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Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

2005; Springer Nature; Volume: 24; Issue: 7 Linguagem: Inglês

10.1038/sj.emboj.7600619

1460-2075

Daniela Barillà, Mark F. Rosenberg, Ulf Nobbmann, Finbarr Hayes,

Bacteriophages and microbial interactions

Article10 March 2005free access Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF Daniela Barillà Daniela Barillà Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Mark F Rosenberg Mark F Rosenberg Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ulf Nobbmann Ulf Nobbmann Malvern Instruments Ltd, Malvern, Worcestershire, UK Search for more papers by this author Finbarr Hayes Corresponding Author Finbarr Hayes Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Daniela Barillà Daniela Barillà Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Mark F Rosenberg Mark F Rosenberg Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ulf Nobbmann Ulf Nobbmann Malvern Instruments Ltd, Malvern, Worcestershire, UK Search for more papers by this author Finbarr Hayes Corresponding Author Finbarr Hayes Faculty of Life Sciences, University of Manchester, Manchester, UK Search for more papers by this author Author Information Daniela Barillà1, Mark F Rosenberg1, Ulf Nobbmann2 and Finbarr Hayes 1 1Faculty of Life Sciences, University of Manchester, Manchester, UK 2Malvern Instruments Ltd, Malvern, Worcestershire, UK *Corresponding author. Faculty of Life Sciences, The University of Manchester, PO Box 88, Sackville Street, Manchester M60 1QD, UK. Tel.: +44 161 200 8934; Fax: +44 161 236 0409; E-mail: [email protected] The EMBO Journal (2005)24:1453-1464https://doi.org/10.1038/sj.emboj.7600619 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The molecular events that promote accurate chromosome segregation in eukaryotes are well understood. In contrast, the processes involved in faithful distribution of bacterial genomes at cell division have been elaborated less thoroughly. Nevertheless, it is clear that bacterial plasmids and chromosomes are partitioned in an orderly fashion that requires the participation of dedicated segregation proteins and cis-acting DNA sequences (Draper and Gober, 2002; Surtees and Funnell, 2003). Plasmid partition systems broadly are of two types, involving either an ATPase with a deviant Walker-type ATP-binding motif (generally denoted ParA), or an ATPase that is a member of the actin protein family (Motallebi-Veshareh et al, 1990; Bork et al, 1992). Proteins of the former class, which are much more prevalent, are also specified by many bacterial chromosomes (Hayes, 2000). The ATPase activity of ParA proteins is required for successful DNA segregation (Davis et al, 1996; Libante et al, 2001). In ParA-type systems, a second protein (often termed ParB) specified by the partition operon binds to the cis-acting partition site located near to the operon (Surtees and Funnell, 2003). The ATPase does not directly contact partition site DNA, but is instead recruited into the partition nucleoprotein complex through interactions with ParB (Bouet and Funnell, 1999; Barillà and Hayes, 2003). For ParA-type systems, recent evidence suggests that plasmid copies align at midcell shortly before cell division and are propelled bidirectionally along the cell axis by the partition apparatus. Subsequent cell division compartmentalizes the plasmids within new daughter cells (Li and Austin, 2002). In contrast, the actin-type protein of plasmid R1 apparently promotes partition by generating filaments that direct replicated plasmids towards the cell poles (Møller-Jensen et al, 2002; van den Ent et al, 2002). It remains to be elucidated whether bacterial DNA segregations mediated by Walker- and actin-type ATPases are mechanistically distinct. The multidrug resistance plasmid TP228 replicates at low copy number in Escherichia coli. The partition cassette of TP228 consists of the parFG genes and upstream noncoding sequences that harbour a series of repeat motifs. ParG (8.6 kDa), which is unrelated evolutionarily to ParB (Hayes, 2000), is a dimer and binds the upstream region (Barillà and Hayes, 2003). ParG consists of an unstructured N-terminal tail and a folded C-terminal domain that contains a ribbon–helix–helix motif that contacts DNA (Golovanov et al, 2003). The ParF protein (22 kDa) interacts with ParG and modulates binding of the latter to the upstream DNA region (Barillà and Hayes, 2003). ParF is the prototype of a phylogenetically distinct subgroup within the ParA superfamily of Walker-type ATPases. In fact, ParF is more closely related to the MinD cell division site-selection protein than to well-characterized ParA partition proteins such as ParA, SopA and Soj specified by the P1 and F plasmids, and the Bacillus subtilis chromosome, respectively (Hayes, 2000). MinD, in conjunction with MinC, prevents placement of the cell division septum at all locations in E. coli. This inhibition is relieved specifically at midcell by the MinE protein thereby allowing proper cell division to proceed. Division inhibition by MinCD is mediated by their rapid oscillation between the cell poles in a MinE-dependent manner (Raskin and de Boer, 1999; Fu et al, 2001; Hu and Lutkenhaus, 2001). Oscillation apparently occurs along a permanent, but dynamic MinCDE spiral framework that extends along the entire cell length (Shih et al, 2003). This oscillation correlates with polymerization of MinD into long filaments in vitro modulated by ATP, phospholipids and MinE (Hu et al, 2002; Suefuji et al, 2002). Here, we show that the purified ParF protein assembles into extensive filaments when incubated with ATP. These filaments are remarkably similar to those of the evolutionarily related MinD protein, but are quite distinct from those of the unrelated FtsZ tubulin homologue involved in cell division and the actin-type partition protein of plasmid R1. By analogy with the Min system, a smaller protein, ParG, modulates the polymerization of ParF as well as stimulating its ATPase activity. These results point to a possible mechanistic parallel between DNA segregation and cell division in prokaryotes, and suggest a molecular mechanism for DNA segregation by ParA-type proteins. Results ParF is a Walker-type ATPase of the ParA superfamily The functionally diverse Walker superfamily of ATPases is characterized by the signature A motif GxxxxGKS/T that is involved in contacts with the triphosphate moiety of ATP (Leipe et al, 2002). A number of subgroups of the Walker family are distinguished by a deviant A box, GKGGhGKS/T (Motallebi-Veshareh et al, 1990). ParF is a member of one of these subgroups, the ParA family, that mediates bacterial plasmid and chromosome partitioning (Hayes, 2000). The ATPase activity of ParF was assessed by thin-layer chromatography (TLC) (Figure 1B–E). The protein has a weak intrinsic ATPase activity like other ParA family members (Davis et al, 1996; Fung et al, 2001; Libante et al, 2001). The K0.5 for ATP is approximately 100 μM, which is in the same range as the KM values for other examined ParA proteins (Davey and Funnell, 1997; Easter and Gober, 2002). 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. Download figure Download PowerPoint ParF assembles into filaments, and polymerization is potentiated by ATP Ultracentrifugation studies hinted that the ParF protein has a propensity to polymerize in vitro, and that ATP enhances this polymerization (Barillà and Hayes, 2003). This was investigated further by conducting sedimentation assays in which ParF (8 μM) was incubated in the presence or absence of nucleotides for 10 min at 30°C, separated by centrifugation into pellet and supernatant fractions, and analysed by SDS–PAGE. If the protein assembles into filaments of sufficiently high molecular weight, it will enter the pellet, whereas unpolymerized or partially polymerized protein will remain in the supernatant. In the absence of nucleotides, ∼30% of ParF was recovered in the pellet (Figure 2A). In contrast, ∼60% of ParF was pelleted after incubation with ATP, indicating that the protein had formed an increased quantity of polymeric species. To assess whether ATP binding was sufficient for polymerization, ParF was incubated with the nonhydrolysable analogue adenosine-5-O-(3-thiotriphosphate) (ATPγS). Virtually all of the input ParF protein (∼95%) sedimented, showing that ATPγS efficiently promotes ParF sedimentation, but that nucleotide hydrolysis is unnecessary for polymerization. Interestingly, the amount of ParF in the pellet was reduced to ∼15% in the presence of ADP, which suggests that this nucleotide antagonized polymerization and perhaps even reversed it (Figure 2A). A corollary of these studies is that the binding of di- or triphosphate nucleotides is likely either to mediate significant conformational changes in ParF or to modify the monomer–monomer interface. 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. Download figure Download PowerPoint The critical concentration required for ParF assembly in the presence of 2 mM ATP was determined to be 0.34±0.05 μM at 14 000 r.p.m. This value was unaltered at 50 000 r.p.m. (Figure 3). As determined from the slopes of these lines, equivalent amounts of the protein enter the pellet fractions at both centrifugation speeds, indicating that ∼60% of ParF assembles into polymers under these reaction conditions. Similarly, a survey of centrifugation speeds showed that, at concentrations above the critical value, ∼60% of ParF entered the pellet fraction at 14 000 r.p.m. This percentage progressively diminished as the speed was reduced, declining to ∼13% at 1000 r.p.m. 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. Download figure Download PowerPoint Dynamic light scattering (DLS), which allows an assessment of both particle abundance and size, was used to further analyse the polymerization of ParF. DLS differs from static light scattering in that it measures the hydrodynamic radius of a protein. The average hydrodynamic size is the diameter of a sphere that has the same diffusion coefficient as that of the particle being measured. In the absence of nucleotides at 30°C, ParF protein (2.16 μM) remained stable for ∼12 min with an average intensity of 70–80 kilocounts/second (kct/s) and a particle size of ∼25 nm (Figure 2D). This average size is likely to represent an oligomeric, prefilamentous form of the protein, which correlates with previous results (Barillà and Hayes, 2003). Subsequently, the intensity of light scattering increased steadily, reaching a final value of ∼2000 kct/s after 40 min (Figure 2D, bottom). In parallel, the size of the particles increased to ∼450 nm (Figure 2D, top). These results indicate that ParF has an intrinsic tendency to polymerize. When ATP (500 μM) was added to ParF, the count rate immediately elevated from 70 to ∼1000 kct/s and then gradually increased to ∼2500 kct/s (Figure 2D, bottom). This was accompanied by an increase in particle size from ∼20 to ∼800 nm (Figure 2D, top). Therefore, ATP stimulates ParF polymerization. In contrast, addition of ADP (500 μM) not only failed to induce polymerization but entirely inhibited it: the intensity of light scattering and particle size remained constant for 40 min (Figure 2D). This result correlates with the outcome of pelleting assays in which ADP decreased the amount of ParF in the pellet fraction (Figure 2A) and indicates that polymerization requires an appropriate nucleotide configuration. The response of the protein to ATPγS was also analysed by DLS (Figure 2D). The addition of ATPγS (500 μM) to ParF triggered instantaneous and extensive polymerization as reflected by a pronounced increase in light scattering intensity from ∼60 to 12 000 kct/s. In parallel, particle size increased 80-fold (Figure 2D, top). Therefore, ATPγS has a more dramatic impact on ParF polymerization compared to ATP. The DLS results mirror those obtained by the pelleting assay: in the presence of ATPγS, virtually all of ParF assembles into stable and numerous filaments, probably because they are irreversibly locked into a polymeric state as ATP hydrolysis cannot occur. In contrast, only a portion of ParF is recovered in the pellet in the presence of ATP and the intensity of light scattering is comparably lower, presumably because filamentation is more dynamic with polymerization and depolymerization occurring simultaneously. In summary, the sedimentation and DLS data conclusively demonstrate that extensive polymerization of ParF occurs in response to ATP. Ultrastructure of ParF polymers The stages of ParF polymerization were investigated by negative-stain electron microscopy (EM). In the absence of ATP, purified precentrifuged ParF (2.16 μM) appeared as globular particles whose size (10–20 nm) is consistent with that of small oligomers (Figure 4A). This reflects the tendency of the protein to assemble into higher order structures even in the absence of exogenous nucleotide (Barillà and Hayes, 2003). ParF was next incubated with ATP (2 mM) at 30°C and aliquots were withdrawn at intervals and applied to EM grids. After 5 min, needle-like projections (∼100 nm long) were visible (Figure 4A). These structures, which are likely to represent an early morphological stage in filament accretion, appeared longer after 10 min. Within 20 min, the fibres had elongated into extensive filament bundles (Figure 4A). These fibres were 400–650 nm in length and 30–70 nm wide. One end of many of the polymers had an irregular, frayed appearance, whereas the opposite end was more compact. Higher magnification images revealed a multistranded ultrastructure of parallel protofilaments (Figure 4B). Each protofilament appeared as a chain of bead-like particles with a cross-sectional diameter of ∼2.5 nm. These results confirm that ParF assembles into filaments extensively in response to ATP. The ParF fibres induced by ATPγS were morphologically identical to those produced with ATP, confirming that nucleotide binding is sufficient for polymerization. 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. Download figure Download PowerPoint Mutations in conserved residues of the Walker motif A in ParF abolish proper plasmid segregation The pivotal role of ATP in ParF polymerization and plasmid segregation was investigated further by constructing mutations in critical residues of the ATP-binding domain of ParF. Residues 10–17 of ParF correspond to the variant Walker A motif. A conserved glycine residue in this motif was changed to valine (G11V) and a conserved lysine to glutamine (K15Q) (Figure 1A). The residue equivalent to G11 in other Walker A proteins is thought to be involved in nucleotide hydrolysis: the glycine-to-valine substitution in Ha-ras-p21 induces perturbations of the catalytic site, resulting in impaired GTPase activity and insensitivity to GTPase-activating protein (GAP) stimulation (Vogel et al, 1988; Maegly et al, 1996). The lysine residue corresponding to K15 in ParF is almost fully conserved among Walker family ATPases (Figure 1A): its side chain forms hydrogen bonds to the β- and γ-phosphate oxygens of ATP (Hayashi et al, 2001). Mutation of this lysine residue into glutamine has been investigated in vivo in a number of ParA family members. For example, both E. coli MinDK16Q and B. subtilis SojK16Q have lost their canonical cell pole localization and are dispersed throughout the cell (Quisel et al, 1999; Hu et al, 2002). The effects exerted by the G11V and K15Q changes in ParF were first assessed in vivo by partition assays. Both mutations entirely abrogated ParF-mediated plasmid segregation: the level of plasmid retention after ∼25 generations in the absence of selective pressure was <1%, just like the level of the stability probe vector, whereas the level of segregational stability conferred by the wild-type parFG cassette was ∼70%. These results indicate that residues G11 and K15 of ParF fulfil a crucial role in DNA segregation. The polymerization kinetics and ATPase activities of ParFK15Q and ParFG11V are perturbed The severe partition defect prompted an investigation of the biochemical properties of the mutated ParF proteins. The ATPase activity of ParFK15Q was reduced compared to that of wild-type ParF at low ATP concentrations (Figure 1E). However, at ATP concentrations of 250 μM and 1 mM, the levels of hydrolysis by ParFK15Q were respectively ∼70% and equivalent to that of ParF (Figure 1B and D). The nucleotide binding impairment predicted for this derivative correlates with the observed paucity of ATPase activity at low ATP concentrations (Figure 1E), that becomes alleviated at high ATP concentrations (Figure 1D). The K0.5 for ATP of ParFK15Q (∼200 μM) is two-fold higher than that of wild-type ParF. The ability of ParFK15Q to polymerize was first assessed by sedimentation assays. The mutant protein remained mostly in the supernatant, failing to respond to ATP or ATPγS after 10 min incubation at 30°C (Figure 2B). In DLS trials, ParFK15Q remained stable in the absence of nucleotides, displaying a constant intensity of light scattering of ∼70 kct/s for 25–30 min after which it began to polymerize with the count rate increasing up to ∼600 kct/s after 40 min. The size of the particles increased in parallel up to a final value of ∼130 nm. When ATP (500 μM) was added, it did not trigger immediate polymerization of ParFK15Q. Instead ParFK15Q only began to polymerize after a lag period of ∼25 min. After a further 15 min, the count rate reached a value of 1200 kct/s, two-fold higher than that attained in the absence of ATP (Figure 2E, bottom). The particle size increased up to ∼200 nm (Figure 2E, top). As this protein displayed a prolonged lag before polymerization, its behaviour in the presence of ATP was followed by DLS for up to 90 min. After the initial lag, ParFK15Q steadily polymerized, reaching a final count rate of ∼4000 kct/s (data not shown). The pattern in the presence of ATPγS (500 μM) was very similar to that seen with ATP. These results show that ParFK15Q is less responsive to ATP and ATPγS than wild-type ParF and that, even though the mutated protein retains the capacity to polymerize, polymerization is less extensive than that of ParF and the fibres formed are smaller. Interestingly, as for wild-type ParF, ADP totally suppressed ParFK15Q polymerization (Figure 2E). To shed light on the ultrastructure of the polymers, the ParFK15Q polymerization process was investigated by EM. Sporadic, isolated fibres of ParFK15Q were visible 10 min after ATP addition, whereas numerous thin-bodied projections were observed after 60 min (data not shown). Although ParFK15Q retains the ability to polymerize, the polymerization is poor and probably incapable of supporting plasmid partition in vivo. Although it has yet to be rigorously shown, we are inclined to believe that ParFK15Q is impaired specifically in ATP binding on the basis of (i) the predicted interaction of this lysine with the β- and γ-phosphates of ATP (Hayashi et al, 2001); (ii) the reduced ATPase activity at low ATP concentrations, which attains wild-type levels at high ATP concentrations; and (iii) the poor responsiveness of this mutant to ATP and ATPγS in polymerization. ParFG11V failed to hydrolyse ATP efficiently and the residual activity was much lower (∼10%) than that of ParF (Figure 1B–D). However, at low ATP concentrations (up to 500 nM), ParFG11V exhibited an ATP hydrolysis rate identical to that of wild-type ParF (Figure 1E). The K0.5 of ParFG11V for ATP (∼40 μM) is slightly lower than that of wild-type ParF. In DLS experiments, ParFG11V displayed a strong tendency to polymerize both in the absence and presence of triphosphate nucleotides (Figure 2F). In the absence of nucleotides, this mutant polymerized promptly without a lag, gradually reaching a plateau of ∼3500 kct/s (compared to ∼2000 kct/s for ParF and ∼600 kct/s for ParFK15Q). ParFG11V still appeared responsive to ATP and ATPγS, which elicited an immediate increase in light scattering intensity. Nevertheless, the final count rate in the presence of ATP or ATPγS was ∼4000 kct/s, similar to that attained by the protein without nucleotides (Figure 2F). However, the nucleotide sensitivity of ParFG11V is less than that of ParF, as illustrated by the response to ATPγS, which was less dramatic for ParFG11V than that observed for the wild-type protein (∼4500 and ∼12 000 kct/s for ParFG11V and ParF, respectively) (Figure 2F and D). As observed for ParF and ParFK15Q mutant, ADP (500 μM) also antagonized the polymerization of ParFG11V: both light scattering intensity (1400 kct/s) and particle size (∼340 nm) were lower compared to those of ParFG11V alone (Figure 2F). At a higher concentration of ADP (5 mM), the inhibition of ParFG11V polymerization was more pronounced, as revealed by a lower count rate (∼270 kct/s) and smaller fibre size (∼140 nm) (data not shown). The behaviour of the ParFG11V protein was also analysed by EM (Figure 5). Isolated, thick and long fibres were visible before ATP addition. This correlates with the pronounced tendency of this mutant to polymerize in the absence of ATP. At the first analysed time point after ATP addition, short projections were observed. These structures were morphologically comparable to those observed for wild-type ParF at the same juncture. The needles grew into longer and thicker filaments within 10 min. Some of the ParFG11V fibres were particularly elongated, twice or three times the length of wild-type polymers (Figure 5B). The cross-sectional diameter of these polymers was also wider compared to wild-type fibres. Interestingly, polymerization of ParFG11V produced an intricate meshwork of overlapping, interconnected filaments within 20 min of exposure to ATP (Figure 5A, and details in Figure 5C). The EM data show that ParFG11V forms fibres and that the protein is prone to hyperfilamentation. However, the monomer–monomer and interprotofilament interactions within these fibres are likely to be intrinsically weaker or structurally different from those of wild-type ParF, as the fibres were never recovered in the pellet fraction in repeated sedimentation assays with and without nucleotides at 14 000 r.p.m. (Figure 2C) and only poorly (∼25%) at 80 000 r.p.m. EM of these experiments also revealed that ParFG11V fibres remained in the supernatant fraction and exhibited a similar morphology as before centrifugation, suggesting that the ultrastructure of these fibres must be different from that of the wild-type protein. 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. Download figure Download PowerPoint ParG plays at least two distinct roles in ParF polymerization dynamics: enhancement of ATP hydrolysis and promotion of filament bundling ParF and ParG interact in vivo and in vitro (Barillà and Hayes, 2003). The effect of ParG on the ATPase activity of ParF was examined at various ParG concentrations. ATP hydrolysis by ParF (0.5 μM) was strongly stimulated (∼30-fold) by ParG, which alone exhibited no nucleotide hydrolysis (Figure 6A). At a higher ParF concentration (5 μM), a similar pattern of stimulation was observed (Figure 6B). Enhancement of nucleotide hydrolysis by a partner regulatory protein is a recurring theme among members of the ParA superfamily, including P1ParA (Davis et al, 1996; Fung et al, 2001), F SopA (Libante et al, 2001), ParA of Caulobacter crescentus (Easter and Gober, 2002) and the MinD cell division protein of E. coli (Hu and Lutkenhaus, 2001; Suefuji et al, 2002), as well as more broadly in the case of Walker-type hydrolases like Ha-ras-p21 (Vogel et al, 1988). The stimulation of ParF ATPase activity by ParG exhibited a sigmoidal behaviour at lower ParG concentrations (Figure 6A and B) analogous to the stimulation of E. coli MinD ATPase activity by MinE (Suefuji et al, 2002). The effect of ParG and DNA together on the ATPase activity of ParF was also examined. Both a cognate DNA fragment, containing the partition site, and an unrelated DNA fragment further augmented ParG stimulation of ATP hydrolysis by ParF by 40–50% (Figure 6C). When ParF was incubated with DNA alone, either cognate or unrelated, no enhancement of ATP hydrolysis was observed (data not shown). 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. Download figure Download PowerPoint The effect of ParG on ATP hydrolysis by the mutated ParF proteins was also investigated. ParG failed to enhance nucleotide hydrolysis by either ParFG11V or ParFK15Q (Figure 6A), suggesting that both mutations ablate the stimulatory response to ParG. Sedimentation assays including both proteins were performed to assess whether ParG associates with ParF polymers (Figure 7A). In either the absence of nucleotides or in the presence of ATP or ATPγS (2 mM), a significant fraction of the input ParG cosedimented with ParF filaments. When ParG was tested alone, very little sedimentation was evident. Therefore, ParG is able to associate with polymeric forms of ParF. Intriguingly, the fact that ParF was recovered mostly in the pellet fraction in the presence of ParG even without added nucleotides suggests a function for the partner protein in pr