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In memory of
John Maynard Smith
Phenotypic variability is omnipresent in nature
It takes all the running you can do to keep in the same place
If you want to get somewhere else, you must run at least twice as fast
Lewis Carroll, 1871
environmentally
induced adaptation
intraspecific
variability
•
A
•B
•A
•B
•A
•B
•
•B
Lamarckian Paradigm
natural
selection
natural
selection
•
A
•
B
•
A
•
B
Darwinian Paradigm
A
Number of copies
Darwinian evolution :
variability, selection, transmission
time
Adaptives mutations :
0
1
2
3
Can be applied to any «amplifiable information»
(Dawkins, 1976, « the selfish gene »)
4
5
6
Different types of MUTATIONS
wildtype
Mutator
 Neutral
mutS+
mutS-
 Lethal
10 -5
10 -3
 Deleterious
10 -4
10 -2
 Adaptives
10 -8
10 -6
Estimated total mutation rate for bacteria
1 mutation / 300 genomes replicated
An invariant in evolution of DNA !? (Drake rule)
Mechanisms controlling the maintenance
of genetic information
nucleotide pool
post-replication control
Fidelity of synthesis
DNA repair
Photoactivation
Repair in E. coli
Exposing UV treated cells to
blue light results in a reversal of
the thymine dimer formation
Enzyme, photoactivation repair
enzyme (PRE) absorbs a photon
of light (from blue light) and is
able to cleave the bond forming
the thymine dimer.
Once bond is cleaved, DNA is
back to normal
Excision Repair
Like other repair system
It is conserved throughout
evolution, conserved from
bacteria (where first discovered)
to man where they are involved
in a variety of disease
Xeroderma Pigmentosum &
Nucleotide Excision Repair
• Xeroderma pigmentosum (XP)- is a rare genetic disorder
that predisposes an individual to skin abnormalities
– Individuals lose the ability to undergo NER
• UV radiation exposure leads to reactions from freckling and skin
ulceration to skin cancer
– Studies suggest many different genes may be involved in excision
repair
– XP-variant is encoding a lesion by-pass DNA polymerase (SOS)
By-pass polymerases
can lead to error free or
error prone (mutagenic)
synthesis depending on
the lesion
Oxidation of guanine lead to transversion
The Mismatch Repair System
CH3
MutS
MutL
GATC-site
MutH
Mismatch site
ExoI, ExoVII, RecJ, UvrD,
PolIII, SSB, Ligase
Mismatch repair system
• corrects replication errors
• ensures global genomic stability
• prevent tumour formation
CH3
1E-5
mutL-
mutSmutS-
Commensals Croatia
Commensals Mali
Commensals France
mutS-
1E-6
mutL-
Frequency of mutations to Rif
R
in mutator strains
High polymorphism of mutation rates in commensal and pathogenic
Escherichia coli natural isolates
haemolytic-uremic
syndrome
neonatal meningitis
pus
bacteremia
UTI
1E-7
1E-8
1E-9
0
5
10
15
20
25
30 35 40 45
Strain number
50
55
60
65
70
I. Matic, M. Radman, F. Taddei, B. Picard, C. Doit, E. Bingen, E. Denamur and J. Elion
Science (1997) vol. 277 p. 1833
The frequencies of mutator
among E. coli vary
with the associated pathologies
Denamur J. bacteriol. 2002
Number of virulence factors correlates with in vivo virulence
only in non-mutator strains
Picard Infect.Immun. 2001
Mutation rates are higher
among strains with
intermediate virulence
Picard Infect.Immun. 2001
Role of mutators during adaptation to a new environment
mutator
wildtype
fitness
mutator
wildtype
adaptative landscape
genotype
Mutator frequency
Modelling mutators frequencies during adaptation to a new environment
Time (generations)
Selecting for mutators is easier in larger population
Tenaillon Genetics (1999)
When mutation is rate limiting large population adapt much faster
Tenaillon Genetics (1999)
log (population size)
Mutator can speed up adaptation (even when rare)
Tenaillon Genetics (1999)
log (population size)
An in vivo model:
an animal with a controled microbial flora
Kiss me
I ’m germfree
Giraud
mutS-
mutS+
log (population size)
Evolution of population size
10,2
mutS-
10,0
9,8
9,6
mutS+
9,4
9,2
9,0
8,8
0
5
10 15 20
days
Mutator bacteria adapt faster to a new environment
Giraud Science 2001
The initial population size influence
the outcome of the competition
5
4
Mean log(mutator/wild type)
3
2
1
mutS-/mutS+
0
-1
mutS-/ 50 mutS+
-2
mutS-/ 50 000 mutS+
-3
-4
-5
0
1
Giraud et al Science 2001
2
3 4 5 6
Time (Days)
7
8
9 10
The population threshold for mutator victory
is 1/(mutation rate)
Le Chat
Mutator victory threshold is frequency independent
The victory is stochastic with a constant expected gain
Le Chat
Once adaptation is achieved the mutator
advantage is reduced
3.5
Mean log(mutator/wild type)
3
2.5
2
1.5
1
0.5
0
-0.5
-1
0 1 2 3 4 5 6 7 8 9 10
Time (Days)
Giraud et al Science 2001
Naive
adapted
&reduced
migration
in vivo of migration
The benefit of theMutators
mutator is
in presence
WT+Mut
Mut
WT
3
2
1
0
Days
-1
0 2 4 6 8 10
Log(mutS-/ mutS+)
Log(mutS-/ mutS+)
3
2
1
0
Days
-1
0 2 4 6 8 10
Controlling migration timing in vitro
WT
Mut
migration
V
migration
V
V
V
media: LB + Spc
Mut : mutS-
0
Le Chat
24 h
12
24 h
15
18
21
24
hours
Mutator population adapt faster
10
9,5
9
8,5
8
7,5
7
6,5
6
5,5
5
mutator
non
mutator
3
log (mutator/WT)
log (CFU)
"migration"
2,5
2
1,5
1
0,5
0
-0,5
9
12 15 18 21 24
9 12 15 18 21 24
The benefit of the mutator disappears
if adaptation is over before migration
Mean % of auxotrophs
Mutator bacteria suffer from genetic amnesia
30
25
20
15
10
5
0
mutS- ancestor
mutS+ ancestor
nd
nd
100
150
200
250
Days post inoculum
Giraud et al Science 2001
300
non mutator
Emerging
mutator
Log (population size)
Impact of antibiotic treatments
on mutation rates
4 mice
11
10
9
8
7
6
5
4
3
2
0
5
10 15 20
fos spc
1 mouse
11
10
9
8
7
6
5
4
3
2
0
5
10 15
fos spc
11
10
9
8
7
6
5
4
3
2
20 0
1 mouse
5
10
15 20
fos spc
population Rif R
1 mouse
11
10
9
8
7
6
5
4
3
2
0
5
11
10
9
8
7
6
5
4
3
2
10 15 20 0
fos spc
Day 0 : inoculation
1 mouse
5
10 15 20
fos spc
fos
spc
streptomycin
Nalidixic acid
time
Measures of population sizes
Impact of mutation rates on
bacterial survival to antibiotic treatments
Log ( population size)
11
10
9
8
7
6
5
mutator
4
3
2
non mutator
0
5
str + nal
10
H ow many antibiotics should be used against mutator bacteria ?
Percentage of treatment failure
Number of antibiotics administered simultaneously
Ancestral genotype
1
2
3
Antimutator (mutS+)
100
0 (+17*)
n.d.
70
0
Mutator (mutS-)
*emerging mutator (2 mutS- )
Giraud AAC (2002)
n.d.
Mutator bacteria are
more likely to become
antibiotic resistant
Denamur J. bacteriol. 2002
Non mutator (A) and mutator (B) phenotypes on antibiograms
Denamur et al J. bacteriol 2002
Mutators are abundant and more antibiotic resistant
among P. aeruginosa infecting Cystic Fibrosis patients
Mutator (CF)
Non-mutator (CF)
Non-CF
Oliver Science (2000)
Probability of increased resistance
Resistance accumulate 3 times faster in patients colonised by mutators
Moumile
Mutator
Non mutator
delay (days)
Cell number
Cell number
Mutator can speed up cellular evolution
time
Adaptives mutations
0 1 2 3 4 5 6
Mutator sub-population
time
The bacterial Red Queen
A network approach of bacterial variability
Why change ?
Population genetics
Who changes ?
Molecular epidemiology
Godelle Gouyon Brown Maynard-Smith
Binguen Denamur Picard Brisabois Berche
B. Toupance
O. Tenaillon
J-B André
Change what?
Bio-informatics
Rocha
Change where ?
Microbial ecology
Fons
Duriez
Moumile
How to change ?
Molecular biology
Who has changed ?
Molecular Phylogeny
Matic Radman Vulic Dionisio Bjedov
Bregeon Leroy Hayakawa Sekiguchi Dukan
Lecointre Darlu
Giraud
Lechat
Bambou
Change when ?
transcriptome analysis
Knudsen Cerf
Phenotypic variability
Life History
Stewart Madden Lindner
Paul Gabriel Fontaine
Depaepe Bredèche Mosser
Diard
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Hyper-recombination
phenotypes of
mismatch repair mutants
Denamur Cell (2000)
Homologous Recombination
• exchange of DNA
1strands to form
heteroduplex DNA
A
B
B
• cleavage of Holliday
junction at A or B
• religation to
recombinant products
A
Holliday junction
+
Splice
A: splice products
B: patch products
+
Patch
The barrier to recombination is DNA sequence divergence
Vulic PNAS (1997)
Homeologous Recombination
mismatch -
mismatch +
• divergent sequences
do not recombine efficiently
• mismatch repair prevents
formation of recombination
intermediates
• in mismatch repair deficient
background homeologous
recombination proceeds to
generate mosaic genes and
genomes
Holliday junction
+
Splice
+
Patch
0
1.5
-1
recAo98 mutS
0.5
-2
-0.5
-3
-1.5
-4
wild type
-2.5
-5
-3.5
-6
lexA3 pmutS,L
-4.5
-7
-5.5
0
10
20
30
40
50
Genomic DNA sequence divergence
60
log (no of mismatch-free blocks/ 4 kb)
log (frequency of recombination)
Effect of Mismatch Repair System on Interspecies
Recombination
Inhibition of Mismatch Repair System
• increases homeologous recombination to the level of
homologous recombination and thus allows
interspecies recombination
• allows broadest genetic variability in vivo
• broad area of applications
• generation of novel low molecular weight entities
• generation of modified and optimised macromolecules
• generation of (micro)organisms with desired properties
Homeologous Recombination In Vivo
Mosaic Genes
Mosaic Genomes
Mosaic Proteins
Mosaic Pathways
A
A´
B
B´
C
D
Novel Products
C´´
D´´
C´
D´
The bacterial Red Queen
A network approach of bacterial variability
Why change ?
Population genetics
Who changes ?
Molecular epidemiology
Godelle Gouyon Brown Maynard-Smith
Binguen Denamur Picard Brisabois Berche
B. Toupance
O. Tenaillon
J-B André
Change what?
Bio-informatics
Rocha
Change where ?
Microbial ecology
Fons
Duriez
Moumile
How to change ?
Molecular biology
Who has changed ?
Molecular Phylogeny
Matic Radman Vulic Dionisio Bjedov
Bregeon Leroy Hayakawa Sekiguchi Dukan
Lecointre Darlu
Giraud
Lechat
Bambou
Change when ?
transcriptome analysis
Knudsen Cerf
Phenotypic variability
Life History
Stewart Madden Lindner
Paul Gabriel Fontaine
Depaepe Bredèche Mosser
Diard
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Most genes in E. coli genome have a common history
Denamur Cell (2000)
Phylogenetic trees of mismatch repair genes show horizontal transfers
Denamur Cell (2000)
Inferred horizontal transfers in mutU gene
Denamur Cell (2000)
Inferred horizontal transfers in mutS gene
Denamur Cell (2000)
Horizontal transfers are more abundant in mismatch repair genes
Denamur Cell (2000)
Hyper-recombination
phenotypes of
mismatch repair mutants
Denamur Cell (2000)
Hyper-rec phenotypes of mutator genes correlate with their sequence mosaicisms
Denamur Cell (2000)
Mean % of auxotrophs
Mutator bacteria suffer from genetic amnesia
30
25
20
15
10
5
0
mutS- ancestor
mutS+ ancestor
nd
nd
100
150
200
250
Days post inoculum
Giraud et al Science 2001
300
non mutator
Emerging
mutator
Role of mutator in adaptive evolution
The bacterial Red Queen
A network approach of bacterial variability
Why change ?
Population genetics
Who changes ?
Molecular epidemiology
Godelle Gouyon Brown Maynard-Smith
Binguen Denamur Picard Brisabois Berche
B. Toupance
O. Tenaillon
J-B André
Change what?
Bio-informatics
Rocha
Change where ?
Microbial ecology
Fons
Duriez
Moumile
How to change ?
Molecular biology
Who has changed ?
Molecular Phylogeny
Matic Radman Vulic Dionisio Bjedov
Bregeon Leroy Hayakawa Sekiguchi Dukan
Lecointre Darlu
Giraud
Lechat
Bambou
Change when ?
transcriptome analysis
Knudsen Cerf
Phenotypic variability
Life History
Stewart Madden Lindner
Paul Gabriel Fontaine
Depaepe Bredèche Mosser
Diard
Quick Time™ et un décompresseur
Photo - JPEG sont requis pour visualiser
cette image.
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How to adapt to
predictable impredictability ?
Localized mutators
x
y
x
x
x
x
Rocha Nucleic Acid Research (2002)
x
y
Close direct repeats
Observed
Observed in 1000
random sequences
of equal length and
3-tuple composition
ObservedExpected = 1.9
Over-represented classes:
• Recombination, repair
• Transcription, RNA degradation
• Translation
• Transport proteins
Close direct repeats
Stress response
genes
All E. coli K12
genes
Rocha NAR (2002)
Rocha NAR (2002)