC1405 V.cholerae VC1298 V.cholerae VC1248 V.cholerae VCA0864 V.cholerae VCA0176 V.cholerae VCA0220 V.cholerae VC1289 V.choleraeVCA1069 V.cholerae VC2439 V.choleraeVC1967 V.cholerae VCA0031 V.cholerae VC1898 V.cholerae VCA0663 V.choleraeVCA0988 V.cholerae VC0216 V.cholerae VC0449 V.cholerae VCA0008 V.cholerae VC1406 V.cholerae VC1535 V.cholerae VC0840 V.cholerae VC0098 V.cholerae VCA1092 V.cholerae VC1403 V.cholerae VCA1088 V.cholerae VC1394 V.cholerae VC0622 NJ ** * * * * * * * * * * *

42. Non homology functional prediction Many genes have homologs in other species but no homologs have ever been studied experimentally Non-homology methods can make functional predictions for these Example: correlated presence/absence of genes across species

43. Most/All Functional Prediction Improves w/ Better Phylogenetic Sampling Better definition of protein family sequence “patterns” Greatly improves “comparative” and “evolutionary” based predictions Conversion of hypothetical into conserved hypotheticals Linking distantly related members of protein families Improved non-homology prediction

44. GEBA Lesson 3 Phylogenetically Guided Selection Can Help Study Uncultured Organisms

45. Great Plate Count Anomaly Culturing Microscope Count

46. Great Plate Count Anomaly Culturing Microscope Count <<<<

47. Great Plate Count Anomaly Culturing Microscope Count <<<< DNA

48. PCR Saves the Day

49. Sequencing and uncultured microbes I: rRNA surveys

50. rRNA: A Phylogenetic Anchor to Determine Who’s Out There Eisen et al. 1992 Biology not similar enough

51. Perna et al. 2003

52. Environmental Shotgun Sequencingshotgun sequence

53. Novel Form of Phototrophy Beja et al. 2000

55. ABCDEFGABCDEFG TUVWXYZTUVWXYZ Binning challenge

56. Glassy Winged Sharpshooter Feeds on xylem sap Vector for Pierce’s Disease Potential bioterror agent

57. Xylem and Phloem From Lodish et al. 2000

58. Wu et al. 2006 PLoS Biology 4: e188.PLoS Biology 4: e188

59. Sharpshooter Shotgun Sequencingshotgun Wu et al. 2006 PLoS Biology 4: e188.PLoS Biology 4: e188 Collaboration with Nancy Moran’s lab

60. Wu et al. 2006 PLoS Biology 4: e188.PLoS Biology 4: e188 Baumannia makes amino acids Sulcia makes vitamins and cofactors

61. ABCDEFGABCDEFG TUVWXYZTUVWXYZ Binning challenge

63. GEBA Lesson 4 We have still only scratched the surface of microbial diversity

64. Protein Family Rarefaction Curves Take data set of multiple complete genomes Identify all protein families using MCL Plot # of genomes vs. # of protein families

66. Mobile Motility Element?

67. Phylogenetic Distribution Novelty: 1st Bacterial Actin Related Protein Haliangium ochraceum DSM 14365

68. Phylogenetic Diversity: Sequenced Bacteria & Archaea

69. Phylogenetic Diversity with GEBA

70. Phylogenetic Diversity: Isolates

71. Phylogenetic Diversity: All

72. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Most phyla with cultured species are sparsely sampled Lineages with no cultured taxa even more poorly sampled Well sampled phyla Poorly sampled No cultured taxa

73. Uncultured Lineages: Technical Approaches Get into culture Enrichment cultures If abundant in low diversity ecosystems Flow sorting Microbeads Microfluidic sorting Single cell amplification

74. GEBA Lesson 6 Need Experiments from Across the Tree of Life too

75. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria As of 2002 Based on Hugenholtz, 2002

76. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Experimental studies are mostly from three phyla As of 2002 Based on Hugenholtz, 2002

77. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Experimental studies are mostly from three phyla Some studies in other phyla As of 2002 Based on Hugenholtz, 2002

78. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Some other phyla are only sparsely sampled Same trend in Eukaryotes As of 2002 Based on Hugenholtz, 2002

79. Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter At least 40 phyla of bacteria Genome sequences are mostly from three phyla Some other phyla are only sparsely sampled Same trend in Viruses As of 2002 Based on Hugenholtz, 2002

80. 0.1 Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter Tree based on Hugenholtz (2002) with some modifications. Need experimental studies from across the tree too

82. MICROBES

83. A Happy Tree of Life

84. GEBA Pilot Project: Components Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen) Project management (David Bruce, Lynne Goodwin et al) Culture collection and DNA prep (DSMZ, Hans-Peter Klenk) Libraries and DNA (Eileen Dalin et al) Sequencing and closure (Susan Lucas, Alla Lapidus et al.) Annotation and data release (Nikos Kyrpides) Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Jenna Morgan, Victor Kunin, Marcel Huntemann, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson) Adopt a microbe education project (Cheryl Kerfeld) Outreach (David Gilbert) $$$ (DOE, Eddy Rubin, Jim Bristow)