Phylogenomics and the Diversification of Microbes, J. A. Eisen at Genentech 1/27/11

Phylogenomics and the Diversity and Diversification of Microbes

Jonathan A. EisenUC Davis

Genentech TalkJanuary 27, 2011

Friday, January 28, 2011

My Obsessions

Jonathan A. EisenUC Davis

UCSD TalkJanuary 19, 2011


Social Networking in Science

Scientist Reveals Secret of the Ocean: It's HimBy NICHOLAS WADE Published: April 1, 2007

Maverick scientist J. Craig Venter has done it again. It was just a few years

ago that Dr. Venter announced that the human genome sequenced by Celera

Genomics was in fact, mostly his own. And now, Venter has revealed a second

twist in his genomic self-examination. Venter was discussing his Global

Ocean Voyage, in which he used his personal yacht to collect ocean water

samples from around the world. He then used large filtration units to collect

microbes from the water samples which were then brought back to his high

tech lab in Rockville, MD where he used the same methods that were used to

sequence the human genome to study the genomes of the 1000s of ocean

dwelling microbes found in each sample. In discussing the sampling methods, Venter let slip his

latest attack on the standards of science – some of the samples were in fact not from the ocean, but

were from microbial habitats in and on his body.

“The human microbiome is the next frontier,” Dr. Venter said. “The ocean voyage was just a cover.

My main goal has always been to work on the microbes that live in and on people. And now that my

genome is nearly complete, why not use myself as the model for human microbiome studies as well.

”

It is certainly true that in the last few years, the microbes that live in and on people have become a

hot research topic. So hot that the same people who were involved in the race to sequence the human

genome have been involved in this race too. Francis Collins, Venter main competitor and still the

director of the National Human Genome Research Institute (NHGRI), recently testified before

Congress regarding this type of work. He said, “There are more bacteria in the human gut than

human cells in the entire human body… The human microbiome project represents an exciting new

research area for NHGRI.” Other minor players in the public’s human genome effort, such as Eric

Lander at the Whitehead Institute and George Weinstock at Baylor College of Medicine are also

trying to muscle their way into studies of the human microbiome.

But Venter was not going to have any of this. “This time, I was not going to let them know I was

coming. There would be no artificially declared tie. We set up a cutting edge human microbiome

sampling system on the yacht, and then headed out to sea. They never knew what hit them. Now I

have finished my microbiome.”

Reactions among scientists range from amusement to indifference, most saying that it is

unimportant whose microbiome was sequenced. But a few scientists expressed disappointment that

Dr. Venter had once again subverted the normal system of anonymity. Recent human microbome

studies by other researchers have all involved anonymous donors. Jeff Gordon, at the Washington

University in St. Louis expressed astonishment, “I have to fill out about 200 forms for every sample.

It takes years to get anything done. And now Venter sails away with the prize. All I can say is, I will

never listen to one of my review boards again.”

Venter had hinted at the possibility that something was amiss in an interview he gave last week for

the BBC News. He said “Most of the samples we studied were from the ocean but a few were from

people.” When the interviewer seemed stunned, Doug Rusch, one of Venter’s collaborators stepped

in and said “Collected with the help of other people.”

Venter was apparently spurred to make the admission today that many of the samples were in fact

from his own microbiome due to a video that surfaced on YouTube showing Jeff Hoffman, the

nytimes.com/sports

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Bacterial evolve


Research Areas


Research Areas

Mechanisms of Origin of New

Functions


Research Areas


Functions

Variation in Mechanisms:

Patterns, Causes and Effects


Research Areas


Functions

Species Evolution




Research Areas

• Study the evolution of function• Make extensive use of genome sequence data• Requires integration of experimental information and genome analysis• Categorize and classify ways that novelty originates (examples)

• Duplication and divergence• Recombination• Simple substitutions• Gene transfer


Research Areas


Functions

• Study the evolution of function• Make extensive use of genome sequence data• Requires integration of experimental information and genome analysis• Categorize and classify ways that novelty originates (examples)

• Duplication and divergence• Recombination• Simple substitutions• Gene transfer


Research Areas

• Patterns of variation• Within species• Between species• Comparative genomics plays important role

• Causes• Variation in RRR• Regulatory complexity

• Effects• Differences in evolvability• Ecological niche• Short and long term genome evolution


Research Areas



• Patterns of variation• Within species• Between species• Comparative genomics plays important role

• Causes• Variation in RRR• Regulatory complexity

• Effects• Differences in evolvability• Ecological niche• Short and long term genome evolution


Research Areas

• Information needed to distinguish convergence from homology• Allows inference of rates and patterns of change• Allows one to determine if something is a “one time” event or a common theme in many lineages


Research Areas

Species Evolution

• Information needed to distinguish convergence from homology• Allows inference of rates and patterns of change• Allows one to determine if something is a “one time” event or a common theme in many lineages


Phylogenomics of Novelty


Functions

Species Evolution




Why do this?

• Discover causes and effects of differences in evolvability

• Improve predictions from genome analysis• Guide interpretation of biological dat


My microbial evolution obsessions

• Introduction• Phylogenomic Stories

– Within genome invention of novelty– Stealing novelty– Community service


Introduction

Genome Sequencing


Fleischmann et al. 1995


From http://genomesonline.orgFriday, January 28, 2011

Human commensals


From http://genomesonline.orgFriday, January 28, 2011

Phylogenomics of Novelty I

Origin of Functions from Within


Origin of New Functions

• Many different processes contribute to the origin of novelty

• Denovo invention of new genes• Simple substitutions within existing genes• Duplication and divergence• Domain swapping• Genome rearrangements• Regulatory changes


Origin of New Functions


Functions

• Many different processes contribute to the origin of novelty

• Denovo invention of new genes• Simple substitutions within existing genes• Duplication and divergence• Domain swapping• Genome rearrangements• Regulatory changes


Phylogenomics of Novelty


Functions

Species Evolution




Example I:Mutation Rates and Functional

Predictions


From Eisen et al. 1997 Nature Medicine 3: 1076-1078.


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9334711&query_hl=2






Blast Search of H. pylori “MutS”

• Blast search pulls up Syn. sp MutS#2 with much higher p value than other MutS homologs

• Based on this TIGR predicted this species had mismatch repair

• Assumes functional constancy

Based on Eisen et al. 1997 Nature Medicine 3: 1076-1078. Friday, January 28, 2011



MutL??

Based on Eisen et al. 1997 Nature Medicine 3: 1076-1078. Friday, January 28, 2011



Phylogenetic Tree of MutS Family

Aquae Trepa

FlyXenlaRatMouseHumanYeastNeucrArath

BorbuStrpyBacsu

SynspEcoliNeigo

ThemaTheaqDeira

Chltr

SpombeYeast

YeastSpombeMouseHumanArath

YeastHumanMouseArath

StrpyBacsu

CelegHumanYeast MetthBorbu

AquaeSynspDeira Helpy

mSaco

YeastCelegHuman

Based on Eisen, 1998 Nucl Acids Res 26: 4291-4300.






MutS Subfamilies

Aquae Trepa

FlyXenlaRatMouse

HumanYeast

NeucrArath

BorbuStrpy

BacsuSynsp

EcoliNeigo

ThemaTheaqDeira

Chltr

SpombeYeast

YeastSpombe

MouseHumanArath

YeastHumanMouse

Arath

StrpyBacsu

CelegHumanYeast

MetthBorbu

AquaeSynsp

Deira Helpy

mSaco

YeastCeleg

Human

MSH4

MSH5 MutS2

MutS1

MSH1

MSH3

MSH6

MSH2







MutS Subfamilies

• MutS1 Bacterial MMR• MSH1 Euk - mitochondrial MMR• MSH2 Euk - all MMR in nucleus• MSH3 Euk - loop MMR in nucleus• MSH6 Euk - base:base MMR in nucleus

• MutS2 Bacterial - function unknown• MSH4 Euk - meiotic crossing-over• MSH5 Euk - meiotic crossing-over


Overlaying Functions onto Tree

Aquae Trepa

Rat

FlyXenla

MouseHumanYeast

NeucrArath

BorbuSynsp

Neigo

ThemaStrpy

Bacsu

Ecoli

TheaqDeiraChltr

SpombeYeast

YeastSpombe

MouseHuman

Arath

YeastHumanMouse

Arath

StrpyBacsu

HumanCeleg

YeastMetthBorbu

AquaeSynsp

Deira Helpy

mSaco

YeastCeleg

Human

MSH4

MSH5MutS2

MutS1

MSH1

MSH3

MSH6

MSH2







Functional Prediction Using Tree

Aquae Trepa

FlyXenlaRatMouse

HumanYeast

NeucrArath

BorbuStrpy

BacsuSynsp

EcoliNeigo

ThemaTheaqDeira

Chltr

SpombeYeast

YeastSpombe

MouseHumanArath

YeastHumanMouse

Arath

MSH1MitochondrialRepair

MSH3 - Nuclear RepairOf Loops

MSH6 - Nuclear RepairOf Mismatches

MutS1 - Bacterial Mismatch and Loop Repair

StrpyBacsu

CelegHumanYeast

MetthBorbu

AquaeSynsp

Deira Helpy

mSaco

YeastCeleg

Human

MSH4 - Meiotic CrossingOver

MSH5 - Meiotic Crossing Over MutS2 - Unknown Functions

MSH2 - Eukaryotic NuclearMismatch and Loop Repair







Evolutionary Functional Prediction

1 2 3 4 5 6

3 5

3

1A 2A 3A 1B 2B 3B

2A 1B

1

1 2

2

2 31

1A 3A

1A 2A 3A

1A 2A 3A

4 6

4 5 6

4 5 6

2B 3B

1B 2B 3B

1B 2B 3B

1A3A

1B 2B3B

12 4

62A

2A

53

5

EXAMPLE BMETHOD

Duplication?

Duplication?

IDENTIFY HOMOLOGS

OVERLAY KNOWNFUNCTIONS ONTO TREE

INFER LIKELY FUNCTIONOF GENE(S) OF INTEREST

ALIGN SEQUENCES

CALCULATE GENE TREE

CHOOSE GENE(S) OF INTEREST

Species 3Species 1 Species 2

ACTUAL EVOLUTION(ASSUMED TO BE UNKNOWN)

EXAMPLE A

Duplication?

Duplication

Ambiguous

Based on Eisen, 1998 Genome Res 8: 163-167.






Example 2: Recent Changes

E.coli gi1787690

B.subtilis gi2633766Synechocystis sp. gi1001299Synechocystis sp. gi1001300Synechocystis sp. gi1652276Synechocystis sp. gi1652103H.pylori gi2313716H.pylori99 gi4155097C.jejuni Cj1190cC.jejuni Cj1110cA.fulgidus gi2649560A.fulgidus gi2649548B.subtilis gi2634254B.subtilis gi2632630B.subtilis gi2635607B.subtilis gi2635608B.subtilis gi2635609B.subtilis gi2635610B.subtilis gi2635882E.coli gi1788195E.coli gi2367378E.coli gi1788194

E.coli gi1789453C.jejuni Cj0144C.jejuni Cj0262c

H.pylori gi2313186H.pylori99 gi4154603C.jejuni Cj1564

C.jejuni Cj1506cH.pylori gi2313163H.pylori99 gi4154575H.pylori gi2313179H.pylori99 gi4154599C.jejuni Cj0019cC.jejuni Cj0951cC.jejuni Cj0246cB.subtilis gi2633374T.maritima TM0014

T.pallidum gi3322777T.pallidum gi3322939T.pallidum gi3322938B.burgdorferi gi2688522T.pallidum gi3322296B.burgdorferi gi2688521T.maritima TM0429T.maritima TM0918T.maritima TM0023T.maritima TM1428T.maritima TM1143T.maritima TM1146P.abyssi PAB1308P.horikoshii gi3256846P.abyssi PAB1336P.horikoshii gi3256896P.abyssi PAB2066P.horikoshii gi3258290P.abyssi PAB1026P.horikoshii gi3256884D.radiodurans DRA00354D.radiodurans DRA0353D.radiodurans DRA0352P.abyssi PAB1189P.horikoshii gi3258414B.burgdorferi gi2688621M.tuberculosis gi1666149

V.cholerae VC0512V.cholerae VCA1034V.cholerae VCA0974V.cholerae VCA0068V.cholerae VC0825V.cholerae VC0282V.cholerae VCA0906V.cholerae VCA0979V.cholerae VCA1056V.cholerae VC1643V.cholerae VC2161V.cholerae VCA0923V.cholerae VC0514V.cholerae VC1868V.cholerae VCA0773V.cholerae VC1313V.cholerae VC1859V.cholerae VC1413V.cholerae VCA0268V.cholerae VCA0658V.cholerae VC1405V.cholerae VC1298V.cholerae VC1248V.cholerae VCA0864V.cholerae VCA0176V.cholerae VCA0220V.cholerae VC1289V.cholerae VCA1069V.cholerae VC2439V.cholerae VC1967V.cholerae VCA0031V.cholerae VC1898V.cholerae VCA0663V.cholerae VCA0988V.cholerae VC0216V.cholerae VC0449V.cholerae VCA0008V.cholerae VC1406V.cholerae VC1535V.cholerae VC0840

V.cholerae VC0098V.cholerae VCA1092


V.cholerae VC1394

V.cholerae VC0622

NJ

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******

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******

*

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• Phylogenomic functional prediction may not work well for very newly evolved functions

• Can we use understanding of origin of novelty to better understand these cases?

• Screen genomes for genes that have changed recently

– Pseudogenes and gene loss– Contingency Loci– Acquisition (e.g., LGT)– Unusual dS/dN ratios– Rapid evolutionary rates


Example II:Duplication


Expansion of MCP Family in V. cholerae

E.coli gi1787690

B.subtilis gi2633766Synechocystis sp. gi1001299

Synechocystis sp. gi1001300Synechocystis sp. gi1652276

Synechocystis sp. gi1652103H.pylori gi2313716H.pylori99 gi4155097C.jejuni Cj1190c

C.jejuni Cj1110cA.fulgidus gi2649560A.fulgidus gi2649548

B.subtilis gi2634254B.subtilis gi2632630B.subtilis gi2635607B.subtilis gi2635608B.subtilis gi2635609

B.subtilis gi2635610B.subtilis gi2635882

E.coli gi1788195E.coli gi2367378E.coli gi1788194

E.coli gi1789453

C.jejuni Cj0144C.jejuni Cj0262c

H.pylori gi2313186H.pylori99 gi4154603

C.jejuni Cj1564

C.jejuni Cj1506cH.pylori gi2313163H.pylori99 gi4154575

H.pylori gi2313179H.pylori99 gi4154599

C.jejuni Cj0019cC.jejuni Cj0951c

C.jejuni Cj0246cB.subtilis gi2633374

T.maritima TM0014

T.pallidum gi3322777T.pallidum gi3322939

T.pallidum gi3322938B.burgdorferi gi2688522T.pallidum gi3322296

B.burgdorferi gi2688521T.maritima TM0429T.maritima TM0918T.maritima TM0023

T.maritima TM1428T.maritima TM1143

T.maritima TM1146P.abyssi PAB1308

P.horikoshii gi3256846P.abyssi PAB1336P.horikoshii gi3256896

P.abyssi PAB2066P.horikoshii gi3258290P.abyssi PAB1026P.horikoshii gi3256884

D.radiodurans DRA00354D.radiodurans DRA0353

D.radiodurans DRA0352P.abyssi PAB1189P.horikoshii gi3258414

B.burgdorferi gi2688621M.tuberculosis gi1666149


V.cholerae VCA0974V.cholerae VCA0068

V.cholerae VC0825V.cholerae VC0282


V.cholerae VCA1056V.cholerae VC1643







V.cholerae VC1248V.cholerae VCA0864V.cholerae VCA0176



V.cholerae VC1967V.cholerae VCA0031V.cholerae VC1898V.cholerae VCA0663

V.cholerae VCA0988V.cholerae VC0216V.cholerae VC0449





V.cholerae VC1394

V.cholerae VC0622

NJ

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Based on Heidelberg et al. 2000 Nature 406:477-483.




Tetrahymena 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

Eisen et al. 2006. PLoS Biology.Friday, January 28, 2011

Phylogenomics of Novelty II

Sometimes, it is easier to steal, borrow, or coopt functions rather than evolve them

anew


Stealing DNA


rRNA Tree of Life

FIgure from Barton, Eisen et al. “Evolution”, CSHL Press.

Based on tree from Pace NR, 2003.

Archaea

Eukaryotes

Bacteria


Perna et al. 2003Friday, January 28, 2011

Network of Life

Figure from Barton, Eisen et al. “Evolution”, CSHL Press.


Archaea

Eukaryotes

Bacteria


Non homology functional prediction methods

• 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: phylogenetic profiling


Phylogenetic profiling basis

• Microbial genes are lost rapidly when not maintained by selection

• Genes can be acquired by lateral transfer• Frequently gain and loss occurs for entire

pathways/processes• Thus might be able to use correlated

presence/absence information to identify genes with similar functions


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)


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 Determined (Wu et al.

2005 PLoS Genetics 1: e65. )


Homologs of Sporulation Genes

Wu et al. 2005 PLoS Genetics 1: e65.


http://www.ncbi.nlm.nih.gov/entrez/utils/lofref.fcgi?PrId=4656&uid=16311624&db=pubmed&url=http://genetics.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pgen.0010065




Carboxydothermus sporulates





Stealing Organisms (Symbioses)


Glassy Winged Sharpshooter

• Feeds on xylem sap

• Vector for Pierce’s Disease

• Potential bioterror agent


Xylem and Phloem

From Lodish et al. 2000


Sharpshooter Shotgun Sequencing

shotgun

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


http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0040188


Higher Evolutionary Rates in Endosymbionts

Wu et al. 2006 PLoS Biology 4: e188. Collaboration with Nancy Moran’ s LabFriday, January 28, 2011



Variation in Evolution Rates

MutS MutL

+ +

+ +

+ +

+ +

_ _

_ _

Wu et al. 2006 PLoS Biology 4: e188. Collaboration with Nancy Moran’ s LabFriday, January 28, 2011



Baumannia is a Vitamin and Cofactor Producing Machine

Wu et al. 2006 PLoS Biology 4: e188.








No Amino-Acid Synthesis


The Uncultured Majority


Great Plate Count Anomaly

Culturing Microscope

CountCount




CountCount <<<<




CountCount <<<<

DNA


PCR Saves the Day


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

Eisen et al. 1992


Uses of rRNA sequences

The Hidden Majority Richness estimates

Bohannan and Hughes 2003Hugenholtz 2002


rRNA data increasing exponentially tooFriday, January 28, 2011

rRNA phylotyping issues

• Massive amounts of data– 1 x 10^6 new partial sequences with new 454– 2 x 10^6 full length sequences in DB

• Alignments of new sequences not always straightforward

• Solutions:– Reliance on similarity scores (bad)– High throughput automated phylogenetic tools

• STAP• WATERs



Eisen et al. 1992



Eisen et al. 1992

Biology not similar enough


Perna et al. 2003Friday, January 28, 2011

Metagenomics

shotgun

clone


How can we best use metagenomic data?

• Many possible uses including:– Improvements on rRNA based phylotyping and

species diversity measurements– Adding functional information on top of

phylogenetic/species diversity information• Most/all possible uses either require or are

improved with phylogenetic analysis


Example I: Phylotyping with rRNA and other genes


0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria

Epsilonproteobacteria

Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes

Major Phylogenetic Group

EFGEFTuHSP70RecARpoBrRNA

Shotgun Sequencing Allows Use of Other Markers

Venter et al., Science 304: 66-74. 2004Friday, January 28, 2011



Functional Diversity of Proteorhodopsins?

Venter et al., 2004Friday, January 28, 2011

Example II: Binning


Metagenomics Challenge


ABCDEFG

TUVWXYZ

Binning challenge


ABCDEFG

TUVWXYZ

Binning challenge

Best binning method: reference genomes


ABCDEFG

TUVWXYZ

Binning challenge

No reference genome? What do you do?


ABCDEFG

TUVWXYZ

Binning challenge

No reference genome? What do you do?

Phylogeny ....Friday, January 28, 2011

No Amino-Acid Synthesis


???????


Commonly Used Binning MethodsDid not Work Well

• Assembly– Only Baumannia generated good contigs

• Depth of coverage– Everything else 0-1X coverage

• Nucleotide composition– No detectible peaks in any vector we looked at


CFB Phyla


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)


Wu et al. 2006 PLoS Biology 4: e188.Friday, January 28, 2011



Essential Amino Acid Synthesis








Baumannia makes vitamins and cofactors

Sulcia makes amino acids




Phylogenomics of Novelty III

Knowing What We Don’t Know


As of 2002


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

As of 2002

Based on Hugenholtz, 2002


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

As of 2002



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

As of 2002



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




• Solution I: sequence more phyla

• NSF-funded Tree of Life Project

• A genome from each of eight phyla

Eisen, Ward, Robb, Nelson, et al


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




• Still highly biased in terms of the tree



Eisen & Ward, PIs


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




• Same trend in Archaea



Eisen & Ward, PIs


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




• Same trend in Eukaryotes



Eisen & Ward, PIs


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




• Same trend in Viruses



Eisen & Ward, PIs


Research Topics


Functions

Species Evolution







• Solution: Really Fill in the Tree

• GEBA• A genomic

encyclopedia of bacteria and archaea

Eisen & Ward, PIs

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


http://www.jgi.doe.gov/programs/GEBA/pilot.htmlFriday, January 28, 2011

GEBA Pilot Project: Components• Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan

Eisen, Eddy Rubin, Jim Bristow)• Project management (David Bruce, Eileen Dalin, Lynne Goodwin)• Culture collection and DNA prep (DSMZ, Hans-Peter Klenk)• Sequencing and closure (Eileen Dalin, Susan Lucas, Alla Lapidus,

Mat Nolan, Alex Copeland, Cliff Han, Feng Chen, Jan-Fang Cheng)• Annotation and data release (Nikos Kyrpides, Victor Markowitz, et

al)• Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Victor

Kunin, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson, Amrita Pati, Natalia N. Ivanova, Athanasios Lykidis, Adam Zemla)

• Adopt a microbe education project (Cheryl Kerfeld)• Outreach (David Gilbert)• $$$ (DOE, Eddy Rubin, Jim Bristow)


GEBA Pilot Project Overview

• Identify major branches in rRNA tree for which no genomes are available

• Identify those with a cultured representative in DSMZ

• DSMZ grew > 200 of these and prepped DNA• Sequence and finish 100+ (covering breadth of

bacterial/archaea diversity)• Annotate, analyze, release data• Assess benefits of tree guided sequencing• 1st paper Wu et al in Nature Dec 2009


GEBA Phylogenomic Lesson 1

The rRNA Tree of Life is a Useful Tool for Identifying Phylogenetically Novel

Genomes



Phylogeny-driven genome selection helps discover new genetic diversity


Network of Life



Archaea

Eukaryotes

Bacteria


Protein Family Rarefaction Curves

• Take data set of multiple complete genomes• Identify all protein families using MCL• Plot # of genomes vs. # of protein families


Wu et al. 2009 Nature 462, 1056-1060


http://www.nature.com/nature/journal/v462/n7276/full/nature08656.html


Synapomorphies exist

Wu et al. 2009 Nature 462, 1056-1060




Structural Novelty

• Of the 17000 protein families in the GEBA56, 1800 are novel in sequence (Wu)

• Structural modeling suggests many are structurally novel too (D'haeseleer)

• 372 being crystallized by the PSI (Kerfeld)


Phylogenetic Distribution Novelty: Bacterial Actin Related Protein

Haliangium ochraceum DSM 14365 Patrik D’haeseleer, Adam Zemla, Victor Kunin

!"#$%&'()*&& !"#$%&'(%()+"#,-.(/01 !"#*+,**'+(

2"#3)&4&*&& !"#*)$*),+%5"#$-.-6&0&1- !"#$%,$-%)(7"#0(1.8-9& !"#$''+-+,',!5"#:1,)*&$/0 !"#&$,%+)+-+

;"#01,&-*0 !"#%*+$--(<"#$-.-3.1%&0 !"#%',&'-+)

2"#$&*-.-1 !"#$'(-%%+&$="#$.1001 !"#-*$+$(&(>"#0$1,/%1.&0 !"#&$**+),)-!;"#01,&-*0 !"#*+,$*'(

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?"#4&0$)&4-/@ !"#''-+&%$-

D"#01(&61 !"#$-&'*)%&+!!"#(C1%&1*1!"#$-%$ $),)

?"#@-%1*)A1(-. !"#$((&+,*-<"#@/0$/%/0 !"#&&'&%'*(,

((

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$++$++

'*

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)*

$++

$++

*$

((),

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(%$++

)%

$++

-)

$++

+/*!

!"#$%

!&'(

!&')

!&'*

+!&'

!&',

!&'-

!&'.

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See also Guljamow et al. 2007 Current Biology. Wu et al. 2009 Nature 462, 1056-1060







Phylogeny driven genome selection (and phylogenetics in general) improves genome annotation


Most/All Functional Prediction Improves w/ Better Phylogenetic Sampling

• Took 56 GEBA genomes and compared results vs. 56 randomly sampled new genomes

• 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 predictionKostas

MavrommatisNatalia Ivanova

Thanos Lykidis

Nikos Kyrpides

Iain Anderson



Improves analysis of genome data from uncultured organisms


0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria


Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes







0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria


Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes




Cannot be done without good sampling of genomes




0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria


Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes



Phylogenetic Binning




0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria


Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes




Cannot be done without good sampling of genomes




0

0.1250

0.2500

0.3750

0.5000

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria


Deltaproteobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Thermus

Euryarchaeota

Crenarchaeota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes




GEBA Project improves metagenomic analysis, but only a little




GEBA Future 1

Need to adapt genomic and metagenomic methods to make use of

GEBA data


Ways to Make Better Use of GEBA Data

• Better phylogenetic methods for short reads• Rebuild protein family models• New phylogenetic markers• Need better phylogenies, including HGT• Improved tools for using distantly related

genomes in metagenomic analysis


iSEEM Project


GEBA Future 2

Need Experiments from Across the Tree of Life too


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


As of 2002



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


• Experimental studies are mostly from three phyla

As of 2002



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


• Experimental studies are mostly from three phyla

• Some studies in other phyla

As of 2002



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




• Same trend in Eukaryotes

As of 2002



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




• Same trend in Viruses

As of 2002



0.1

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

Tree based on Hugenholtz (2002) with some modifications.

Need experimental studies from across the tree too


0.1

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

Tree based on Hugenholtz (2002) with some modifications.

Adopt a Microbe


GEBA Future 3

We have still only scratched the surface of microbial diversity


rRNA Tree of Life




Phylogenetic Diversity: Sequenced Bacteria & Archaea

From Wu et al. 2009 Nature 462, 1056-1060












Phylogenetic Diversity with GEBA

From Wu et al. 2009 Nature 462, 1056-1060












Phylogenetic Diversity: Isolates

From Wu et al. 2009 Nature 462, 1056-1060Friday, January 28, 2011



Phylogenetic Diversity: All

From Wu et al. 2009 Nature 462, 1056-1060Friday, January 28, 2011



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



• Most phyla with cultured species are sparsely sampled

• Lineages with no cultured taxa even more poorly sampled

Well sampled phylaPoorly sampledNo cultured taxa


Uncultured Lineages:Technical Approaches

• Get into culture• Enrichment cultures• If abundant in low diversity ecosystems• Flow sorting• Microbeads• Microfluidic sorting• Single cell amplification


Conclusion

• Phylogenetic sampling of genomes improves our understanding of microbial diversity in many ways

• Still need– More biogeography– More phenotypic/experimental data– Deeper phylogenetic sampling


MICROBES


A Happy Tree of Life


Phylogenomics and the Diversification of Microbes, J. A. Eisen at Genentech 1/27/11

Education

Transcript of Phylogenomics and the Diversification of Microbes, J. A. Eisen at Genentech 1/27/11