A model species for agricultural pest genomics: the genome ...
LGT & Genome EvolutionLGT & Genome Evolution Genomics ......• Genomics ofGenomics of Endosymbionts...
Transcript of LGT & Genome EvolutionLGT & Genome Evolution Genomics ......• Genomics ofGenomics of Endosymbionts...
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
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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
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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
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Salmonella Typhimurium LT2
Esch
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hia
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K12
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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
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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
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Bu
Bu
St-StySt-Sty
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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)
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Science 314:267
(Oct 13, 2006)
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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
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Ener
gy P
rodu
ctio
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Cel
l Div
isio
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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
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Mem
bran
e B
ioge
n
Secr
etio
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Inor
g Io
n M
etab
Seco
ndar
y M
etab
Sign
al T
rand
uctio
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Pred
icte
d/U
nkno
wn
Cha
pero
ne/P
rote
ase
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15
Sum
% S
igna
l Int
ensi
ty
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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
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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
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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
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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.