David Bikard dbikard@pasteur.fr Didier Mazel’s lab

David Bikarddbikard@pasteur.fr

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