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Jonathan A. Eisen

U. C. Davis

Famous Arrowhead Quotes 2006

• Publications, student degrees, etc. • Not trying to say anything bad about anyone• The human guts are a real milieu• Where’s you evening gown?• You better kiss everybody• This is how you do metagenomics on 50

dollars, and that’s Canadian dollars

Lessons Learned at Arrowhead 2006

• Open availability of genome sequences has revolutionized microbiology

• Open Source software tools can help allow non informatics specialists to make use of genome data

• Open genome databases can make sense of disparate information

Lessons Not Yet Learned

• What about Open Access to publications?

• Open Access can revolutionize the use of the literature just as Genbank revolutionized the use of sequence data

Outline

• Introduction

• Three endosymbiont genomics tales– Wolbachia– Sharpshooter– Calyptogena

• Where next?

Sources for the Origin of Novelty

• New biochemical functions– Evolution of new genes– Old genes with new functions– Mixing and matching existing genes

• Changes in uses of existing genes– Targeting– Regulation

• Acquisition of new functions– Recombination– Lateral gene transfer– Symbioses

Endosymbioses Drove Eukaryotic Evolution

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But ….

• Key questions unresolved– Was the pre-organelle ancestor free-living?– What the ancestor a mutualist? a parasite?– What happened early in the evolution of the symbiosis?

• The problems with organelles– Symbioses were so long ago that it is nearly impossible

to figure out what the early events were.– May represent frozen accidents

• Solution?– Study more recent and more diverse symbioses

Wolbachia pipientis wMel

• Wolbachia are obligate, maternally transmitted intracellular symbionts

• Wolbachia infect many invertebrate species– Many cause male specific deleterious effects– Model system for studying sex ratio changes in hosts– Some are mutualistic (e.g., in filarial nematodes)

• wMel selected as model system because it infects Drosophila melanogaster

Wolbachia Metagenomic Sequencing

shotgunshotgun

sequencesequence

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Analysis led by Matin Wu in collaboration with lab of Scott O’Neill

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Genome Completed

Wu et al., PLOS Biology 2004

alanine/glycine

Na+

alanine/glycine

Na+

alanine/glycine

Na+

proline/betaine

H+

proline/betaine

H+WD0168

WD0414

WD1046

WD1047

WD0330

Na+

glutamate/aspartate

Na+

WD0211

WD0229

glutamate/aspartate

ornithine

putrescineWD0957

H+ Na+H+ Na+

WD0316 WD0407

H+

WD1107WD1299WD1300WD1391WD0816WD0765

Mg2+

WD0375

H+ Zn2+/Cd2+

WD1042

ATPADP

Zn2+

WD0362WD0938WD0937

ATPADP

Fe3+

WD1136WD0153WD0897

glycerol-3-phosphate/hexose-6-phosphate

phosphateWD0619

H+

drugs

H+

drugs

WD0056

WD0248

H+

drugs

H+

drugs

WD1320

WD0384

H+

?

H+

?

WD0621

WD0099

H+

metabolite?

WD0470

H+

metabolite?

WD1033

H+WD0249

metabolite?

ATPADP

heme

WD0411WD1093WD0340

K+

WD1249

Na+H+

drugsATP

ADP WD0400

phosphate

ATPADP

ORF00100ORF00714ORF00927ORF00940

(2?)

H+

F-type ATPase

ATP ADP

WD1233WD0203WD0204WD0427WD0428WD0429WD0655WD0656

phosphoenolpyruvate

1,3-bisphosphoglycerate

3-phosphoglycerate

2-phosphoglycerate

pyruvate

acetyl-CoA

citrate

isocitrate

oxaloacetate

suc-CoAsuccinate

fumarate

malate

oxaloacetate

TCA CYCLE

glyceraldehyde-3P

fructose-1,6-P2

dihydroxyacetone-P

WD1238

WD0091

WD0451

WD1167

WD0868

WD0494

WD0690

WD0105

WD0791

WD1309WD0544WD0751

WD1209WD1210

WD0437WD0727WD1221WD1222

WD0492

WD1121

mannose-1P mannose-6PWD0695

MALATE WD0488 WD1177WD0416WD0473WD0751WD0325

Non-oxidative Pentose Phosphate Pathway

xylulose-5P

glyceraldehyde-3P

sedoheptulose-7P

fructose-6P

ribose-5P

ribulose-5P

glyceraldehyde-3P

WD0551WD0387

WD0387

WD0712

erythrose-4P

WD1151

glycerol-3P

WD0731

Amino Acid catabolism

GLUTAMATE glutamineWD1322

GLUTAMINE glutamateWD0535

CYSTEINE alanineWD0997

THREONINE glycineWD0617,WD0617

PROLINE glutamateWD0103

SERINE glycineWD1035

Fatty Acid Biosynthesis WD0985, WD0650, WD1083, WD1170, WD0085

PRPP

WD0036

Thiamine metabolismWD1109,WD0763,WD0029,WD0913,WD1018,WD1024

AMP,ADP,dAMP, dADP,ATP,dATP,ITP,dITP,IMP,XMP,GMP,GDP,dGDP,dGTP,dGMP

WD1142WD1305WD1023WD0786WD0867WD0337WD0786

WD0661WD1183WD0197WD0089WD0195WD0439WD0197

adenylosuccinate WD0786

Purine Metabolism

UMPUDP

WD0684WD1295WD0895WD0230WD1239WD0228WD0461

aspartate semialdehydeaspartateWD1029 WD0960 WD0954

Mitochondrial Origin Unresolved

Wolbachia Evolutionary Rate is Accelerated

Endosymbiont Trends

• Compared to free-living relatives– Smaller genomes– Lower GC content– Higher pIs– Higher rates of sequence evolution

• Wolbachia shows ALL of these– However ….

Explanations for Endosymbiont Differences with Free-Living Relatives

• Repair hypothesis– Loss of DNA repair genes leads to increased mutation rate

– Trends are the direct and indirect result of this increased mutation rate

• Population genetics hypothesis– Smaller effective population size leads to more genetic drift

– Trends are mostly the result of accumulation of slightly deleterious mutations

• PopGen explanations favored– Wolbachia has full suite of repair genes

Wolbachia Overrun by Mobile ElementsRepeatClass

Size(Median)

Copies Protein motifs/families IS Family Possible Terminal Inverted Repeat Sequence

1 1512 3 Transposase IS4 5’ ATACGCGTCAAGTTAAG 3’2 360 12 - New 5’ GGCTTTGTTGCAT CGCTA 3’3 858 9 Transposase IS492/IS110 5’ GGCTTTGTTGCAT 3’4 1404.5 4 Conserved hypothetical,

phage terminaseNew 5’ ATACCGCGAWTSAWTCGCGGTAT 3’

5 1212 15 Transposase IS3 5’ TGACCTTACCCAGAAAAAGTGGAGAGAAAG 3’6 948 13 Transposase IS5 5’ AGAGGTTGTCCGGAAACAAGTAAA 3’7 2405.5 8 RT/maturase -8 468 45 - -9 817 3 conserved hypothetical,

transposaseISBt12

10 238 2 ExoD -11 225 2 RT/maturase -12 1263 4 Transposase ???13 572.5 2 Transposase ??? None detected14 433 2 Ankyrin -15 201 2 - -16 1400 6 RT/maturase -17 721 2 transposase IS63018 1191.5 2 EF-Tu -19 230 2 hypothetical -

Wu et al. 2004

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Wu et al. PLoS Biology 2006

Glassy Winged Sharpshooter Symbiont

• Vector for Pierce’s disease in grapes

• Potential bioterror agent• Feeds on nutrient poor

xylem sap• Needs to get amino-

acids and other nutrients from symbionts like aphids

Sharpshooter Shotgun Sequencing

shotgunshotgun

sequencesequence

400,000

100,000

200,000

300,000

500,000

600,000

1

Genome Helps Resolve Phylogeny

Higher Evolutionary Rates in Clade

Endosymbiont Trends

• Compared to free-living relatives– Smaller genomes– Lower GC content– Higher pIs– Higher rates of sequence evolution

• Baumannia shows ALL of these

Explanations for Endosymbiont Differences with Free-Living Relatives

• Repair hypothesis

• Population genetics hypothesis

• PopGen explanations favored

Variation in Evolution RatesCorrelated with Repair Gene Presence

MutS MutL

+ +

+ +

+ +

+ +

_ _

_ _

Explanations for Endosymbiont Differences with Each Other

• Repair hypothesis

• Population genetics hypothesis

• Repair explanations favored

Polymorphisms in Metapopulation

• Data from ~200 hosts– 104 SNPs– 2 indels

• PCR surveys show that this is between host variation

• Much lower ratio of transitions:transversions than in Blochmannia

• Consistent with absence of MMR from Blochmannia

Baumannia Predicted Metabolism

No Amino-Acid Synthesis

???????

Binning by Phylogeny

• Identified putative genes• Built phylogenetic trees of genes• Examined and classified trees

Binning by Phylogeny

• Four main “phylotypes”– Gamma proteobacteria (Baumannia)– Arthropoda (sharpshooter)– Bacteroidetes (Sulcia)– Alpha-proteobacteria (Wolbachia)

Binning by Phylogeny

• Four main “phylotypes”– Gamma proteobacteria (Baumannia)– Arthropoda (sharpshooter)– Bacteroidetes (Sulcia) - only a.a. genes here– Alpha-proteobacteria (Wolbachia)

Finished 130 kb of Sulcia

Co-Symbiosis?

Metagenomics, Symbionts and Binning

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• Understanding multisymbiont ecosystems is greatly aided by binning

• Multiple methods can be combined for better signal– Assembly

– Composition

– Phylogeny

– Depth of coverage

ABCDEFG

TUVWXYZ

Binning in More Complex Systems?

Venter et al., 2004

What is Next I:Autotrophic Endosymbioses

Calyptogena magnifica symbionts

C. magnifica symbiont sequencing

• Collaboration between Cavanaugh Lab at Harvard (Irene Newton led analysis), Eisen lab, and JGI (Woyke and others).

• Funded by DOE through CSP program and sequencing and closure done at JGI

• Annotation and analysis involved DOE (JGI, ORNL), Harvard, TIGR, Davis, et al.

A Streamlined Chemoautotrophic Machine

What is Next II+?

• Vertical vs. environmental transmission – Two chemosymbioses– P.I. C. M. Cavanaugh, Harvard

• Chordate symbionts– Prochloron - tunicate– P.I. Jacques Ravel, TIGR.

• Epibionts– AMH epibiont of Alvinella worm– P.I. Craig Cary. U. Delaware

• Commensals

TIGRTIGR

Other peopleOther people

Mom and DadMom and Dad

H. OchmanH. OchmanF. RobbF. Robb

J. BattistaJ. Battista

E. OriasE. Orias

D. BryantD. BryantS. O’NeillS. O’Neill

M. EisenM. Eisen

N. MoranN. Moran

R. MyersR. Myers

C. M. CavanaughC. M. Cavanaugh

P. HanawaltP. Hanawalt

J. HeidelbergJ. HeidelbergN. WardN. Ward

J. VenterJ. Venter

C. FraserC. Fraser

S. SalzbergS. Salzberg

I. PaulsenI. Paulsen

$$$$$$

NSFNSFDOEDOE

NIHNIH

M. WuM. Wu

D. WuD. Wu

S. ChatterjiS. Chatterji

H. HuseH. Huse

A. HartmanA. Hartman

MooreMoore

VIVI

D. RuschD. Rusch

A. HalpernA. Halpern

Eisen Eisen GroupGroup

J. MorganJ. Morgan

JGIJGI

E. EisenstadtE. Eisenstadt

M. FrazierM. Frazier

T. WoykeT. Woyke

E. RubinE. Rubin

Correlation of Endosymbiont Features

• Correlation makes it difficult to tease apart cause and effect

• Need examples where properties are decoupled

• May be the case in Baumannia with genome size

Ruthia magnifica

Sulcia Role Categories

Long Term Effects of Repair Loss

• Endosymbionts are model systems for understanding the consequences of loss of repair activities

• RecA lost in Buchnera and Blochmannia but kept in Baumannia and Wigglesworthia

• MutSL loss mentioned previously• RecBCD present even in species without RecA• Mfd present in many species without UvrABCD

Famous Arrowhead 2004 Quotes

• Space-time continuum of genes and genomes

• Gene sequences are the wormhole that allows one to tunnel into the past

• The human mind can conceive of things with no basis in physical reality

• Thoughts can go faster than the speed of light