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Comparative Study
, 24 (7), 1453-64

Bacterial DNA Segregation Dynamics Mediated by the Polymerizing Protein ParF

Affiliations
Comparative Study

Bacterial DNA Segregation Dynamics Mediated by the Polymerizing Protein ParF

Daniela Barillà et al. EMBO J.

Abstract

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.

Figures

Figure 1
Figure 1
ParF harbours a deviant Walker A motif and exhibits ATPase activity. (A) Schematic representation of the ParF protein displaying the Walker A and B motifs with an alignment of Walker A boxes of ParF and other ParA superfamily members. Amino-acid residues subjected to mutagenesis are numbered and indicated by asterisks. (B, C) ATP hydrolysis plotted as a function of high (B) and low (C) protein concentration with ATP at 250 μM. (D, E) ATPase experiments performed with proteins (4 μM) at 50–1000 μM (D) and 0.05–1.0 μM (E) ATP concentration.
Figure 2
Figure 2
ParF polymerizes and mutations in the Walker A box perturb the polymerization profile. ParF (A), ParFK15Q (B) and ParFG11V (C) were incubated in the absence (−) or presence of nucleotides and the reactions were then centrifuged. In all, 100 and 33%, respectively, of the pellet (P) and supernatant (S) fractions were resolved on a 12% SDS gel and stained with Coomassie blue. The percentages of ParF protein detected in the pellet fractions are shown. (D) ATP stimulates ParF assembly into polymeric structures. ParF polymerization was detected by DLS. The bottom panels illustrate the increase in light scattering intensity, expressed as kct/s, upon nucleotide addition (arrow). The top panels show the corresponding augmentation in polymer average size (nm). Black, no nucleotide added; green, ADP (500 μM); blue, ATP (500 μM); red, ATPγS (500 μM). (E, F) ParFK15Q and ParFG11V analysed as described for ParF in panel D. Note the difference in vertical scale in the three panels.
Figure 3
Figure 3
Critical concentration for ParF polymerization. Different protein concentrations were analysed by sedimentation assays at 14 000 r.p.m. (diamonds) or 50 000 r.p.m. (squares) and the amount of ParF in the pellet fractions was plotted against total ParF in the reaction. Extrapolating the Y value to 0 gives the critical concentration for ParF assembly into polymers.
Figure 4
Figure 4
Ultrastructure of ParF filaments observed by EM. (A) ParF was examined before and after addition of ATP (2 mM) at the indicated time points. In the ‘no ATP' panel, arrowheads point to globular structures likely to be nucleation seeds (bar=100 nm). In the following panels, the scale bar is 500 nm. (B) Higher magnification, reverse contrast image highlighting details of ParF fibres. Bar=100 nm.
Figure 5
Figure 5
ParFG11V exhibits a perturbed polymerization pattern. (A) EM time course of ParFG11V polymerization upon addition of ATP. Arrowheads indicate short growing projections. Bar=500 nm. (B) Higher magnification image of another field of the ParFG11V grid revealing particularly elongated filaments. Bar=1 μM. (C) Details of the intricate meshwork of highly interlaced filaments produced by ParFG11V after 20 min exposure to ATP. Bar=500 nm.
Figure 6
Figure 6
ParG stimulates ParF ATPase activity. (A) Levels of ATP hydrolysis driven by ParF, ParFG11V and ParFK15Q as a function of ParG concentration. ParF proteins were used at 0.5 μM. Diamonds, ParF; squares, ParFK15Q; triangles, ParFG11V. (B) Stimulation of ATP hydrolysis by ParF as a function of ParG concentration, using 5 μM ParF. The inset shows an expanded version of the early points of the curve. (C) Effect of DNA on the ATPase activity of ParF in the presence of ParG. The ParF protein was used at 0.5 μM, ATP at 50 nM and DNA at 500 ng per reaction. Filled circles, ParF+ParG; open circles, ParF+ParG+partition DNA; squares, ParF+ParG+non-partition DNA. The partition DNA was a PCR fragment containing the 259 bp upstream of parF start codon and the non-partition DNA was a similarly sized fragment comprising rna-15 Saccharomyces cerevisiae gene.
Figure 7
Figure 7
ParG has a stabilizing/remodelling role in filament bundling. (A) ParG cosediments with ParF in the pellet fraction. ParF (5.8 μM) was incubated with ParG (7.17 μM, dimer) either in the absence or presence of nucleotides and the reactions processed and analysed on a 15% SDS gel. (B) ParG partially rescues ParFG11V polymerization as detected by sedimentation assay. ParFG11V (7 μM) and ParG (7 μM) were coincubated and processed as in (A). (C) ParG rejuvenates ParFK15Q polymerization behaviour. ParFK15Q (3.62 μM) was mixed with ParG (3.62 μM, dimer) and the reactions processed as in (A). (D) ParG ratio-dependent modulation of ParF polymerization. ParF (10 μM) and ParG (varying molarity) were mixed in different ratios in the presence of ATP (100 μM) and the reactions processed and analysed as described in (A). In panels A–D, 100% of the pellet (P) and 33% of the supernatant (S) fractions were loaded on the gels. The amount of protein recovered in the pellets is shown at the bottom of each gel.
Figure 8
Figure 8
ParG remodels ParF fibres. EM reverse contrast images of ParF (A) or ParFK15Q (B) fibres assembled in the presence of ParG. Bar=210 nm.
Figure 9
Figure 9
Molecular models for ParF-mediated plasmid segregation. In both models, paired nucleoprotein partition complexes assemble at midcell. Complexes consist of the ParF (red rectangles) and dimeric ParG (green ovals) proteins arranged on the plasmid-located partition sites (yellow circles). A conformational change in ParF resulting from ATP binding induces polymerization (indicated by the square-to-diamond shape change). In bidirectional pulling, a pair of ParF filaments extends from midcell towards opposite cell poles where the filaments are anchored by a hypothetical host factor (blue half circles). Concerted depolymerization of the filaments from midcell draws plasmids away from the cell centres. In bidirectional pushing, the filaments extend from a tethered midcell position (orange oval) and plasmids are propelled towards the cell poles by the polymerization process.

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