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How does the ParABC system segregate low copy number plasmids in bacteria?

How does the ParABC system segregate low copy number plasmids in bacteria?. Martin Howard. Dept of Systems Biology. John Innes Centre. Norwich, UK. Patterning in Bacteria. Traditional view of bacterial organization: “randomly filled bag” But this isn’t true at all!

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How does the ParABC system segregate low copy number plasmids in bacteria?

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  1. How does the ParABC system segregate low copy number plasmids in bacteria? Martin Howard Dept of Systems Biology John Innes Centre Norwich, UK

  2. Patterning in Bacteria • Traditional view of bacterial organization: • “randomly filled bag” • But this isn’t true at all! • Many processes where proteins are precisely localized in space and time • Several key examples have emerged, including: • Chemotaxis (polar localization of chemotactic receptors) • Min system (how to divide accurately) • How is precise positioning achieved? • Self-organization:Howard & Kruse, J. Cell Biol. 168 533 (2005)

  3. MinCDE Oscillations MinD Oscillations Hale, Meinhardt, de Boer: EMBO J. 20 1563 (2001) • MinE stimulates coherent pole to pole oscillations of MinCDE • Centre of cell marked by minimum MinC/MinD concentration

  4. MinCDE Oscillations MinE Oscillations Hale, Meinhardt, de Boer: EMBO J. 20 1563 (2001) • Formation of oscillating MinE ring structure Fu, Shih, Zhang, Rothfield: PNAS 98 980 (2001) • Centre of cell marked by minimum MinC/MinD concentration

  5. MinD in Filamentous Cells • Raskin & de Boer: PNAS (1999) • Induce filamentous cells by deleting FtsZ • Clear evidence for characteristic wavelength

  6. Modelling the Min System • Many mathematical models of the Min system • Howard et al: Phys. Rev. Lett. (2001) Meinhardt et al: PNAS (2001) Kruse: Biophys J. (2002) • All have common basis: oscillations result from a dynamical instability resulting from intrinsic interactions of Min proteins • Howard & Kruse: JCB (2005) Kruse, Howard & Margolin: Mol. Microbiol. (2007) • Models can explain characteristic wavelength of Min patterning important in identifying underlying mechanism

  7. Introduction to Plasmids • DNA not on main chromosomes that can replicate independently • Sometimes present at very low copy numbers • Causes a problem at cell division: how to ensure plasmid are transmitted reliably to both daughter cells? • Importance: • Encode important functions e.g. antibiotic resistance or virulence • Method of segregation prior to cell division is a primitive ancestor of mitotic apparatus in eukaryotes

  8. Par Dynamics and Plasmid Segregation • How are low copy number plasmids segregated in bacteria? • Often through oscillatory dynamics of ParABC system • Without oscillations plasmids are not segregated • Somehow Par oscillations generate force that moves plasmids! • What is the mechanism behind the oscillations? ParA dynamics in E. coli plasmid pB171 Ebersbach & Gerdes: Mol. Microbiol. (2004)

  9. The ParABC System • ParA: ATPase that binds nonspecifically to DNA • polymerizes on nucleoid surface • ParB: protein that binds to DNA at parC regions • together form “partition complex” • ParB interacts with ParA functioning as “adaptor” between ParA andparC • Dynamics of plasmids/Par proteins occurs along nucleoid surface

  10. Par and Min: Similarities • ParA/MinD both form polymers • ParA/MinD are both ATPases • ParA/MinD both undergo spatiotemporal oscillations, though in difference locations (nucleoid vs membrane) • ParA oscillations require ParB and parC centromere-like site • MinD oscillations require MinE • ParB/MinE stimulate ATPase activity of ParA/MinD • Are Par dynamics also driven by reaction-diffusion dynamic instability? As in Adachi et al: JMB (2006) • Is there a characteristic wavelength for Par oscillations?

  11. Par and Min Oscillations Arise From Different Mechanisms • Position of plasmid foci seem to depend on the number of plasmids • For Min-like dynamic instability, number of foci would depend on the length of the nucleoid • Mechanism of oscillation could be fundamentally different in Par vs Min Niki et al: Mol. Microbiol. (2007) Ebersbach et al: Mol. Microbiol. (2006)

  12. Pushing or Pulling? • Do ParA filaments push (similar to ParM) or pull? • No evidence that plasmids directly nucleate ParA • How are plasmids close together separated? • More likely to be by pulling Plasmid: redParA: green

  13. Conclusions • Many similarities between Min and Par • Nevertheless, new mechanism may be needed for Par dynamics • Pulling force generated by depolymerisation of ParA filaments by ParB/parC

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