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

Study of integron recombination mechanism and Integron use as a genetic shuffling device for biotechnological purpose. David Bikard dbikard@pasteur.fr Didier Mazel’s lab. 29.09.10 – Institut Pasteur. Multiple resistances : why so fast ?.

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

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  1. Study of integron recombination mechanism and Integron use as a genetic shuffling device for biotechnological purpose David Bikard dbikard@pasteur.fr Didier Mazel’s lab 29.09.10 – Institut Pasteur

  2. Multiple resistances : why so fast ? Isolation frequency of multiple resistant Shigella (%) Production of antibiotics in Japan Resistance to 4antibiotics simultaneously Year Mitsuhashi S. et al., Jpn J Exp Med (1961)

  3. Integrons Site attC n Site attC n+1 attC Stable platform Variable cassette array ORF ORF Pc Cassette n+1 attI IntI 2 major types of integrons • Multiresistant integrons • Chromosomal superintegrons Hall R., Stokes HW., Mol. Microbiol. (1989)

  4. 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)

  5. Chromosomal superintegrons 175 cassettes 3% genome Vibrio cholerae O1 N16961 Mazel et al., Science (1998); Mazel, Nat Rev Microbio (2006)

  6. Integron recombination sites The primary recombination site: attI The cassette recombination site: attC Cambray G., Guerout AM, Mazel D., Ann. Rev. Genet. (2010)

  7. The integrase recognizes single stranded attC sites Double stranded substrate Single stranded substrate attC attI IntI IntI IntI IntI Francia MV. et al., J. Bact. (1999)

  8. Conjugation assay Bouvier M., Demarre G., Mazel D., EMBO J. (2005)

  9. The attC recombination site attCaadA7ds attCaadA7bs

  10. The attC site Bouvier M. et al., Plos Genet. (2005)

  11. Unconventional model of cassette Integration ? M. Bouvier, G. Demarre and D. Mazel, EMBO J, (2005)

  12. How and when do attC sites fold ? C. Loot*, D. Bikard*, et al., EMBO J (2010)

  13. VCR derivatives

  14. attC folding probability base1 base2 P UNAFold Software Markham NR, Zuker M, Methods Mol. Biol., 2008 A17

  15. Conjugation assay Bouvier M., Demarre G., Mazel D., EMBO J. (2005)

  16. Conjugation assay (log) C. Loot*, D. Bikard*, et al., EMBO J (2010)

  17. Conjugation assay Replication in the recipient cell _ + Replication can induce recombination

  18. The replication fork

  19. Leading / Lagging strand 10 10 10 • When the bottom strand is on the lagging strand, most recombination events happen during replication C. Loot*, D. Bikard*, et al., EMBO J (2010)

  20. Orientation of chromosomal integrons

  21. Recombination on the leading strand?Cruciforms? Free energy a ~ Ea dGc

  22. Size of the loop

  23. Recombination on the leading strand?Cruciforms? ~ a

  24. Energy landscapes pVCR-GAA pSW97a Kcal/mol Kcal/mol 10 10 20 20 30 40 30 window size (bp) 50 60 40 70 50 80 90 60 position (bp) position (bp) Free energy of cruciform formation window size (bp)

  25. Parasite structures

  26. Analysis of Covariance log(F) = μ + α log(VTSsize) + β log(A17) + ε μ = - 0.29 α = - 0.017 β = 0.48 R2 = 0.825 P-value = 4.7 10-9

  27. Cruciforms formation in vitroS1 nuclease sensitivity C. Loot*, D. Bikard*, et al., EMBO J (2010)

  28. Cruciforms formation in vitroS1 nuclease sensitivity

  29. Mapping the S1 cleavage sites

  30. Cruciform formation in vivo attC attI R6K P15A Pir-

  31. Influence of superhelicity on attC folding -2

  32. 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

  33. ssDNA folding on the leading strand template: sensor of DNA damage ? D. Bikard et al., MMBR (2010)

  34. Synthetic Biology:Engineering life In silico system design DNA synthesis Working system

  35. Engineering approach Standardization Abstraction Decoupling

  36. Engineering approach Standardization Abstraction Decoupling

  37. Bottom-up engineering approach Danino et al., Nature (2010)

  38. Combinatorial approaches Directed evolution MAGE: Wang & al. 2009

  39. Reconstruction of functional tryptophan operons Integrase expression Selection on tryptophan-free medium D. Bikard et al., NAR (2010)

  40. Recombination Frequencies Deletion of “useless” cassettes 3*10-3 D. Bikard et al., NAR (2010)

  41. Recombination Frequencies Reordering event ~10-4

  42. Recombination Frequencies Second reordering event ~10-5

  43. Recombination histories D. Bikard et al., NAR (2010)

  44. Tryptophan production TRP - TRP producer Fluorescence measurement

  45. Combinations Phenotypes D. Bikard et al., NAR (2010)

  46. Plasmid Shuffling

  47. Cassette delivery through conjugation

  48. Protein domain shuffling Polyketide synthetase attC site  good protein linker

  49. attC linker algorithm

  50. attC linker algorithm

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