Talk by Jonathan Eisen for For U. C. Davis MCB Department Seminar Series. October 2006.

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Phylogenomics and the
Diversification of Microbes
Jonathan A. Eisen
October 12, 2006
MCB Seminar

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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

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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

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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

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

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Comparative vs. Evolutionary
Approaches
• Comparative approaches involve
documenting similarities and differences
• Evolutionary approaches involve
documenting how and why the similarities
and differences arose

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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

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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

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Phylogenomics I:
Carboxydothermus and the
Prediction of Gene Function

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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)

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

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CO Metabolism
• Streamlined genome may contribute to
efficient growth
• Five homologs of CooS found in the
genome
• CooS and relatives are carbon monoxide
dehydrogenases

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CooS Homologs are Divergent
Wu et al. 2005
PLoS Genetics
1(5): e65

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Prediction of Functions for CooSs
Wu et al. 2005 PLoS Genetics 1(5): e65

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Evolutionary
Method
PHYLOGENENETIC PREDICTION OF GENE FUNCTIONIDENTIFY HOMOLOGSOVERLAY KNOWN
FUNCTIONS ONTO TREE
INFER LIKELY FUNCTION
OF 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.

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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 …

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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

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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)

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Homologs of Sporulation Genes
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Wu et al. 2005
PLoS Genetics
1(5): e65

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

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Predicting Novel Sporulation Genes
Wu et al. 2005
PLoS Genetics
1(5): e65

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Phylogenomics II:
Tetrahymena and the Mechanisms of
Diversification

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Tetrahymena thermophila
Macronuclear 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

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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

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Consensus Eukaryotic Tree of Life
Eisen et al.
PLoS
Biology
4(9): e286

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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

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Genome Processing in Ciliates

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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

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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

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Lineage Specific Duplications

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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

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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. elegans
N. crassa
S. cerevisiae
S. pombe E. cuniculi
P. falciparum
ABC
MFS
VIC
P-ATPase

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Even Tubulins are Expanded

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Phylogenomics III:
The Hidden Majority and Microbial
Forensics

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Great Plate Count Anomaly
Culturing Microscope
CountCount

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Great Plate Count Anomaly
Culturing Microscope
CountCount <<<<

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Who is Out There?
rRNA PCR and the Uncultured
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Phylotyping Diversity Indices
Bohannan and
Hughes 2003
Hugenholtz 2002

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

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What are they Doing?
rRNA Anchors and Metagenomics
Beja et al. 2000

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Limits of Large Insert Approach
• Large insert libraries less random and less
representative than small inserts
• Lower throughput
• Requires some thinking

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shotgunshotgun
sequencesequence
Warner Brothers, Inc.Warner Brothers, Inc.
The Final Frontier
Environmental Shotgun Sequencing

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Who is Out There?

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rRNA Phylotyping in Sargasso Sea
Metagenomic Data
Venter et al., 2004

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GSSEA rRNA Phylotypes
0
5
10
15
20
25
30
35
Unclassified
AcidobacteriaActinobacteria
CFB Group
Cyanobacteria
Firmicutes
OP11
Planctomyces
Proteobacteria-unassigned
Proteobacteria-AlphaProteobacteria-BetaProteobacteria-Delta
Proteobacteria-Gamma
Thermomicrobia
Phylogenetic group
% of Clones
Venter et al., 2004

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Shotgun Sequencing Allows Use of
Alternative Anchors (e.g., RecA)
Venter et al., 2004

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Sargasso Phylotypes
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
AlphaproteobacteriaBetaproteobacteria
GammaproteobacteriaEpsilonproteobacteria
Deltaproteobacteria
Cyanobacteria
Firmicutes
Actinobacteria
Chlorobi
CFB
Chloroflexi
SpirochaetesFusobacteria
Deinococcus-Thermus
EuryarchaeotaCrenarchaeota
Major Phylogenetic Group
Weighted % of Clones
EFG
EFTu
HSP70
RecA
RpoB
rRNA
Other Markers Give Similar Phylotpyes
Venter et al., 2004

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What Are They Doing?

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Diversity of Proteorhodopsins by
Metagenomics
Venter et al.,
2004

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Linking Who and What is Still
Challenging With Metagenomic Data

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Phylogenomics IV:
Symbioses and the Acquisition of
Function
Symbionts as model metagenomic
systems

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Endosymbioses Drove Eukaryotic Evolution
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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

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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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Completed Genome Allows Detailed
Analysis of Uncultured Species
Wu et al., PLoS Biology 2004

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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
putrescine
WD0957
H+
Na+
H+
Na+
WD0316 WD0407
H+
WD1107
WD1299
WD1300
WD1391
WD0816
WD0765
Mg2+
WD0375
H+
Zn2+
/Cd2+
WD1042
ATPADP
Zn2+
WD0362
WD0938
WD0937
ATPADP
Fe3+
WD1136
WD0153
WD0897
glycerol-3-phosphate/
hexose-6-phosphate
phosphate
WD0619
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
WD0411
WD1093
WD0340
K+
WD1249
Na+
H+
drugs
ATP
ADP WD0400
phosphate
ATPADP
ORF00100
ORF00714
ORF00927
ORF00940
(2?)
H+
F-type
ATPase
ATP ADP
WD1233
WD0203
WD0204
WD0427
WD0428
WD0429
WD0655
WD0656
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
WD1309
WD0544
WD0751
WD1209
WD1210
WD0437
WD0727
WD1221
WD1222
WD0492
WD1121
mannose-1P mannose-6P
WD0695
MALATEWD0488 WD1177
WD0416
WD0473
WD0751
WD0325
Non-oxidative Pentose Phosphate Pathway
xylulose-5P
glyceraldehyde-3P
sedoheptulose-7P
fructose-6P
ribose-5P
ribulose-5P
glyceraldehyde-3P
WD0551
WD0387
WD0387
WD0712
erythrose-4P
WD1151
glycerol-3P
WD0731
Amino Acid catabolism
GLUTAMATE glutamine
WD1322
GLUTAMINE glutamateWD0535
CYSTEINE alanine
WD0997
THREONINE glycine
WD0617,WD0617
PROLINE glutamate
WD0103
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
WD1142
WD1305
WD1023
WD0786
WD0867
WD0337
WD0786
WD0661
WD1183
WD0197
WD0089
WD0195
WD0439
WD0197
adenylosuccinate WD0786
Purine Metabolism
UMP
UDP
WD0684
WD1295
WD0895
WD0230
WD1239
WD0228
WD0461
aspartate semialdehydeaspartateWD1029 WD0960 WD0954

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Mitochondrial Origin Unresolved

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Wolbachia Evolutionary Rate is Accelerated

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Endosymbiont Trends
• Compared to free-living relatives
– Smaller genomes
– Lower GC content
– Higher pIs
– Higher rates of sequence evolution
• Wolbachia shows ALL of these

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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

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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 ….

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Wolbachia Overrun by Mobile Elements
Repeat
Class
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’ GGCTTTGTTGCATCGCTA 3’
3 858 9 Transposase IS492/IS110 5’ GGCTTTGTTGCAT 3’
4 1404.5 4 Conserved hypothetical,
phage terminase
New 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,
transposase
ISBt12
10 238 2 ExoD -
11 225 2 RT/maturase -
12 1263 4 Transposase ???
13 572.5 2 Transposase ??? None detected
14 433 2 Ankyrin -
15 201 2 - -
16 1400 6 RT/maturase -
17 721 2 transposase IS630
18 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

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Sharpshooter Shotgun Sequencing
shotgunshotgun
sequencesequence

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400,000
100,000
200,000
300,000
500,000
600,000
1

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Higher Evolutionary Rates in Clade

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Endosymbiont Trends
• Compared to free-living relatives
– Smaller genomes
– Lower GC content
– Higher pIs
– Higher rates of sequence evolution
• Baumannia shows ALL of these

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Explanations for Endosymbiont
Differences with Free-Living Relatives
• Repair hypothesis
• Population genetics hypothesis
• PopGen explanations favored

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Variation in Evolution Rates
Correlated with Repair Gene Presence
MutS MutL
+ +
+ +
+ +
+ +
_ _
_ _

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Explanations for Endosymbiont
Differences with Each Other
• Repair hypothesis
• Population genetics hypothesis
• Repair explanations favored

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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

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Baumannia Predicted Metabolism

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No Amino-Acid Synthesis

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???????

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Binning by Phylogeny
• Identified putative genes
• Built phylogenetic trees of genes
• Examined and classified trees

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

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

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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

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Symbionts as a model for studying
uncultured microbes

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Finished 130 kb of Sulcia

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Co-Symbiosis?

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A
B
C
D
E
F
G
T
U
V
W
X
Y
Z
Binning in More Complex Systems?

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Venter et al.,
2004

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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

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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

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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

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are needed to see this picture.
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:
sequence more
phyla

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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

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TIGRTIGR
Other peopleOther people
Mom and DadMom and Dad
H. OchmanH. OchmanF. RobbF. Robb
J. BattistaJ. Battista
E. OriasE. Orias
D. BryantD. Bryant
S. O’NeillS. O’Neill
M. EisenM. Eisen
N. MoranN. Moran
R. MyersR. Myers
C. M. CavanaughC. M. Cavanaugh
P. HanawaltP. Hanawalt
J. HeidelbergJ. Heidelberg
N. WardN. Ward
J. VenterJ. Venter
C. FraseC. Fraser
S. SalzbergS. Salzberg
I. PaulsenI. Paulsen
$$$$$$
NSFNSF
DOEDOE
NIHNIH
M. WuM. Wu
D. WuD. Wu
S. ChatterjiS. Chatterji
H. HuseH. Huse
A. HartmanA. Hartman
MooreMoore
VIVI
D. RuschD. Rusch
A. HalpernA. Halpern
EisenEisen
GroupGroup
J. MorganJ. Morgan
JGIJGI
E. EisenstadtE. Eisenstadt
M. FrazierM. Frazier
T. WoykeT. Woyke
E. RubinE. Rubin