phylogenomics and the diversification of microbes

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Phylogenomics and the Diversification of Microbes

Jonathan A. Eisen

October 12, 2006

MCB Seminar



Outline

• Introduction– Origin of novelty

– Phylogenomics

• Phylogenomic tales– Carboxydothermus and functional predictions

– Tetrahymena and genome diversification

– Mutualisic symbioses and the acquisition of function

– The hidden majority and phylogenomic forensics

• Conclusions



Eisen Lab Research

• Origin of new functions and processes– Evolution of new genes

– Change in function of existing genes

– Acquisition of new functions

• Evolvability– What are the constraints on the origin of novelty?

– Role of DNA metabolism in the origin of novelty

– Variation within and between taxa



Eisen Lab Model Systems

• Extremophiles– How far can novelty be pushed?– Parallel origins of each extremophily allows the

identification of “rules” – Many applied uses of the information

• Mutualistic Symbioses– Perhaps the most straightforward mechanism for

novelty to originate– Also many parallel origins allowing rules to be

identified– Key role in modern life and evolutionary history



“Nothing in biology makes senseexcept in the light of evolution.”

T. H. Dobzhansky (1973)



Comparative vs. Evolutionary Approaches

• Comparative approaches involve documenting similarities and differences

• Evolutionary approaches involve documenting how and why the similarities and differences arose



Comparative vs. Evolutionary

Topic Comparative Evolutionary

Structure prediction for rRNA

Conserved regions Correlated changes along tree

Gene presence vs. phenotpye

Presence and absence of genes

Gain and loss, lateral transfer

Selection Degree and pattern of conservation

HKA, Ds/Dn

Functional prediction

Ranking by level of similarity

Predicting function from trees



Eisen Lab Methods: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



Phylogenomics I:Carboxydothermus and the

Prediction of Gene Function



Carboxydothermus hydrogenoformans

• Isolated from a Russian hotspring

• Thermophile (grows at 80°C)

• Anaerobic

• Grows very efficiently on CO (Carbon Monoxide)

• Produces hydrogen gas

• Low GC Gram positive (Firmicute)



Genome Completed

Wu et al. 2005 PLoS Genetics 1(5): e65



CO Metabolism

• Streamlined genome may contribute to efficient growth

• Five homologs of CooS found in the genome

• CooS and relatives are carbon monoxide dehydrogenases



CooS Homologs are Divergent




Prediction of Functions for CooSs




EvolutionaryMethod

PHYLOGENENETIC PREDICTION OF GENE FUNCTIONIDENTIFY HOMOLOGSOVERLAY KNOWNFUNCTIONS ONTO TREE

INFER LIKELY FUNCTIONOF GENE(S) OF INTEREST

1234563531A2A3A1B2B3B2A1B1A3A1B2B3BALIGN SEQUENCESCALCULATE GENE TREE1246CHOOSE GENE(S) OF INTEREST2A2A53Species 3Species 1Species 211222311A3A1A2A3A1A2A3A464564562B3B1B2B3B1B2B3B ACTUAL EVOLUTION(ASSUMED TO BE UNKNOWN)

Duplication?EXAMPLE AEXAMPLE BDuplication?Duplication?Duplication5 METHODAmbiguous

Eisen, 1998.



Phylogenetic Prediction of Function

• Termed phylogenomics (Eisen, et al 1997)

• Greatly improves accuracy of functional predictions compared to similarity based methods (e.g., blast)

• Somewhat challenging to automate

• Automated methods now available– Eddy, Brenner, Sjolander, etc.

• But …



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



Non-Homology Predictions: Phylogenetic Profiling

• Step 1: Search all genes in organisms of interest against all other genomes

• Ask: Yes or No, is each gene found in each other species

• Cluster genes by distribution patterns (profiles)



Homologs of Sporulation Genes

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Carboxydothermus sporulates




Predicting Novel Sporulation Genes




Phylogenomics II:Tetrahymena and the Mechanisms of

Diversification



Tetrahymena thermophilaMacronuclear Genome Project

• Collaboration between TIGR (Eisen), UCSB (Orias) and Stanford (Cherry)

• Shotgun sequencing of the MAC genome

• Annotation and analysis

• Creation of TGD, the Tetrahymena Genome Database at http://www.ciliate.org

• Closure and EST sequencing under way as well

• SAB made up of 15 members of the Tetrahymena research community

Supported by NSF, NIGMS

http://www.ciliate.org/



Tetrahymena as a Model Organism

• Good genetic tools available

• Relatively easy to grow and work with

• Has been used for many fundamental discoveries– Telomeres and telomerase– Dynein motors– Histone acetylation– Catalytic RNA



Consensus Eukaryotic Tree of Life

Eisen et al. PLoS Biology 4(9): e286



Tetrahymena’s two nuclear genomes

Micronucleus (MIC) Germline Genome (Silent) 5 pairs of chromosomes

Macronucleus (MAC) Somatic genome (Expressed) 250-300 chromosomes @ ~45 copies each 1 chromosome at > 5000

copies



Genome Processing in Ciliates



Mac Chosen for Shotgun Sequencing

• MAC benefits– Less repetitive DNA– Site of gene expression– Assortment can be used to reduce

polymorphisms

• MAC drawbacks– 200+ chromosomes– Not all in equal copy numbers– Excised DNA could be interesting



Evolution and Genome Processing

• Probably exists as a defense mechanism• Analogous to RIPPING and heterochromatin silencing• Presence of repetitive DNA in MAC but not TEs suggests

the mechanism involves targeting foreign DNA• Thus unlike RIPPING ciliate processing does not limit

diversification by duplication



Lineage Specific Duplications



Lineage Specific Gene Family Expansions

• Lineage specific expansions frequently associated with adaptive evolution

• Most large expansions in T. thermophila are in families with roles in sensing and responding to environment– Signal transduction– Transport– Proteolysis– Cytoskeletal structure and function



Transporter Expansion in T. thermophila

Comparisons of major transporter families in Tetrahymena and other eukaryotes

0

50

100

150

200

250

300

350

T. thermophila

H. sapiens A. thaliana

D. melanogaster

C. elegansN. crassa

S. cerevisiae

S. pombe E. cuniculi

P. falciparum

ABC

MFS

VIC

P-ATPase



Even Tubulins are Expanded



Phylogenomics III:The Hidden Majority and Microbial

Forensics



Great Plate Count Anomaly

Culturing Microscope

CountCount



Great Plate Count Anomaly

Culturing Microscope

CountCount <<<<



Who is Out There?rRNA PCR and the Uncultured



Phylotyping Diversity Indices

Bohannan and Hughes 2003

Hugenholtz 2002



rRNA: A Phylogenetic Anchor to Determine Who’s Out There

Eisen et al. 1992

Biology not similar enough



What are they Doing?rRNA Anchors and Metagenomics

Beja et al. 2000



Limits of Large Insert Approach

• Large insert libraries less random and less representative than small inserts

• Lower throughput

• Requires some thinking



shotgunshotgun

sequencesequenceWarner Brothers, Inc.Warner Brothers, Inc.

The Final FrontierEnvironmental Shotgun Sequencing



Who is Out There?



rRNA Phylotyping in Sargasso Sea Metagenomic Data

Venter et al., 2004



GSSEA rRNA Phylotypes

0

5

10

15

20

25

30

35

UnclassifiedAcidobacteriaActinobacteria

CFB GroupCyanobacteria

Firmicutes

OP11

Planctomyces

Proteobacteria-unassigned

Proteobacteria-AlphaProteobacteria-BetaProteobacteria-DeltaProteobacteria-Gamma

Thermomicrobia

Phylogenetic group

% of Clones

Venter et al., 2004



Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA)

Venter et al., 2004



Sargasso Phylotypes

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

AlphaproteobacteriaBetaproteobacteriaGammaproteobacteriaEpsilonproteobacteria

Deltaproteobacteria

CyanobacteriaFirmicutes

Actinobacteria

Chlorobi

CFB

ChloroflexiSpirochaetesFusobacteria

Deinococcus-Thermus

EuryarchaeotaCrenarchaeota

Major Phylogenetic Group

Weighted % of Clones

EFG

EFTu

HSP70

RecA

RpoB

rRNA

Other Markers Give Similar Phylotpyes

Venter et al., 2004



What Are They Doing?



Diversity of Proteorhodopsins by Metagenomics

Venter et al., 2004



Linking Who and What is Still Challenging With Metagenomic Data



Phylogenomics IV:Symbioses and the Acquisition of

FunctionSymbionts as model metagenomic

systems



Endosymbioses Drove Eukaryotic Evolution





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







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



Completed Genome Allows Detailed Analysis of Uncultured Species

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



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



Endosymbiont Trends


• Wolbachia shows ALL of these

• However ….



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



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



Higher Evolutionary Rates in Clade



Endosymbiont Trends


• 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




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




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



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



Symbionts as a model for studying uncultured microbes



Finished 130 kb of Sulcia



Co-Symbiosis?



ABCDEFG

TUVWXYZ

Binning in More Complex Systems?



Venter et al., 2004



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

FirmicutesFusobacteria Actinobacteria

Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira

SynergistesDeferribacteres

Thermudesulfobacteria

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• At least 40 phyla of bacteria



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter


• Genome sequences are mostly from three phyla



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter



• Some other phyla are only sparsely sampled



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter



• Some other phyla are only sparsely sampled

• Solution: sequence more phyla



What is Next?

• More endosymbioses– Diversity of host species– Diversity of symbionts– Diversity of biology

• Epibionts and other obligate symbioses• Commensals

– Human gut– Hotspring mats



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

phylogenomics and the diversification of microbes

Science

tiff lzw decompressor

prediction of functions

homology functional

likely function of genes

evolutionary history

absence of genes

genome coos

species cluster genes