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Jonathan Eisen talk on "Phylogenomics of Microbes" at Lake Arrowhead Small Genomes Meeting 2004
1. The Axis of Evol:
Uncultured Organisms,
Phylogenetic Anchors
and the Tree of Life
TIGR
2. 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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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4. QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
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5. Outline
• Three phylogenomic tales, each with
discovery and woe
– Uncultured organisms I: complete genomes of
symbionts
– Uncultured organisms II: incomplete genomes
and phylogenetic anchors
– Predicting gene function
• One approach that can help with all the woe
TIGR
9. Wolbachia Primer
• Intracellular bacteria related to Rickettsias (e.g., agent of
Typhus)
• Found in many invertebrate species including insects,
Arachnids, isopods, and nematodes
• Transmitted vertically from mother to offspring, like
mitochondria
• Many Wolbachia inhibit male survival or reproduction, to
promote transmission through females.
• Wolbachia that infect parasitic filarial nematodes (e.g.,
Brugia malayi) are needed by the host. Treatment of people
infected with these nematodes with bacterial antibiotics kills
the Wolbachia and helps kill the nematode.
TIGR
16. Selection Apparently Inefficient in wMel
• Likely not due to higher mutation rate
– Full suite of DNA repair genes
• Likely not due to low amounts of homologous
recombination
– RecA present
– Population studies suggest homologous recombination occurs
• Wolbachia has multiple types of bottlenecks
– Maternal transmission like obligate mutualists
– Infectious sweeps of cytoplasmic incompatibility like pathogens
TIGR Wu et al., 2004
17. Baumannia cicadellinicola genome project:
1° symbionts of the Glassy-winged Sharpshooter
• Sap feeding insect
•Carriers of Xylella
fastidiosa that causes
Pierce’s disease of
grapevines
•Listed as potential Agro-
Terrorism Agent
•There are >20000
sharpshooter species,
Glassy-winged Sharpshooter within which intracellular
symbiotic bacteria are
wildspread
TIGR
18. Baumania Genome Completed
1
600,000
100,000
500,000
200,000
400,000
300,000
Collaboration between Jonathan Eisen and Nancy Moran (U.
TIGR Arizona). Analysis led by Dongying Wu in Eisen’s group.
26. ArgA ArgB ArgC ArgD ArgE AarAB ArgF ArgG ArgH
Glutamate Omithine Arg
IvHI IlvC IlvD IlvE
Pyruvate Val
Ile
Pyruvate
Threonine IlvA +
Alpha-Ketobutyrate
HisC Phe
PheA
PEP + AroH AroB AroD AroE AroK AroA AroC
Erythrose 4- Chorismate
phosphate
TrpE
G TrpD
TrpC Trp
TrpA
B
C
ThrA Asd ThrA ThrB Thr Thr
Aspartate Homoserine
Dap MetB
A Dap MetC
B D MetE Met
apD
Dap
C Dap
E D
apF
Lys Lys
A
HisG HisI HisA HisHF HisB HisC HisB HisD
PRPP+ATP His
Essential amino acid biosynthetic pathways
TIGR
27. Another Symbiont Present
9359 clones that are not included in the final assembly
Run_TA
7152 assembles (400 have been assembled)
<1kb 6996
1kb-2kb 125
2kb-3kb 18
3kb-4kb 6
4kb-5kb 3
5kb-6kb 2
6kb-7kb 1
7kb-8kb 1
TIGR 150 Bacteroides/Chlorobi (njtree/blast)
28. C
ThrA Asd ThrB Thr Thr
Aspartate Homoserine
Dap MetB
A Dap MetC
B D MetE Met
apD
Dap
C Dap
E D
apF
Lys Lys
A
TIGR
29. Phylogenetic Anchors and the Sargasso
Sea Shotgun Sequencing
Warner Brothers, Inc.
shotgun
sequence
TIGR
30. rRNA as a Phylogenetic Anchor
TIGR Venter et al., 2004
37. DNA Repair Genes in D.
radiodurans Complete Genome
Process Genes in D. radiodurans
Nucleotide Excision Repair UvrABCD, UvrA2
Base Excision Repair AlkA, Ung, Ung2, GT, MutM, MutY-Nths,
MPG
AP Endonuclease Xth
Mismatch Excision Repair MutS, MutL
Recombination
Initiation RecFJNRQ, SbcCD, RecD
Recombinase RecA
Migration and resolution RuvABC, RecG
Replication PolA, PolC, PolX, phage Pol
Ligation DnlJ
dNTP pools, cleanup MutTs, RRase
Other LexA, RadA, HepA, UVDE, MutS2
TIGR
38. Problem:
List of DNA repair gene homologs
in D. radiodurans genome is not
significantly different from other
bacterial genomes of the similar size
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39. Proteobacteria
TM6
OS-K
~40 Phyla
Acidobacteria
Termite Group
OP8
of Bacteria
Nitrospira
Bacteroides
Chlorobi
Fibrobacteres
Marine GroupA
WS3
Gemmimonas
Firmicutes
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes 0.1
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae Tree based on
Thermudesulfobacteria
Hugenholtz (2002)
TIGR
Thermotogae
OP1 with some
OP11 modifications.
40. Proteobacteria
TM6
OS-K
Acidobacteria Most DNA
Termite Group
OP8
Nitrospira metabolism
Bacteroides
Chlorobi
Fibrobacteres studies in
Marine GroupA
WS3
Gemmimonas
two Phyla
Firmicutes
Fusobacteria
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes 0.1
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae Tree based on
Thermudesulfobacteria
Thermotogae Hugenholtz (2002)
TIGR OP1
OP11
with some
modifications.
41. Proteobacteria
TM6
OS-K
Acidobacteria Deinococcus
Termite Group
OP8
Nitrospira is very distant
Bacteroides
Chlorobi
Fibrobacteres from well
Marine GroupA
WS3
Gemmimonas
studied
Firmicutes
Fusobacteria groups
Actinobacteria
OP9
Cyanobacteria
Synergistes
Deferribacteres
Chrysiogenetes
NKB19
Verrucomicrobia
Chlamydia
OP3
Planctomycetes
Spriochaetes 0.1
Coprothmermobacter
OP10
Thermomicrobia
Chloroflexi
TM7
Deinococcus-Thermus
Dictyoglomus
Aquificae Tree based on
Thermudesulfobacteria
Thermotogae Hugenholtz (2002)
TIGR OP1
OP11
with some
modifications.
43. Non-Homology Prediction:
Phylogenetic Profiles
• 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)
TIGR
44. Carboxydothermus hydrogenoformans
• Isolated in Yellowstone
• Thermophile (grows at 80°C)
• Anaerobic
• Grows on CO (Carbon Monoxide)
• Produces hydrogen gas
• Low GC gram postive species
• Many Archaeal-like genes
TIGR
45. QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
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47. Reminders
1.Phylogenetic anchors don’t work if you do
not have data from across the tree (e.g.,
Sargasso study limited)
2.Non homology methods rely on diverse
genomes
3.Novel processes need to be studied in novel
lineages
TIGR
48. Biased Sampling of Bacterial Genomes
Hugenholtz 2002
• Of 40 bacterial phyla, most
genome sequences come from
only 3 groups
• Sargasso Sea Study Limited
By Poor Sampling of Species
for Data in Genbank
• Difficult to figure out what
types of species may be
present except when analyzing
rRNA genes
TIGR
49. Coprothmermobacter
Dictyoglomus
Thermomicrobia
Chrysiogenetes
Thermudesulfobacteria
Deferribacteres
Synergistes
Nitrospira
OP11
OP1
TM7
OP10
OP3
NKB19
OP9
WS3
Marine Group
OP8
TermiteGroupA
OS-K based
TM6 project
Thermotogae
Aquificae
Deinococcus-Thermus
Spriochaetes
Chlamydia
Cyanobacteria
Actinobacteria
Fusobacteria
Firmicutes
Chlorobi
Proteobacteria
Chloroflexi
Planctomycetes
Verrucomicrobia
Gemmimonas
Fibrobacteres
Bacteroides lineage
Acidobacteria on
Tree
Uncultured
In progress
Published
This
0.1
Hugenholtz (2002)
• Solution: TIGR Tree of with some
modifications.
Life Project
– Eisen and Ward, Co-PIs
– Selecting genome
projects to increase
phylogenetic
diversity
– Supported by NSF Tree
of Life Program
– Genomes from 6 new
phyla in closure
– More information at
http://www.tigr.org/tol
TIGR
50. QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
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51. TIGR
$$$ J. C. Venter Fraser
C. Frase
Other people DOE
M-I Benito
ONR NSF
N. Moran H. Tettelin N. Ward
NIH
F. Robb H. Ochman T.Read
S. Salzberg
J. Battista
E. Orias J. Heidelberg
D. Bryant R. Myers
S. O’Neill O. White
M. Eisen
W. Martin I. Paulsen
P. Hanawalt
Eisen Group
C. M. Cavanaugh
Martin Wu
Dongying Wu
Mom and Dad James Sakwa
TIGR Jonathan Badger
52. Duplication?SEQUENCES
Species 3ACTUAL
6 IDENTIFY HOMOLOGS PREDICTION
5 peciesMETHOD EVOLUTION
4B EXAMPLE A B
3A CALCULATE GENE KNOWN
2CHOOSE GENE(S) OF FUNCTION
1ALIGNINFER LIKELY INTEREST
1EXAMPLE
Ambiguous
Duplication
S
1A
3B
2B
1B
3A
2A 2
PHYLOGENENETIC OVERLAY TREE OF GENE FUNCTION
(ASSUMED TO BEOFONTO TREE
OF GENE(S) UNKNOWN)
FUNCTIONS INTEREST
Evolutionary
Method
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Shotgun genome sequencing works by breaking a genome apart into millions of little pieces and then sequencing those pieces randomly
Functional prediction using a gene tree is just like predicting the biology of a species using a species tree
Shotgun genome sequencing works by breaking a genome apart into millions of little pieces and then sequencing those pieces randomly
This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems. clone from the Sargasso Sea. This shows that this
Samples are unbiased
Phil Hugenholtz wrote an excellent review paper in Genome Biology tracing which Phyla had genome sequences available. Proteobacteria have the most with Firmicutes next (these are the low GC gram positives like B. subtilis) and Actinobacteria (the high GC gram positives like M. tuberculosis) third.
In Red are the Phyla of bacteria with cultured species but no genome sequences that we are sequencing as part of a NSF Tree of Life project. NOTE **** In White are the Phyla with no cultured representatives. ALSO NOTE **** This project is contingent upon having a good tree of bacteria.