Download Functional evolution of bacterial genome

Functional evolution of
bacterial genome
The Escherichia coli case
Catherine Schouler, june 2006
1
Plan
Presentation of E. coli
 Genomes sequenced
 Genomic island

 Definition
 Roles
 Examples
Tools for functional analysis
 Conclusion

Catherine Schouler, june 2006
2
Escherichia coli


Theodor Escherich (18571911), a German pediatrician
and bacteriologist discovered
Escherichia coli, which was
named after him in 1919
Gram-negative bacilli,
facultatively anaerobic, non
motile or motile by
peritrichous flagella
Catherine Schouler, june 2006
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E. coli : the gram-negative model
organism


Catapulted to prominence
by the discovery of strain
K-12’s ability to carry out
genetic recombination by
conjugation (Tatum,
1946) and by generalized
transduction (Lennox,
1955)
Easy to rear, small,
cheap, rapid growth
cycle, short lived,
genetically manipulable
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E. coli habitat




Resident of animal
intestinal tracts
Well adapt to life in rivers,
oceans and soils
Can be found living at 2°C in McMurdo Bay
(Antarctica)
Indicator of fecal pollution
and water contamination
Number of E. coli/ml water
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E. coli : a versatile bacteria



Commensal
Use a probiotic
Could be virulent
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E. coli as a commensal
Intestinal tract of humans and many
animal species
 Source of vitamin K and B-complex
vitamins
 One of the first bacterial species to
colonize the infant’s intestine

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E. coli as a pathogen

Intra intestinal infections : iPEC

Extra intestinal infections : ExPEC
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E. coli as a probiotic



E. coli strain Nissle
1917 (O6:K5:H1)
forms the basis of the
probiotic preparation
Mutaflor
used for treatment of
various intestinal
disorders (Crohn’s
disease…)
successful colonizer
of the human gut
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iPEC
ETEC - enterotoxigenic E. coli (Cholera-like)
 EIEC - enteroinvasive E. coli (Shigella-like)
 EHEC - enterohemorrhagic E. coli
(Hamburger disease)
 EPEC - enteropathogenic E. coli (Neonatal
diarrhea)
 EAEC - Enteroaggregative E. coli *
 DAEC - Diffuse adhering E. coli

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ExPEC
UPEC : uropathogenic E. coli
 MENEC : new born meningitis causing E. coli
 SEPEC : septicemia-causing E. coli
 APEC : avian pathogenic E. coli

Catherine Schouler, june 2006
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Serotyping

Serotyping scheme
based on three types of
antigen :




the somatic (O) antigen
the capsular (K) antigen
the flagellar (H) antigen
Over 700 antigenic types
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E. coli genomes sequences
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K-12 genome sequences
Independent efforts by American and
Japanese groups using two different
strains of K-12 : MG1655 and W3110
 These strains were diverged from the
same ancestral strain about 50 years ago
 Slight but significant differences including
the large inversion involving the ribosomal
RNA genes

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Genome sequencing

First genome of E. coli
that has been
sequenced : MG1655, a
commensal strain in
1997(The complete genome sequence of
Escherichia coli K-12, Science 277 (5331), 1453-
)
Determination of the
complete E. coli
sequence has required
almost 6 years
1474 (1997)

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General features of MG1655
genome
4,639,221-base pair
 4288 protein-coding genes annotated
 Protein-coding genes account for 87.8% of
the genome

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Status of annotation of E. coli gene
products (2006)
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Genome of E. coli sequenced


UPEC strain
Welch et al., 2002,
PNAS
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Genome of E. coli sequenced

EHEC strains

EDL933 (Perna et al.,
2001, Nature)

SaKai (Hayashi et al.,
2001, DNA Research)
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Current E. coli genomes
sequenced or in progress
Strains
type
B03
Number
of genes
commensal 4387
Length (bp) Numbers
of contigs
4,629,810 1
MG1655
commensal 4,254
4,639,675
1
W3110
commensal 4,390
4,641,433
1
HS
commensal 3,689
4,643,538
1
101NA1
EAEC
4,238
4,880,380
70
536
UPEC
~4800
~4,900,000 1
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Current E. coli genomes
sequenced or in progress
Strains
type
ETEC
Number
of genes
4,254
Length (bp) Numbers
of contigs
4,980,187 1
E24377A
E2348
EPEC
4,594
5,072,200
4
RS218
NMEC
~4,900
5,089,235
1
B7A
ETEC
4,637
5,202,558
198
F11
UPEC
4,467
5,206,906
88
H10407
ETEC
~5,000
~5,208,000 225
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Current E. coli genomes
sequenced or in progress
Strains
type
UPEC
Number
of genes
5,379
Length
Numbers
(bp)
of contigs
5,231,428 1
CFT073
042
EAEC
4,899
5,241,977 2
53638
EIEC
4,783
5,289,471 119
B171
EPEC
4,467
5,299,753 159
E110019
Atypical
EPEC
4,746
5,384,084 119
Catherine Schouler, june 2006
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Current E. coli genomes
sequenced or in progress
Strains
type
EHEC
Number
of genes
5,361
Length
Numbers
(bp)
of contigs
5,498,450 1
SAKAI
E22
EPEC
4,788
5,516,160 109
EDL933
EHEC
5,349
5,289,471 1
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Undergoing project :
Coliscope
Commensal : IAI1 (serogroup O8)
 EAEC : 55989
 UPEC : IAI39 (serogroup 07)
 UPEC : UMN026 (serogroup 017)
 Commensal : ED1a (serogroup O81)
 MNEC : S88 (serogroup O45)

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Average genome size
From 4.62 Mb (Commensal isolates)
 To 5.28 Mb (Virulent isolates)

Much of this diversity comes from bacteriophages
BUT :
Presence of foreign DNA : concept of
genomic island (GEIs)
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Genomic islands
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Definition of genomic island

Concept of PAI (pathogenicity island) was
founded in the late 1980s by Jörg Hacker and
colleagues in Werner Goebel's group at the
University of Würzburg, Würzburg, Germany
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Common features of genomic
island





presence of virulence genes
Specific presence in pathogens, absence in benign relatives
Large distinct chromosomal regions (10 to 200 kb)
Characteristic base composition different from core genome
Insertion of PAI adjacent to tRNA genes
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Common features of genomic
island

Frequent association with mobile genetic elements, i.e., presence of:






DR
Cryptic or functional integrase or transposase
IS elements
Chromosomally integrated conjugative transposons, plasmids, and phages
Genetic instability (if functional mobility elements are present)
Mosaic structures of several acquisitions
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GEIs can be involved in
Pathogenicity
 Symbiosis
 Fitness
 Metabolism
 Resistance to xenobiotics

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Comparison between
EHEC and K-12
Common linear backbone of 4.1 Mb
 1.34 Mb specific to EHEC (O-islands)
 0.53 Mb specific to K-12 (K-islands)

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O-islands larger than 15 kb
encoding factors
LEE (locus of enterocyte effacement)
 Macrophage toxin and ClpB-like
chaperone (IAHP island)
 RTX-like exoprotein and a transport
system

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Contribution of mobile genetic elements to
the evolution of pathogenic E. coli
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One of the best studied PAI : The
LEE



LEE : locus of enterocyte effacement
Present in enteropathogenic and
enterohaemmoragic strains
Encode a type III secretory system that injects
proteins into host cells to modulate function.
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A/E lesions caused by EPEC
QuickTime™ et un
décompresseur Vidéo 1 Microsoft
sont requis pour visionner cette image.
Catherine Schouler, june 2006
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A/E lesions caused by EPEC
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décompresseur Vidéo 1 Microsoft
sont requis pour visionner cette image.
Catherine Schouler, june 2006
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A/E lesions caused by EPEC
QuickTime™ et un
décompresseur Vidéo 1 Microsoft
sont requis pour visionner cette image.
Catherine Schouler, june 2006
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The TTSS

Secretion and
translocation of
bacterial effector
proteins into and
through the host cell
membrane
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Identification and
characterization of a genomic
island from an avian ExPEC
strain
tRNA target screening
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Avian ExPEC
Localized infection
 Respiratory diseases
 Some virulence factors identified
 Pathogenicity not fully understood

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tRNA target screening
Design of primers after comparison of 4
completely sequenced strains (MG1655,
CFT073, EDL933 and Sakai)
 PCR analysis
 Focus on some loci to identify the EcDNA
inserted (arbitrary PCR followed by a
screening of a genomic library)

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selC : an integration hot spot
Strain
PAI
Length (kb)
function
E.coli 536 (UPEC)
PAI-I536
70
hemolysin
E.coli CFT073 (UPEC)
GEI
68
E.coli E2348/69
(EPEC)
LEE
35
Type III secretion,
invasion
E.coli O157:H7
(EHEC)
LEE
43
Type III secretion,
invasion
E.coli (ETEC)
Tia PAI
46
invasion
S.flexneri
SHI-2
ND
Aerobactin synthesis,
transport
S.enterica
SPI-3
17
Invasion, survival in
monocytes
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AGI-3
Tnase ISEc8
DR2
(ISEc8) Tnase
(ISEc8)
DR2
DR1
selC
IS2 orfB
IS1 orfA
1 (int)
5
6 7 8
IRL
IRR
c4881
IS911 orfA
10
9
12
11
1314
16
yicL
yicLbis
IS911
delta orf IS1222
IS1 orfB
2 3 4
IS100Tnase
Tnase
IS100
IS2 fA
putative iso IS1
DR1
18 19
22
23
20
15
17
28 32
24 26 29
21
30
nlpA
fin yicM
25
3133
27

49600pb inserted at selC. Mosaic structure : 5
modules of genes

Presence of mobile genetic elements such as IS
elements, integrase gene and direct repeats
Catherine Schouler, june 2006
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Features of aec35 to 37
DR2
DR1
DR2
DR1
selC
33 34 35
36
yicM
81 nlpA
37
yicL

aec35 : 360 aa putative transcriptional regulator of the LacI regulator
family (99% CFT073 C4494, 74% Yersinia pestis C092, Yersinia
pestis Kim, Yersinia pseudotuberculosis)

aec36 : 452 aa putative MFS superfamily hexuronate transporter
(100% CFT073 C4495, 85% Yersinia pseudotuberculosis)

aec37 : 795 aa putative α-glucosidase family 31 (100% CFT073
C4496 and C4497, 83% Yersinia pseudotuberculosis)
Catherine Schouler, june 2006
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Prevalence in other bacteria
strain
Escherichia coli
BEN2908
Escherichia coli
CFT073
Erwinia carotovora
subsp. atroseptica
SCRI1043
Yersinia
pseudotuberculosis
IP 32953
Yersinia pestis biovar
Orientalis CO92
Yersinia pestis biovar
Mediaevalis KIM
Yersinia pestis biovar
Mediaevalis 9100
Burholderia cepacia
R18194
Ralstonia
solanacearum
GMI1000
Similarity % with Aec35, 36 et 37
Aec35
Aec36
Aec37
C4494
99%
C4495
100%
C4496 and C4497
100%
ECA1966
75%
ECA1967
89%
ECA1968
84%
YPTB3091
74%
YPTB3092
85%
YPTB3093
83%
YP00846
74%
Y3231
74%
YP3543
74%
YP00847
85%
Y3232
85%
YP3544
85%
Bcepa03004976
62%
YP00848
83%
Y3233
83%
YP3545
83%
Bcepa03004977
59%
RSc1080
62%
RSc1081
61%
-
-
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Role in virulence for chicken
DR2
DR2
DR1
selC
IRR
52
40
39
50
IRL
47
49
33 34
42 43 44 45 46
48
53
DR1
57
60
76
66
56 59
55
62 63
58
64 65
70
68 69
75
78
74 77
38
yicM
81 nlpA
79
80
kanaR
41
51
54
61
67
71
yicL
72
73



Deletion of 3 orfs : aec35, aec36 and aec37 (5kb) (Datsenko and
Wanner, 2000)
Experimental reproduction of colibacillosis
2 inocula preparations :
 cullture at 37°C with shaking since OD600nm 0.6, frozen at -70°C
 culture at 37°C without neither shaking nor freezing
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Experimental procedure






Inoculation of 5.106 CFU (shaken inoculum) or
107 CFU (non shaken inoculum) to 3 weeks old
SPF chickens (12/set randomly constituted) into
the right thoracic air sac
Blood sampling at 24h and 48h p.i. and bacterial
numeration
48h p.i., euthanasia, necropsy
Lesion score determination
Liver sampling and bacterial numeration
Statistical analysis : Wilcoxon-Mann-Withney
(non parametric test)
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Bacteraemia
24h PI
shaken
non shaken
48h PI
both
shaken
non shaken
both
Log CFU/ml blood
7
6
5
4
3
2
1
0
The mutant derivative is less bacteraemic than the wt strain
Catherine Schouler, june 2006
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Liver colonization
shaken
non shaken
both
7
Log CFU/g of liver
6
5
4
3
2
1
0
The mutant derivative less colonized the liver in comparison to the wt strain
Catherine Schouler, june 2006
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Lesions scores
shaken
non shaken
both
8
7
score/8
6
5
4
3
2
1
0
No lesion score difference
Catherine Schouler, june 2006
50
Conclusion (1)

The Δaec35 to aec37 mutant induces same
lesions as the wt but is less bacteriaemic and is
less able to colonize liver

Involvement of these three orfs in the
pathogenicity of BEN2908 for chickens

Functional analysis of these three orfs
(determination of the sugar involved....)
Catherine Schouler, june 2006
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Phenotypic analysis of aec35,
aec36 and aec37

Reminder

Protein identity




aec35 : putative transcriptional regulator LacI family
aec36 : putative MFS superfamily hexuronate transporter
aec37 : putative glucosidase family 31 of glycosyl hydrolases
Phenotype Microarrays (Biolog Inc.)




minimal media
190 carbon sources
aerobic conditions 37°C
OD590 mesures (ELISA reader)
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Phenotypic micro arrays results
1.8
Metabolisme (DO590 )
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
D-Trehalose
D-Xylose
D-Mannitol
D, L-Malic acid α-D-Glucose
α- D - Lactose
D - Galacturonic negative control
acid
Hydrates de carbone
sauvage
complémenté
mutant
mutant +vecteur de complémentation
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Growth in minimal media



M9 Supplemented by
trehalose or
galacturonate
Growth followed
during 24h in a
Bioscreen apparatus
The mutant has a
two-fold lower growth
rate compared to the
wt strain
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Conclusion (2)
AGI-3 has all the features of a
pathogenicity island
 Is involved in virulence
 Played a role in carbohydrate metabolism

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Tools for E. coli functional
analysis
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E. coli K-12 one of the best-characterized
organisms in molecular biology
 Many key ressources for functional
genomics and systems biology of E. coli
are still lacking

Catherine Schouler, june 2006
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Tools available for K-12 strains
DNA microarrays : transcriptome
(available for K-12, UPEC and
development of patho-arrays)
 The Keio collection mutants : 3985
deletions (in duplicate) of the K-12 strain
strain BW25113
 The ASKA library (A complete Set of E.
coli K-12 ORF Archive)
 Metabolome

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Transcriptome



Transcriptome of K-12
in various growth
conditions
Transcriptome of
uropathogenic
Escherichia coli
during urinary tract
infection
Transcriptome of
carbon utilisation in
the mouse intestine
Catherine Schouler, june 2006
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Proteome




Many in vitro studies
for K-12 strains :
Proteomic analysis of
extracellular proteins
from E. coli W3110
carbon source
variation
Response to
stress……
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Metabolome
Definition : the full complement of
metabolites of an organism
 The E. coli metabolome has been
characterized using the two-dimensional
structures of 745 metabolites, obtained
from the EcoCyc and KEGG databases.

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Conclusion
E. coli is a versatile bacteria
 Many genomes of several E. coli strains
are available
 Tools for functional analysis are developed
 A lot of data are available
 Three worlds exist in the field of E. coli
research (non pathogenic, intestinal
pathogenic and extra intestinal pathogenic
strains)

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