In memory of

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In memory of John Maynard Smith

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Phenotypic variability is omnipresent in nature
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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
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Darwinian evolution variability, selection,
transmission
Number of copies
time
Adaptives mutations
0
1
2
3
4
5
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Can be applied to any amplifiable  information
(Dawkins, 1976,  the selfish gene )
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Different types of MUTATIONS

• Neutral
• Lethal
• Deleterious
• Adaptives

Estimated total mutation rate for bacteria 1
mutation / 300 genomes replicated An invariant
in evolution of DNA !? (Drake rule)
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Mechanisms controlling the maintenance of
genetic information
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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

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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
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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)

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By-pass polymerases can lead to error free or
error prone (mutagenic) synthesis depending on
the lesion
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Oxidation of guanine lead to transversion
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The Mismatch Repair System
CH3
MutS MutL
GATC-site
MutH
ExoI, ExoVII, RecJ, UvrD, PolIII, SSB, Ligase

• Mismatch repair system
• corrects replication errors
• ensures global genomic stability
• prevent tumour formation

CH3
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The frequencies of mutator among E. coli vary
with the associated pathologies
Denamur J. bacteriol. 2002
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Number of virulence factors correlates with in
vivo virulence
Picard Infect.Immun. 2001
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Mutation rates are higher among strains with
intermediate virulence
Picard Infect.Immun. 2001
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Modelling mutators frequencies during adaptation
to a new environment
Mutator frequency
Time (generations)
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Selecting for mutators is easier in larger
population
Tenaillon Genetics (1999)
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When mutation is rate limiting large population
adapt much faster
log (population size)
Tenaillon Genetics (1999)
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Mutator can speed up adaptation (even when rare)
log (population size)
Tenaillon Genetics (1999)
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An in vivo model an animal with a controled
microbial flora
Kiss me I m germ-free
Giraud
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Evolution of population size
10,2
10,0
9,8
log (population size)
9,6
9,4
mutS
9,2
9,0
8,8
days
0
5
10
15
20
Mutator bacteria adapt faster to a new environment
Giraud Science 2001
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The initial population size influence the
outcome of the competition
5
4
3
2
1
0
Mean log(mutator/wild type)
-1
-2
-3
-4
-5
0
1
2
3
4
5
6
7
8
9
10
Time (Days)
Giraud et al Science 2001
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Le Chat
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The victory is stochastic with a constant
expected gain
Le Chat
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Once adaptation is achieved the mutator advantage
is reduced
3.5
3
2.5
2
1.5
1
Mean log(mutator/wild type)
0.5
0
-0.5
-1
0
1
2
3
4
5
6
7
8
9
10
Time (Days)
Giraud et al Science 2001
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Mutators migration in vivo
The benefit of the mutator is reduced in presence
of migration
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Controlling migration timing in vitro
migration
migration
WT
Mut
V
V
V
V
media LB Spc Mut mutS-
24 h
24 h
0
12
24
15
18
21
hours
Le Chat
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Mutator population adapt faster
mutator
non mutator

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Mutator bacteria suffer from genetic amnesia
30
25
20
Mean of auxotrophs
15
10
mutS- ancestor
5

mutS ancestor
0
100
150
200
250
300
Days post inoculum
Giraud et al Science 2001
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Impact of antibiotic treatments on mutation rates
4 mice
11
10
9
8
7
6
Log (population size)
5
4
3
2
10
15
0
5
20
fos
spc
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streptomycin Nalidixic acid
fos
Day 0 inoculation
spc
time
Measures of population sizes
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Impact of mutation rates on bacterial survival
to antibiotic treatments

str nal
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Giraud AAC (2002)
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Mutator bacteria are more likely to become
antibiotic resistant
Denamur J. bacteriol. 2002
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Non mutator (A) and mutator (B) phenotypes on
antibiograms
Denamur et al J. bacteriol 2002
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Mutators are abundant and more antibiotic
resistant among P. aeruginosa infecting Cystic
Fibrosis patients
Mutator (CF)
Non-mutator (CF)
Non-CF
Oliver Science (2000)
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Resistance accumulate 3 times faster in patients
colonised by mutators
Non mutator
Mutator
Probability of increased resistance
delay (days)
Moumile
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Mutator can speed up cellular evolution
Adaptives mutations
0
1
2
3
4
5
6
Mutator sub-population
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The bacterial Red Queen
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Moumile
Diard
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www.necker.fr/tamara/
Join Fun Science in Paris
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Hyper-recombination phenotypes of mismatch
repair mutants
Denamur Cell (2000)
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Homologous Recombination
A

• exchange of DNA
• 1strands to form
• heteroduplex DNA
• cleavage of Holliday
• junction at A or B
• religation to
• recombinant products
• A splice products
• B patch products

B
B
Holliday junction
A

Splice

Patch
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The barrier to recombination is DNA sequence
divergence
Vulic PNAS (1997)
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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
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Effect of Mismatch Repair System on
Interspecies Recombination
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• 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

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Homeologous Recombination In Vivo
Mosaic Genes
Mosaic Genomes
Mosaic Proteins Mosaic Pathways
A
A
B
B
C
C
C
D
D
D
Novel Products
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The bacterial Red Queen
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Moumile
Diard
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www.necker.fr/tamara/
Join Fun Science in Paris
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Most genes in E. coli genome have a common history
Denamur Cell (2000)
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Phylogenetic trees of mismatch repair genes show
horizontal transfers
Denamur Cell (2000)
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Inferred horizontal transfers in mutU gene
Denamur Cell (2000)
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Inferred horizontal transfers in mutS gene
Denamur Cell (2000)
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Horizontal transfers are more abundant in
mismatch repair genes
Denamur Cell (2000)
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Hyper-recombination phenotypes of mismatch
repair mutants
Denamur Cell (2000)
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Hyper-rec phenotypes of mutator genes correlate
with their sequence mosaicisms
Denamur Cell (2000)
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Mutator bacteria suffer from genetic amnesia
30
25
20
Mean of auxotrophs
15
10
mutS- ancestor
5

mutS ancestor
0
100
150
200
250
300
Days post inoculum
Giraud et al Science 2001
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Role of mutator in adaptive evolution
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The bacterial Red Queen
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Moumile
Diard
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www.necker.fr/tamara/
Join Fun Science in Paris
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How to adapt to predictable impredictability ?
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Localized mutators
x
y
x
x
y
x
x
x
Rocha Nucleic Acid Research (2002)
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Close direct repeats
Observed

• ObservedExpected 1.9
• Over-represented classes
• Recombination, repair
• Transcription, RNA degradation
• Translation
• Transport proteins

Observed in 1000 random sequences of equal
length and 3-tuple composition
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Close direct repeats
Stress response genes
All E. coli K12 genes
Rocha NAR (2002)
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Rocha NAR (2002)