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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! • 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) MinCDE Oscillations • MinE stimulates coherent pole to pole oscillations of MinCDE • Centre of cell marked by minimum MinC/MinD concentration MinD Oscillations Hale, Meinhardt, de Boer: EMBO J. 20 1563 (2001) MinCDE Oscillations • Formation of oscillating MinE ring structure Fu, Shih, Zhang, Rothfield: PNAS 98 980 (2001) • Centre of cell marked by minimum MinC/MinD concentration MinE Oscillations Hale, Meinhardt, de Boer: EMBO J. 20 1563 (2001) MinD in Filamentous Cells Raskin & de Boer: PNAS (1999) • Induce filamentous cells by deleting FtsZ • Clear evidence for characteristic wavelength 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 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 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) 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 and parC • Dynamics of plasmids/Par proteins occurs along nucleoid surface 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? Par and Min Oscillations Arise From Different Mechanisms • Position of plasmid foci seem to depend on the number of plasmids Niki et al: Mol. Microbiol. (2007) • 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 Ebersbach et al: Mol. Microbiol. (2006) 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: red ParA: green 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