Systems Microbiology Monday Oct 29 - Andersson and Moran readings

Genome Evolution & Ecology •• LGT & Genome EvolutionLGT & Genome Evolution •• Genomics ofGenomics of EndosymbiontsEndosymbionts•• Environmental GenomicsEnvironmental Genomics

Mechanisms and consequences of Lateral Gene Transfer

Redfield, Nat. Rev. Genet. 2001

A B C

Organismal phylogeny

A B C

gene phylogeny

Transduction: via a virus (bacteriophage)

Transformation: integration of free DNA

Conjugation: direct contact (plasmid)

Microscopic photographs of phage and bacterial conjugation removed due to copyright restrictions.

DNA transfer by transduction

DNA transfer by conjugation

Phage-infected donor

Recipient

Recipient

Recipient

Donor

Common

Rare

Hfr

Donor

Dead Donor Competent recipient

Gene transfer by competence

F

A

B

C

Figure by MIT OCW.

POPULATION GENETICS OF PATHOGENS

UROPATHOGENIC & ENTEROHAEMORRHAGIC “HOT-SPOTS” Welch, R. A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 17020-17024

5857.6%

MG1655 (K-12)Non-Pathogenic

CFT073 Uropathogenic

EDL933 (O157:H7)Enterohaemorrhagic

5146.7%

2042.6%

162321.2%

134617.6%

1932.5%

299639.2%

Total proteins = 76382996 (39.2%) in all 3911 (11.9%) in 2 out of 33554 (46.5%) in 1 out of 3

Figure by MIT OCW.

POPULATION GENETICS OF PATHOGENS

UROPATHOGENIC & ENTEROHAEMORRHAGIC “HOT-SPOTS”

Welch, R. A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 17020-17024

100 Kb aspV

aspV

thrW

serX

serXserW

serU

serU

argW

argW

ssrA

ssrA

metV

selC (pap)pheU

selC(LEE)

leuX

leuX

(pap-hly)pheV

pheV

asnT,V

thrW

**

*

* * * * * **

100 Kb

CFT073

EDL933

Figure by MIT OCW.

1. Construct multiple alignment of sequences

2. Construct table listing all pairwise differences (distancematrix)

3. Construct tree from pairwise distances

Gorilla : ACGTCGTA Human : ACGTTCCT Chimpanzee: ACGTTTCG

-Ch

2-Hu

44-Go

ChHuGo

Go

Hu

Ch

2

1 1

1

Molecular Phylogenetics : Inferring Evolutionary Relationships

Lateral Gene Transfer and molecularphylogenetics

“Gene tree” assumes similarity by descent

But what if there is extensive lateral genetransfer between bacteria ???

“true” tree based on Gene ‘Y’

Gene “X” laterally transfered

Gene “X” tree looks like this

Example of genome dynamics over time due to Lateral Gene Transfer

Gene uptakeIn gamma-Proteobacteria E. Lerat et al 03

S. typhimurium

E. coli

Y. pestis KIM

Y. pestis CO92

W. brevipalpis

B. aphidicola

P. multocida

H. influenzae

V. cholerae

P. aeruginosa

X. fastidiosa

X. campestris

X. axonopodis

Many genes in most genomes arrived via LGT after the common ancestor.

Most genes arriving via LGT come from distant sources (not in this group)

Many persist as vertically transmitted genes within the descendant clade. ---but many are lost quickly (many present only in tips of tree)

Detecting Horizontal Transfers

1. Unexpected ranking of sequence similarity among homologs 2. Unexpected phylogenetic tree topology 3. Unusual phyletic pattern 4. Conservation of gene order 5. Anomalous DNA composition

“All criteria for identifying probable horizontal gene transfer, or more precisely acquisition of foreign genes by a particular genome, inevitably rely on some unusual feature(s) of subsets of genes that distinguishes them from the bulk of genes in the genome.” Koonin et al. 2001

• Direct proofs are unavailable • Indications of horizontal transfers remain probabilistic

Koonin et al. ARM (2001) 55 709-42

aphid, Acyrthosiphon pisum

Image of an aphid (Acyrthosiphon pisum) removed due to copyright restrictions.

Essential amino acids - not in the bugs diet !

*

*

Plasmids for essential amino acid biosynthesis found in aphid symbionts

trpG

trpG trpG

trpG

trpE

trpE

trpE

trpE

Chorismate

Anthranilate Tryptophan

(trpEG)14-15

ORF-B ORF-A P14trpD trpB trpA

ORF-V

ORF-VItrpC(F)8.4 kt

pBAtrpEG

14.3 kb

Figure by MIT OCW.

Moran et al., PNAS 100:14545 (2003)

Aphid Host Aphid Clone/Population leuABCD trpEG

A. pisum

Diuraphis noxia

Rhopalosiphum maidis

S. graminum

Uroleucon ambrosiae

12 United Kingdom clonesN. A. Moran lab clone 5A (Madison, Wl)P. Baumann lab clone (Lincoin, NE)South Africa population

N. A. Moran lab clone (Tucson)

Biotype B (K. A. Shufran lab clone)Biotype E (T. Mittler lab clone)Biotype E (P. Baumann lab clone)Biotype E (N. A. Moran lab clone)Biotype E (K.A. Shufran lab clone)Biotype G (K.A. Shufran lab clone)Biotype SC (K.A. Shufran lab clone)

86 individuals, 15 U.S. populations

4.81.80.4

0.3

0.514.52.11.52.62.40.5

0.3-1.9

0.60.90.3

23.51.41.91.60.5

0.5-2.8

2.4-16.2*

The ratios of copies of plasmid-borne amino acid biosynthetic genes (leuABCD, trpEG) to chromosomalgene copies for Buchnera of different aphid species and strains.

Figure by MIT OCW.

Symbiont phylogeny mirrors insect host phylogeny - co-evolution

Ruminobacter amylophilus

Proteus vulgarisEscherichia coli

Sl. chinensisMe. rhois

P. betae

Mi. victoriaC. viminalis

D. noxiaA. pisum

U. sonchiM. persicae

S. graminumR. maidisR. padi

Aphidini

Macrosiphini

Chaitophorini

Mindarini

Pemphigini

Fordini

Tribe Family

Aphididae

Drepanosiphidae

Mindaridae

Pernphigidae

25 Base changes

rrs (165 rDNA)

BUCHNERA PHYLOGENY APHID PHYLOGENY

Figure by MIT OCW.

Figures removed due to copyright restrictions.

UNCHARACTERIZED GENOMIC ANALYSES ? NATIVE TAXA

Figures removed due to copyright restrictions.

Haemophilus influenzaeFleischmann,R.D et al. 1995 Science 269: 496-512

Two basic metagenomic approaches 1. Extract DNA from environmental sample

2. Construct library

3. Screen

large insert (cosmid or BAC) library (up to 200 kb), allowssampling of whole operons

conventional small insert (<10kb) library

Perform functional screens: directly test for somebiochemical property in thecloning host

Sequence DNA or RNA,look for genes with functionsof interest

Limitations:Possible problems with efficienttranscription of the cloned fragment,translation, secretion of the product,correct chaperones for folding of theproduct

Limits search to genes withdetectable (evolutionary)homology to functionallycharacterized genes:Sequence or structural homology

10.0

5.0

1.0

0.5

0.1100 500 1000 5000 10000

Nanoarchaeum equitansMycoplasma genitalium Wigglesworthia glossinidia

Rickettsia conoriiPelagibacter ubique

Ehrlichia ruminantiumBartonella quintanaThermoplasma acidophilum

Bartonella henselaeCoxiella burnetii

Prochlorococcus marinus MED4Prochlorococcus marinus SS120

Prochlorococcus marinus MIT9313

Synechococcus sp.WH8102

Silicibacter pomeroyi

Rhodopirellula baltica

Streptomyces coelicolor

Mesoplasma florum

Free-livingHost-associated

Obligate symbionts/parasitesPelagibacter ubique

Number of protein encoding genes

Gen

ome

size

(Mbp

)

Figure by MIT OCW.

Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS Shuji Shigenobu, Hidemi Watanabe, Masahira Hattori, Yoshiyuki Sakaki, Hajime Ishikawa

Nature 407, 81-86 (07 Sep 2000)

Figure removed due to copyright restrictions.

Science 314:267(Oct 13, 2006)

Figure removed due to copyright restrictions.

/Ap divergence

Diagram showing Buchnera gene loss from a reconstructed enteric ancestor removed due to copyright restrictions.

2000 genes lost from ancestor, to Sg/Ap divergenceMust have been rapid evolution and gene loss !

COMPARATIVE GENOMICS

SCIENCE 296:2376 (2002)

Genome size (bp) 641, 454 640, 681

Genic G + C content (%) 26.2 26.3

Intergenic G + C content (%) 14.8 16.1

Protein coding genes (no.) 545 564

Pseudogenes (no.) 38 13

Avg. gene length (bp) 978 985

Avg. intergenic length (bp) 118 127

Feature

Comparison of Genome Features for B. aphidicola (Sg) and B. aphidicola (Ap)

B. aphidicola (Sg) B. aphidicola (Ap)

Figure by MIT OCW.

COMPARATIVE GENOMICS

SCIENCE 296:2376 (2002) Buchnera aphidicola the bacterial symbiont, was compared (strain-wise) between the aphids Schizaphus graminum (Sg) versus Acyrthosiphon pisum (Ap)

After 70 million years, no chromosomal rearrangements or gene acquisitions

But considerable sequence divergence, and substantial gene loss(9e-9 synonymous substitutions/yr; 1.65e-9 non-synonymous substitutions/yr)

In comparison, E coli vs. S. typhyimurium: 2000X more labile in gene content/order

Gene plot, ncbi

00

2000

4000

2000

Salmonella Typhimurium LT2

Esch

eric

hia

coli

K12

4000 00

200

400

200

Buchnera aphidicola str. APS (Acyrthosiphon pisum)

No inversions, translocations, duplications, or gene acquisitions !

Buc

hner

a ap

hidi

cola

str.

Sg

(Sch

izap

his

gram

inum

)

400 600

Figure by MIT OCW.

0.1

0.1

0.2

0.2

0.3 0.4 0.50.0

0.6

flgE

flgD

flgN

fliJ

fliN

fliM

Ka [Buchnera (Sg)-Buchnera (Ap)]

Ka

[E. c

oli-S

. typ

hi]

Ka = nonsynomymous substitutions/site

0

~450 genes

Figure by MIT OCW.

Figure by MIT OCW.

S. typhi

Free-living

0.065100

100

99

100

100

100

100

100

100

100

y-ProteobacteriaBuchnera (Sg)Buchnera (Ap)

S. typhimurium

E. coli K12E. coli 0157

R. prowazekii

B. halodurans

M. pneumoniaeM. genitalium

C. pneumoniae AR39C .muridarumC. trachomatis

R. conorii

B. subtilis

H. pylori 26695H. pylori J99

Obligate Symbiont

00 0

11

1

11

22

22

23

3

3

3

4

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4

4

5

5

5

5

6

6

65

6

01

23

45

6

Bu

Bu

St-StySt-Sty

7 78 89 9

K12-O157 K12-O157

Genome size (

Mb)

Genome size (

Mb)

Repeats (kb)

log(

Rea

rran

gem

ent/K

a)

25 50 75 100 125 150 00

Repeats (kb)25 50 75 100 125 150

3,4lo

g(In

del/K

a)

100

Science 314:267

(Oct 13, 2006)

00 1 2 3 4 5 6 7 8 9

10

20

30

40

50

60

70

Chromosomal size (Mb)

GC

con

tent

(%)

Carsonella

Carsonella

50 µm

Figure by MIT OCW.

GENOME DYNAMICS IN Buchnera• Enhanced stability of genome architecture in obligate symbionts,

despite substantial sequence divergence

• Prominence of pseudogenes, loss of DNA repair mechanisms (So how is genome stability maintained ???)

• Gene transfer elements are greatly reduced/eliminated (Reduced phage, exchange with other genomes, repeat seqs, transposons)

• Lack of recombination mechanisms (no recA and recF) - lowers rearrangement/gene acquisitions

• Lowered freqs of recombin., likely renders it neutrally selective => genome stasis

Prepare (or buy)microarray carrying"probes" of interest

Isolate RNA from cells

Generate cDNA and label"targets"

Samples will be labeledwith two differentfluorescent dyes

Incubate this hybridizationmix with DNA microarrays

Scan microarrays todetect bound cDNA. Storedata

Analyze data

A. RNA IsolationE. Imaging

Sample A

Sample B > A

Sample B

C. Labeling of ProbeB. cDNA Generation

D. Hybridizationto Array

Reverse Transcriptase

FloorescentTags

Sample A > BSample A = B

+

Figure by MIT OCW.

Consequences of reductive evolution for gene expression in an obligate endosymbiont.Mol Microbiol. 2003 Jun;48(6):1491-500.

Graphs removed due to copyright restrictions.

PNAS 100:14543 (2003)

5

10

15

% G

enom

e C

onte

nt 20

25 Buchnera(Sg) : 541 GenesE. coli MG1655: 3587 Genes

5

Ener

gy P

rodu

ctio

n

Cel

l Div

isio

n

Am

ino

Aci

d Sy

n

Nuc

leot

ide

Syn

Car

bohy

d M

etab

Coe

nzym

e M

etab

Lipi

d M

etab

Tran

slat

ion

Tran

scrip

tion

DN

A R

eplic

atio

n

Mem

bran

e B

ioge

n

Secr

etio

n

Inor

g Io

n M

etab

Seco

ndar

y M

etab

Sign

al T

rand

uctio

n

Pred

icte

d/U

nkno

wn

Cha

pero

ne/P

rote

ase

10

15

Sum

% S

igna

l Int

ensi

ty

20

25Buchnera Favorable PlantBuchnera Resistant PlantE. coli Rich MediumE. coli Minimal Medium

Figure by MIT OCW.

PNAS 100:14543 (2003)

2.0

Essential Amino AcidsNon-Essential

4

# O

rthol

ogs C

onsi

dere

d in

Pat

hway

8

12

16

ARG

ILE/

LEU

/VA

L

LEU

TRP/

PHE

TRP

PHE

PHE/

HIS

HIS

LYS

LYS/

THR

MET

GLY

CYS

Sum

% S

igna

l, A

djus

ted

for #

Gen

es

4.0

6.0

8.0

10.0

12.0

14.0

16.0

Buchnera Favorable PlantBuchnera Resistant PlantE. coli Rich MediumE. coli Minimal Medium

Figure by MIT OCW.

Metabolic Complementarity and Genomics of the Dual Bacterial Symbiosis of Sharpshooters Dongying Wu, et al. PLoS Biology 4:(6) e188 (2006)

Two different bacterial endsymbionts in glassy-winged sharpshooter bacteriome

Microscopic image of Baumannia and Sulcia removed due to copyright restrictions.

-

Metabolic Complementarity and Genomics of the Dual Bacterial Symbiosis of Sharpshooters Dongying Wu, et al. PLoS Biology 4:(6) e188 (2006)

Sharpshooters are also a vector for extracellular Xyllela a pathogen on grapes

100

100

10051

100

100

100

94

100

100

40

100

95 99

10095

98

96

100

51

Xylella fastidiosa Temeculal

Xanthomonas axonopodis citriVerrucomicrobium spinosum

Fibrobacter succinogenes

Chlorobium tepidum

Sulcia muelleri

Bacteroides forsythus

Porphyromonas gingivalis

Prevotella ruminicola

Bacteroides thetaiotaomicro

Bacteroides fragilis YCH46

Bacteroides fragilis NCTC

Pseudomonas aeruginosa PAOIVibrio cholerae

Pasteurella multocida

Haemophilus influenzae Rd

Baumannia cicadellinicola

Buchnera aphidicola Bp

Buchnera aphidicola str APS

Buchnera aphidicola str Sg

Yersinia pestis KIM

Escherichia coli K12

Salmonella typhimurium LT2Ralstonia solanacearum

Blochmannia floridanus

Wigglesworthia glossinidia

GammaproteobacteriaBacteroidetes

Figure by MIT OCW.