Movement and equipositioning of plasmids by ParA filament disassembly - PubMed (original) (raw)

Movement and equipositioning of plasmids by ParA filament disassembly

Simon Ringgaard et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Bacterial plasmids encode partitioning (par) loci that confer stable plasmid inheritance. We showed previously that, in the presence of ParB and parC encoded by the par2 locus of plasmid pB171, ParA formed cytoskeletal-like structures that dynamically relocated over the nucleoid. Simultaneously, the par2 locus distributed plasmids regularly over the nucleoid. We show here that the dynamic ParA patterns are not simple oscillations. Rather, ParA nucleates and polymerizes in between plasmids. When a ParA assembly reaches a plasmid, the assembly reaction reverses into disassembly. Strikingly, plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution of the plasmids. Mathematical modeling of ParA and plasmid dynamics support these interpretations. Mutational analysis supports a molecular mechanism in which the ParB/parC complex controls ParA filament depolymerization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

par2_-carrying plasmids trail retracting ParA on the nucleoid. (A and B) Intracellular localization of nucleoid (Hoechst stain, green) and plasmids (red) in strain SR1 harboring (A) par_− plasmid pSR230 and (B) par2+ plasmid pSR233. (C) Time lapse showing the subcellular localization of ParA-GFP and par2+ plasmid pSR233 (R1 par2+ p_lac::parA::gfp tetO120) in strain SR1 (KG22Δ_pcnB) also carrying the pBR322-based pSR124 plasmid (pBAD_::tetR::mCherry_). Numbers on the side indicate minutes in the time lapse. (Ca) Overlay of phase-contrast images and TetR-mCherry (red). (Cb) Intracellular localization of ParA-GFP. (Ac) Overlay of ParA-GFP (green) and TetR-mCherry (red). (Cd) 2D deconvolved images of ParA-GFP. (Ce) Overlay of deconvolved ParA-GFP (green) and TetR-mCherry (red). (Cf) Overlay of deconvolved ParA-GFP (green), TetR-mCherry (red), and nucleoid (blue). (Cg) Kymograph of plasmid (red) and maximum intensity of ParA-GFP signal in (Cb). A movie of the time lapse is presented in

Movie S1

. (D) 3D surface intensity plot of the dashed region of minutes 15–29 in (Cc).

Fig. 2.

Fig. 2.

Simulated kymographs and plasmid foci distributions generated by mathematical modeling. (A–C) Simulated kymographs of ParA/focus dynamics for (A) 1, (B) 2, and (C) 3 foci cases, a movie of (C) is presented in

Movie S7

. (D) Simulated kymograph of one focus splitting into 2 and being segregated. (E-G) Simulated foci distributions for (E) 1, (F) 2, and (G) 3 foci cases. For all simulated foci distributions, the distributions were built up over 7,500 min of simulated time, with sampling every 7.5 min, and means and error bars constructed from 40 independent runs. (H) Plasmid travel distance is ParA filament length dependent. (Ha) Schematics showing how the focus travel distance was measured relative to initial ParA filament length. (Hb) The plot shows the focus travel distance, Δ_c_, as function of initial ParA filament length, a. (Hc) The plot shows the ratio between focus travel distance and initial ParA filament length, Δ_c_/a, as function of initial ParA filament length, a.

Fig. 3.

Fig. 3.

Perpetual cycles of ParA assembly/disassembly move and position plasmids. (A–C) Time lapses showing the subcellular localization of ParA-GFP and par2+ plasmids (A and B) in a cephalexin-treated cell. Numbers on the side indicate minutes in the time lapse. Experimental setup as in Fig. 1_C_. Videos of the time lapse in (A–C) are presented in

Movies S4

, S5, and S6, respectively. (Aa, Ba, and Cc) Overlay of phase-contrast images and TetR-mCherry (red). (Ab, Bb, and Cc) Intracellular localization of ParA-GFP. (Ac, Bc, and Cc) Overlay of ParA-GFP (green) and TetR-mCherry (red). (Ad) Nucleoid stained with Hoechst. (Ae) 3D surface intensity plot of (Ac). (Af) Kymograph of plasmid (red) and maximum intensity of ParA-GFP signal (green) in (Ac). Dotted blue lines indicate the average foci positions during the time lapse. (Bd and Cg) Kymograph of plasmid (red) and highest-intensity ParA-GFP signal in (Bb) and (Cb). (C) Focus splitting and segregation by ParA filaments. (Cd) Deconvolved images of ParA-GFP. (Ce) Overlay of deconvolved ParA-GFP (green) and TetR-mCherry (red). (Cf) Overlay of deconvolved ParA-GFP (green) and TetR-mCherry (red) and nucleoid (blue).

Fig. 4.

Fig. 4.

Molecular model showing how plasmid movement is generated by dynamic ParA filaments. See Discussion for a detailed description of the molecular model.

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