uc davis eve161 lecture 13 by @phylogenomics

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

Lecture 13:

EVE 161:Microbial Phylogenomics

!Lecture #13:

Era III: Genome Sequencing and Phylogenomic Analysis

!UC Davis, Winter 2014

Instructor: Jonathan Eisen

!1


Where we are going and where we have been

• Previous lecture: !12: Guest Lecture

• Current Lecture: !13: Genome Sequencing III

• Next Lecture: !14: Metagenomics

!2


Phylogenomics I:Major Evolutionary Transitions


• Analysis of S. pombe genome by Wood et al 2002

• Compared the genomes of eukaryotes to those of prokaryotes

• “Are there genes found in all eukaryotes with no obvious homologs in any prokaryote?”

Phylogenomics I:Major Evolutionary Transitions


Evolutionary Model

BacteriaArchaea

Eukaryotes

Giardia

Trichomonas

Naegleria

Trypanosoma

Euglena

Plasmodium

Tetrahymena

Phytophthora

Arabidopsis

Chlamydomonas

Dictyostelium

HumansFly

Worm

Encephalatozoon

S. cerevisiaeS. pombe


Eukaryotic Specific Genes

• >200 genes found including: – Cytoskeleton components: tubulin,

ankyrin, myosin – Protein degradation: ubiquitin, proteases – Chromatin and DNA packaging

• Of the 200 many had no known function: could encode novel eukaryotic wide processes


Multi- vs. Single-Cellular Eukaryotes

• Further analysis of S. pombe genome • Compared multi-cellular vs. single-cellular eukaryotes

(animals and plants vs. yeast) • “Are there genes in all multi-cellular and not in any single-

cellular?” • Found only 3 • Concluded that the genetic basis of multi-cellularity was

likely to be gene regulation and not invention of new genes


Multiple Origins of Multicellularity


Phylogenomics II:Endosymbiont Evolution


Endosymbiont Evolution

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

• Baumannia shows ALL of these


Uses of Whole Genome Trees


Wolbachia Evolution


Variation Between Endosymbionts and Free Living

• Repair hypothesis !

• Population genetics hypothesis !


Variation Between Endosymbionts and Free Living



• PopGen explanations favored


Variation Among Endosymbionts



MutS MutL

+ +

+ +

+ +

+ +

_ _

_ _




• Repair explanations favored



Phylogenomics III:Lateral Gene Transfer


Vertical Evolution

From C. Darwin, origin of species, via W. F. Doolittle


Vertical Inheritance - Binary Bission


Lateral inheritance I: Competence


Lateral inheritance II: Conjugation


Agrobacterium conjugation


Figure 7.19 - Ab transfer


Lateral inheritance III: Transduction


Pathogenicity Island


Steps in Lateral Gene Transfer (LGT)A B C D



1 Gene acquires host features




2

Transfer




2

Transfer

3-5 Integration, selection, spread




2

Transfer

6 Amelioration3-5 Integration, selection, spread


How to Infer Gene Transfers

• Unusual distribution patterns !

• Unusual nucleotide composition !

• High sequence similarity to supposedly distantly related species !

• Unusual gene trees !

• Observe transfer events


Case Study I: Aphids

Fig. 1 Coloration and carotenoids in the pea aphid. Typical green (A) and red (B) aphid clones, (C) 5AY, a green mutant clone arising from the red clone 5A. (D) Profiles of carotenoids in red (5A, LSR1), mutant redgreen (5AY, two samples), and green (8-10-1, 7-2-1) pea aphid clones. Torulene and a related red compound are restricted to red clones; the mutant 5AY clone lacks these and displays an elevation in their predicted precursor, -carotene.


Table 1 Genes in the A. pisum genome with closest homology to carotenoid biosynthetic enzymes, including scaffold of origin and matching EST sequences. Similar color indicates that the gene is on the same scaffold. The 3' end of scaffold NW_001925130 overlaps with the 5' end of NW_001923501 for 5400 base pairs, and PCR demonstrated continuity of these scaffolds. Pink row is the gene corresponding to torR and conferring red color (see text). Protein length, amino acids; ESTs are those present in GenBank, mostly from clone LSR1.



Fig. 2 Phylogenetic relations of inferred carotenoid biosynthetic enzymes from the pea aphid genome. (A) Carotenoid desaturases and (B) carotenoid cyclase–carotenoid synthases. Sequences are from aphids, bacteria, plants, and fungi; no homologs were detectable in other sequenced animal genomes. Bootstrap support greater than 50% is indicated on branches.

!



Case Study II: GEBA


Tree of Life

Figure from Barton, Eisen et al. “Evolution”, CSHL Press based on Baldauf et al Tree


Genomes Poorly Sampled



TIGR Tree of Life Project


Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !41


Genomes Still Poorly Sampled



Genomic Encyclopedia of Bacteria & Archaea

Wu et al. 2009 Nature 462, 1056-1060


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


GEBA Lesson 1: rRNA utility in IDing novel genomes

From Wu et al. 2009 Nature 462, 1056-1060!45



GEBA Lesson 2: rRNA Tree is not perfect

Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026.

16s WGT, 23S

!46

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15879228&query_hl=2


GEBA Lesson 3: Phylogenetic sampling improves annotation

• 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 prediction

!47


GEBA Lesson 4 : Metadata Important

!48


GEBA Lesson 5:Improves discovering new genetic diversity

!49


Protein Family Rarefaction Curves

• Take data set of multiple complete genomes

• Identify all protein families using MCL

• Plot # of genomes vs. # of protein families

!50

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014Wu et al. 2009 Nature 462, 1056-1060

!51



Synapomorphies exist

Wu et al. 2009 Nature 462, 1056-1060!52



Phylogenetic Distribution Novelty: Bacterial Actin Related Protein

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

A. cliftonii gi14269497U. pertusa gi50355609

C. boidinii gi57157304S. cerevisiae gi14318479L. starkeyi gi166080363 S. japonicus gi213407080

H. sapiens gi4501889M. cerebralis gi46326807

C. cinerea gi169844021N. crassa gi85101929I. scapularis gi215507378 H. sapiens gi5031569

S. japonicus gi213404844S. cerevisiae gi6320175D. melanogaster gi24642545G. gallus gi45382569C. neoformans gi58266690S. cerevisiae gi6322525D. melanogaster gi17737543H. sapiens gi5031573 H. ochraceum gi227395998

P. patens gi168051992 A. thaliana gi18394608

S. cerevisiae gi1008244

D. melanogaster gi17737347

D. hansenii gi218511921S. cerevisiae gi6323114

S. japonicus gi213408393 S. cerevisiae gi1301932

D. discoideum gi66802418

O. sativa gi182657420 A. thaliana gi1841 1737

D. melanogater gi19920358M. musculus gi226246593

99

67

100100

65

100

100

75

100

100

51

9973

10097

94100

74

100

87

100

0.5

ACTIN

ARP1

ARP2

ARP3

BARP

ARP4

ARP5

ARP6

ARP7

ARP10

See also Guljamow et al. 2007 Current Biology.



GEBA Cyanobacteria

Shih et al. 2013. PNAS 10.1073/pnas.1217107110

light-harvesting strategies. The majority of cyanobacteria absorblight mainly with soluble pigment–protein complexes calledphycobilisomes, in contrast to eukaryotes, which use membrane-bound light-harvesting complexes (LHCs). However, an increasingnumber of transmembrane proteins involved in cyanobacteriallight harvesting are being identified, such as Pcb and IsiA (22, 23).These proteins are analogous in function to eukaryotic LHCs.Because of the growing number of proteins and names, an over-arching nomenclature has been proposed to name this proteinfamily the chlorophyll binding proteins (CBPs), which are char-acterized by six transmembrane helices and the ability to bindchlorophyll (24).With the increase in number and diversity of genomes, we find

that CBPs are widely distributed across the cyanobacterial phy-lum: 67% (84 of 126) of cyanobacterial genomes have, in addi-tion to the phycobilisomes, genes that putatively function asmembrane-bound light-harvesting proteins. In our phylogeneticanalysis, the increase in sequence diversity reveals strong supportfor various subclades that we have provisionally named CBPIV,-V, and -VI (Fig. 3A and SI Appendix, Fig. S5). Although not yetexperimentally demonstrated, members of CBPIV, -V, and -VIare expected to bind chlorophyll because they contain position-ally conserved histidine and glutamine residues that ligate chlo-rophyll in confirmed chlorophyll-binding CBPs (SI Appendix, Fig.S6). Some of these proteins, such as CBPIV, have previously

been annotated as PsbC homologs (25), because all CBP pro-teins are thought to have a common evolutionary origin with thepsbC gene (24). Because of the vast enrichment of cyanobacterialprotein sequences, the increase from two to six known CBPVIsequences augments phylogenetic resolution (bootstrap supportof 85%), allowing us to more confidently assert that there isa separate and distinct CBPVI subfamily. On the basis of ourphylogenetic analysis of the CBP family, and consistent withprevious studies (26), there seems to be a substantial amount ofgene duplication and horizontal gene transfer among CBPIV,-V, and -VI. In some genomes, CBPIV and CBPV are found ina gene cluster with other CBP proteins, including IsiA (Fig. 3C),suggestive of the potential for lateral transfer of gene clustersencoding light-harvesting proteins, as documented in marinecyanobacteria (27). Interestingly, many proteins of the CBPVclade also contain a C-terminal extension (SI Appendix, Fig. S7)with homology to the PsaL subunit of photosystem I (PSI).Notably, two distinct subclades within the CBPV family seem tohave independently lost the PsaL domains, reflecting the mod-ularity of this C-terminal extension. Homology modeling andinsertion of the PsaL-like domain into the PSI structure (Fig. 3Band SI Appendix, Fig. S8) suggests how the CBPV protein couldtheoretically be incorporated as an ancillary light-harvestingpolypeptide into a monomeric, but not trimeric, PSI. Althoughscattered observations of members of these CBP protein clades

0.3

B1

B2

C1

Paulinella

Glaucophyte

GreenRed

Chromalveolates

C2C3

AE

FG

B3D

A

B

Fig. 2. Implications on plastid evolution. (A) Maxi-mum-likelihood phylogenetic tree of plastids and cya-nobacteria, grouped by subclades (Fig. 1). The red dot(bootstrap support = 97%) represents the primaryendosymbiosis event that gave rise to the Arch-aeplastida lineage, made up of Glaucophytes (orange),Rhodophytes (red), Viridiplantae (green), and Chro-maleveolates (brown). The independent primary en-dosymbiosis in the amoeba Paulinella chromatophorais shown in purple. (B) Number of predicted eukary-otic, nuclear genes transferred from a cyanobacterialendosymbiont. Colors correspond to the lineageorganisms as above. Light and dark shades of colorsrepresent before and after adding the CyanoGEBAgenomes, respectively.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1217107110 Shih et al.

http://www.pnas.org/cgi/doi/10.1073/pnas.1217107110


Haloarchaeal GEBA-like

Lynch et al. (2012) PLoS ONE 7(7): e41389. doi:10.1371/journal.pone.0041389


The Dark Matter of Biology

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


Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014!57

Number of SAGs from Candidate Phyla

OD

1

OP

11

OP

3

SA

R4

06

Site A: Hydrothermal vent 4 1 - -Site B: Gold Mine 6 13 2 -Site C: Tropical gyres (Mesopelagic) - - - 2Site D: Tropical gyres (Photic zone) 1 - - -

Sample collections at 4 additional sites are underway.

Phil Hugenholtz

GEBA Uncultured


JGI Dark Matter Project

environmental samples (n=9)

isolation of singlecells (n=9,600)

whole genomeamplification (n=3,300)

SSU rRNA gene based identification

(n=2,000)

genome sequencing, assembly and QC (n=201)

draft genomes(n=201)

SAK

HSM ETLTG

HOT

GOM

GBS

EPR

TAETL T

PR

EBS

AK E

SM G TATTG

OM

OT

seawater brackish/freshwater hydrothermal sediment bioreactor

GN04WS3 (Latescibacteria)GN01

!"#$%&'$LD1

WS1PoribacteriaBRC1

LentisphaeraeVerrucomicrobia

OP3 (Omnitrophica)ChlamydiaePlanctomycetes

NKB19 (Hydrogenedentes)WYOArmatimonadetesWS4

ActinobacteriaGemmatimonadetesNC10SC4WS2

Cyanobacteria()*&2

Deltaproteobacteria

EM19 (Calescamantes)+,-*./'&'012345678#89/,-568/:

GAL35Aquificae

EM3Thermotogae

Dictyoglomi

SPAMGAL15

CD12 (Aerophobetes)OP8 (Aminicenantes)AC1SBR1093

ThermodesulfobacteriaDeferribacteres

Synergistetes

OP9 (Atribacteria)()*&2

CaldisericaAD3

Chloroflexi

AcidobacteriaElusimicrobiaNitrospirae49S1 2B

CaldithrixGOUTA4

*;<%0123=/68>8?8,6@98/:Chlorobi

486?8,A-5BTenericutes4AB@9/,-568/Chrysiogenetes

Proteobacteria

4896@9/,-565BTG3SpirochaetesWWE1 (Cloacamonetes)

C=1ZB3

=D)&'EF58>@,@,,AB&CG56?ABOP1 (Acetothermia)Bacteriodetes

TM7GN02 (Gracilibacteria)

SR1BH1

OD1 (Parcubacteria)

(*1OP11 (Microgenomates)

Euryarchaeota

Micrarchaea

DSEG (Aenigmarchaea)Nanohaloarchaea

Nanoarchaea

Cren MCGThaumarchaeota

Cren C2Aigarchaeota

Cren pISA7

Cren ThermoproteiKorarchaeota

pMC2A384 (Diapherotrites)

BACTERIA ARCHAEA

archaeal toxins (Nanoarchaea)

lytic murein transglycosylase

stringent response (Diapherotrites, Nanoarchaea)

ppGpp

limitingamino acids

SpotT RelA

(GTP or GDP)+ PPi

GTP or GDP+ATP

limitingphosphate,fatty acids,carbon, iron

DksA

Expression of components for stress response

sigma factor (Diapherotrites, Nanoarchaea)

!4

"#$#"%

!2!3 !1

-35 -10

&'()

&*()

+',#-./0123452

oxidoretucase

+ +e- donor e- acceptor

H

'Ribo

ADP

+

'62

O

Reduction

OxidationH

'Ribo

ADP

'6

O

2H

',)##$#6##$#72#####################',)6+ + -

HGT from Eukaryotes (Nanoarchaea)

Eukaryota

O68*62

OH

'6

*8*63

OO

68*62

'6

*8*63

O

tetra-peptide

O68*62

OH

'6

*8*63

OO

68*62

'6

*8*63

O

tetra-peptide

murein (peptido-glycan)

archaeal type purine synthesis (Microgenomates)

PurFPurD9:3'PurL/QPurMPurKPurE9:3*PurB

PurP

?

Archaea

adenine guanine

O

6##'2

+'

'62

'

'

H

H

'

'

'

H

HH' '

H

PRPP ;,<*,+

IMP

,<*,+

A*

GUA *G U

GU

A

*

GU

A UA * U

A * U

Growing AA chain

=+',>?/0@#recognizes

UGA1+',

UGA recoded for Gly (Gracilibacteria)

ribosome

Woyke et al. Nature 2013.



A Genomic Encyclopedia of Microbes (GEM)



GEBA Lesson 6: Improves analysis of metagenomic data

!61


Sargasso Phylotypes

Wei

ghte

d %

of C

lone

s

0.000

0.125

0.250

0.375

0.500

Major Phylogenetic Group

Alphapro

teobacteria

Betap

roteobacteria

Gamm

aproteobacteria

Epsilo

nproteobacteria

Deltapro

teobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Th

ermus

Euryarchaeota

Crenarchaeota

EFGEFTuHSP70RecARpoBrRNA

Other Markers

GEBA Project improves metagenomic analysis

Venter et al., Science 304: 66-74. 2004 !62



Sargasso Phylotypes

Wei

ghte

d %

of C

lone

s

0.000

0.125

0.250

0.375

0.500

Major Phylogenetic Group

Alphapro

teobacteria

Betap

roteobacteria

Gamm

aproteobacteria

Epsilo

nproteobacteria

Deltapro

teobacteria

Cyanobacteria

Firmicutes

Actinobacteria

Chlorobi

CFB

Chloroflexi

Spirochaetes

Fusobacteria

Deinococcus-Th

ermus

Euryarchaeota

Crenarchaeota

EFG EFTurRNA

But not a lot

Venter et al., Science 304: 66-74. 2004

Other Markers

!63



rRNA Tree of Life

Figure from Barton, Eisen et al. “Evolution”, CSHL Press. 2007.

Based on tree from Pace 1997 Science 276:734-740

Archaea

Eukaryotes

Bacteria

!64


PD: Genomes


!65




PD: Genomes + GEBA

!66



PD: Isolates

From Wu et al. 2009 Nature 462, 1056-1060 !67



PD: All

From Wu et al. 2009 Nature 462, 1056-1060 !68



Uncultured Lineages:Technical Approaches

• Get into culture

• Enrichment cultures

• If abundant in low diversity ecosystems

• Flow sorting

• Microbeads

• Microfluidic sorting

• Single cell amplification

!69

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !70

Number of SAGs from Candidate Phyla

OD

1

OP

11

OP

3

SA

R4

06

Site A: Hydrothermal vent 4 1 - -Site B: Gold Mine 6 13 2 -Site C: Tropical gyres (Mesopelagic) - - - 2Site D: Tropical gyres (Photic zone) 1 - - -

Sample collections at 4 additional sites are underway.

Phil Hugenholtz

GEBA uncultured

uc davis eve161 lecture 13 by @phylogenomics

Education

uc davis eve161 course

jonathan eisen winter

jonathan eisenslides

lateral inheritance

lateral gene transferslides

population genetics

lateral gene transfer

free living repair hypothesis