david bikard [email protected] didier mazel’s lab
DESCRIPTION
Study of integron recombination mechanism and Integron use as a genetic shuffling device for biotechnological purpose. David Bikard [email protected] Didier Mazel’s lab. 29.09.10 – Institut Pasteur. Multiple resistances : why so fast ?. - PowerPoint PPT PresentationTRANSCRIPT
David [email protected]
Didier Mazel’s lab
29.09.10 – Institut Pasteur
Study of integron recombination mechanism
and
Integron use as a genetic shuffling device for biotechnological purpose
Introduction attC site folding The Synthetic Integron Conclusion
Multiple resistances : why so fast ?
Mitsuhashi S. et al., Jpn J Exp Med (1961)
Iso
lati
on
fre
qu
ency
of
mu
ltip
le r
esi
sta
nt
Sh
igel
la
(%)
Year
Pro
du
ction
of an
tibio
tics in Jap
an
Resistance to 4 antibiotics simultaneously
Introduction attC site folding The Synthetic Integron Conclusion
Variable cassette arrayStable platform
Integrons
IntI
attI
Pc
Site attC n Site attC n+1
ORF
Cassette n+1 ORF
attC 2 major types of integrons• Multiresistant integrons• Chromosomal superintegrons
Hall R., Stokes HW., Mol. Microbiol. (1989)
Introduction attC site folding The Synthetic Integron Conclusion
Multiresistant integrons
Bear antibiotic resistances (>130) Small : up to 8 cassettes Mobile : located on transposons and plasmids
Partridge et al., JR FEMS Microbiol Rev (2009)
Introduction attC site folding The Synthetic Integron Conclusion
175 cassettes 3% genome
Chromosomal superintegrons
Mazel et al., Science (1998); Mazel, Nat Rev Microbio (2006)
Vibrio cholerae O1 N16961
Introduction attC site folding The Synthetic Integron Conclusion
Integron recombination sites
The primary recombination site: attI
The cassette recombination site: attC
Cambray G., Guerout AM, Mazel D., Ann. Rev. Genet. (2010)
Introduction attC site folding The Synthetic Integron Conclusion
The integrase recognizes single stranded attC sites
Double stranded substrate Single stranded substrate
Francia MV. et al., J. Bact. (1999)
attC attI
IntI IntI IntI IntI
Introduction attC site folding The Synthetic Integron Conclusion
Conjugation assay
Bouvier M., Demarre G., Mazel D., EMBO J. (2005)
Introduction attC site folding The Synthetic Integron Conclusion
The attC recombination site
attCaadA7bs
attCaadA7ds
Introduction attC site folding The Synthetic Integron Conclusion
The attC site
Bouvier M. et al., Plos Genet. (2005)
Introduction attC site folding The Synthetic Integron Conclusion
Unconventional model of cassette Integration
?
M. Bouvier, G. Demarre and D. Mazel, EMBO J, (2005)
Introduction attC site folding The Synthetic Integron Conclusion
How and when do attC sites fold ?
C. Loot*, D. Bikard*, et al., EMBO J (2010)
Introduction attC site folding The Synthetic Integron Conclusion
VCR derivatives
Introduction attC site folding The Synthetic Integron Conclusion
attC folding probability
base1 base2 P
UNAFold Software
Markham NR, Zuker M, Methods Mol. Biol., 2008
A17
Introduction attC site folding The Synthetic Integron Conclusion
Conjugation assay
Bouvier M., Demarre G., Mazel D., EMBO J. (2005)
Introduction attC site folding The Synthetic Integron Conclusion
Conjugation assay
C. Loot*, D. Bikard*, et al., EMBO J (2010)
(log)
Introduction attC site folding The Synthetic Integron Conclusion
Conjugation assay
Replication can induce recombination
Replication in the recipient cell+ _
Introduction attC site folding The Synthetic Integron Conclusion
The replication fork
Introduction attC site folding The Synthetic Integron Conclusion
Leading / Lagging strand
When the bottom strand is on the lagging strand, most recombination events happen during replication
10
10
10
C. Loot*, D. Bikard*, et al., EMBO J (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Orientation of chromosomal integrons
Introduction attC site folding The Synthetic Integron Conclusion
Recombination on the leading strand?Cruciforms?
dGc
Free energy
Ea
~a
Introduction attC site folding The Synthetic Integron Conclusion
Size of the loop
Introduction attC site folding The Synthetic Integron Conclusion
Recombination on the leading strand?Cruciforms?
~a
Introduction attC site folding The Synthetic Integron Conclusion
pVCR-GAA
position (bp)
win
dow
siz
e (b
p)
Kcal/mol10
20
30
40
50
60
10
pSW97a
position (bp)
Kcal/mol
20
30
40
50
60
70
80
90
Energy landscapes
Free energy of cruciform formation
win
dow
siz
e (b
p)
Introduction attC site folding The Synthetic Integron Conclusion
Parasite structures
Introduction attC site folding The Synthetic Integron Conclusion
Analysis of Covariance
log(F) = μ + α log(VTSsize) + β log(A17) + ε
μ = - 0.29
α = - 0.017
β = 0.48
R2 = 0.825P-value = 4.7 10-9
Introduction attC site folding The Synthetic Integron Conclusion
Cruciforms formation in vitroS1 nuclease sensitivity
C. Loot*, D. Bikard*, et al., EMBO J (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Cruciforms formation in vitroS1 nuclease sensitivity
Introduction attC site folding The Synthetic Integron Conclusion
Mapping the S1 cleavage sites
Introduction attC site folding The Synthetic Integron Conclusion
Cruciform formation in vivo
Pir-
attC
attIR6K
P15A
Introduction attC site folding The Synthetic Integron Conclusion
Influence of superhelicity on attC folding
-2
Introduction attC site folding The Synthetic Integron Conclusion
Replication / Cruciforms
Replication is the “easiest” way to fold
Natural chromosomal integrons are on the “leading strand template”
attC sites can recombine as cruciforms,
Cruciforms fold more frequently than expected
Introduction attC site folding The Synthetic Integron Conclusion
ssDNA folding on the leading strand template: sensor of DNA damage ?
D. Bikard et al., MMBR (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Synthetic Biology:Engineering life
In silico system design
DNA synthesis Working system
Introduction attC site folding The Synthetic Integron Conclusion
Engineering approach
• Standardization• Abstraction • Decoupling
Introduction attC site folding The Synthetic Integron Conclusion
Engineering approach
• Standardization• Abstraction • Decoupling
Introduction attC site folding The Synthetic Integron Conclusion
Bottom-up engineering approach
Danino et al., Nature (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Combinatorial approaches Directed evolution
MAGE: Wang & al. 2009
Introduction attC site folding The Synthetic Integron Conclusion
Reconstruction of functional tryptophan operons
Integrase
expression
Selection on
tryptophan-free medium
D. Bikard et al., NAR (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Recombination Frequencies
Deletion of “useless” cassettes
3*10-3
D. Bikard et al., NAR (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Recombination Frequencies
Reordering event
~10-4
Introduction attC site folding The Synthetic Integron Conclusion
Recombination Frequencies
Second reordering event
~10-5
Introduction attC site folding The Synthetic Integron Conclusion
Recombination histories
D. Bikard et al., NAR (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Tryptophan production
TRP producer
TRP -
Fluorescence measurement
Introduction attC site folding The Synthetic Integron Conclusion
Combinations Phenotypes
D. Bikard et al., NAR (2010)
Introduction attC site folding The Synthetic Integron Conclusion
Plasmid Shuffling
Introduction attC site folding The Synthetic Integron Conclusion
Cassette delivery through conjugation
Introduction attC site folding The Synthetic Integron Conclusion
Protein domain shuffling
Polyketide synthetase
attC site good protein linker
Introduction attC site folding The Synthetic Integron Conclusion
attC linker algorithm
Introduction attC site folding The Synthetic Integron Conclusion
attC linker algorithm
Introduction attC site folding The Synthetic Integron Conclusion
Perspectives
Potential applications in the generation of: random regulatory networks, new metabolic pathways…
Easy genome engineering by cassette delivery through conjugation
Custom attC sites performing various cellular functions: promoters, transcriptional terminators…
Introduction attC site folding The Synthetic Integron Conclusion
General Conclusion
attC site folding:• during replication on the lagging strand template• as cruciforms• During repair? Sensors of stress?• Importance of parasite structures in natural attC sites?• Design of attC sites with predictable recombination
frequencies
First demonstration that site-specific recombination can be used to generate large number of combinations in vivo
Introduction attC site folding The Synthetic Integron Conclusion
Thank you for your attention !
All the Bacterial Genome Plasticity team
The BIG boss : Didier Mazel
All those whom encouraged me…
Acknowledgements
Introduction attC site folding The Synthetic Integron Conclusion
Introduction attC site folding The Synthetic Integron Conclusion
The discovery of integrons
Transposons on plasmid
Recombinase & ORFs
First ORFs are highly variables
Transposition genes mer genes
Tn21
Multi-Resistant Integron (MRI)
IRi IRt
Introduction attC site folding The Synthetic Integron Conclusion
Cassette functions
Boucher & al., Trends in microbiology (2007)
Introduction attC site folding The Synthetic Integron Conclusion
Introduction attC site folding The Synthetic Integron Conclusion
High-throughput screening
Introduction attC site folding The Synthetic Integron Conclusion
Folding proba distribution
Introduction attC site folding The Synthetic Integron Conclusion
Influence on recombination?
0 50 100 150
-5-4
-3-2
-1
Index
attC
s[or
g ==
leve
ls(o
rgf)[
22],
"pro
ba"]
Cassette n°
Pro
babi
lity
to f
old
prop
erly
V.cholerae El Tor cassette distribution
Introduction attC site folding The Synthetic Integron Conclusion
ssDNA, stress and genome plasticity
Introduction attC site folding The Synthetic Integron Conclusion
Cruciform formation pathways
Introduction attC site folding The Synthetic Integron Conclusion
Introduction attC site folding The Synthetic Integron Conclusion