Fig. 1. (A) Genetic structure of the par locus from plasmid R1. parC is a centromere-like region that contains the par promoter and 10 direct repeats (indicated by small repeated arrows) to which ParR binds. ParR thereby autoregulates transcription of the par operon. The broken arrow indicates the par operon promoter. The parM (320 codons) and parR (117 codons) genes are shown as hatched and open boxes, respectively. Opposing arrows indicate a transcriptional terminator downstream of parR. The first enlargement shows the five regions in ParM that exhibit similarity to analogous regions present in all proteins with the actin fold (Bork et al., 1992). Numbers are amino acids in ParM. The second enlargement shows an alignment of the phosphate2 regions of actin (from yeast), MreBs from T.maritima, E.coli, B.subtilis and T.acidophilum, and ParMs from plasmids R1 and pSK41. Amino acid changes in the phosphate2 region of ParM of R1 analysed here are indicated with arrows. Conserved amino acids are shown in bold. Ruler units at the top of the figure represent 200 bp of DNA. (B) Phylogram showing the evolutionary relationships of actin, MreB and ParM proteins. The ParM proteins are located on plasmids, except for ParM of E.coli O157:H7 prophage CP-933T. The MreB proteins are encoded by prokaryotic chromosomes. Horizontal lines indicate relative evolutionary distances. The scale bar indicates arbitrary evolutionary distance. Bootstrap values are given at each branch point in the dendrogram. ParM and MreB form two distinct clades, whereas MreB from T.acidophilum and ParM from pSK41 group together. The phylogram was generated by ClustalX. Entrez accession Nos (GI): Actin-Yeast: 113309; MreB-B.subtilis: 7444226; MreB-E.coli: 1171017; MreB-T.maritima: 7444232; MreB-T.acidophilum: 10639751; ParM-pSK41: 3676423; ParR-pSK41: 3676424; ParM-ColIbP9: 9507465; ParR-ColIbP9: 9507466; ParM-R1: 78284; ParR-R1: 208645; ParM-CP-933T: 15802331; ParR&-CP-933T: 15802332; ParM-pB171: 10955418; ParR-pB171: 10955417; ParM-pWR501: 13449145; ParR&--pWR501: 13449144; ParM-R27: 10957204; ParR-R27: 10957203; ParM-R478: 1695865; ParR-R478: 1695866.

Fig. 3. (A) ParM polymers visualized by electron microscopy. Typical ParM filaments formed by incubation in 30 mM Tris–HCl pH 7.5, 100 mM KCl, 2 mM MgCl₂, 1 mM DTT and 2 mM ATP-γ-S. Filament formation required Mg²⁺ and occurred in the presence of ATP or its non-hydrolysable analogue, ATP-γ-S, but not in the presence of ADP. Polymers have a cross-sectional diameter of ∼7 nm. Scale bar: 50 nm. (B) Nucleotide-dependent polmerization of ParM. Polymerization of 10 µM ParM was assayed in 30 mM Tris–HCl pH 7.5, 100 mM KCl and 1 mM DTT. MgCl₂ (2 mM), EDTA (10 mM) and nucleotides (2 mM) were added as indicated. The protein was centrifuged immediately after mixing at 100 000 g for 15 min at ambient temperature. Supernatant and solubilized pellets were analysed by SDS–PAGE and Coomassie Blue staining.

Fig. 5. ParM ATPase exhibits cooperativity. The ATPase activity of ParM (mol ATP hydrolysed/h/mol ParM) depicted as a function of the ParM concentration. Activities are averages of four independent determinations.

Fig. 2. (A) ParM forms intracellular actin-like filaments. ParM localization was visualized by combined phase-contrast and immunofluorescence microscopy. Cells of E.coli MC1000 containing different plasmids were fixed and stained as described in Materials and methods. (a) Control cell containing the par^– plasmid pOU82. The scale bar represents 2 µm. (b–r) Cells containing plasmid pKG491 carrying the wild-type par locus of R1. (r) Filamentous cell of MC1000/pKG491 obtained by addition of 10 µg/ml cephalexin to the growth medium 90 min prior to fixation. (B) ParM filamentation depends on the ParR–parC complex. ParM localization in the absence of other par components (a–d), in the presence of parR (e–h) or parR–parC on a second plasmid (i–l). In (a–d), ParM was expressed from pPH138 (ptac::parM) in the presence of pOU82 (par^– control plasmid); in (e–h) in the presence of pMD1326 (parR⁺ plasmid); and in (i–l) in the presence of pDD1509 (parR⁺/parC⁺ plasmid). (C) Mutation of the ParM ATPase active site changes filament dynamics and morphology. (a–c) Cells containing pRBJ337 expressing ParM D170A in an otherwise par⁺ genetic context; (d–f) pRBJ338 expressing ParM D170E in a par⁺ context; (g and h) cells expressing ParM D170A and D170E in the absence of ParR and parC (from pRBJ212 and pRBJ213, respectively); (i) filamentous cell of MC1000/pRBJ338 obtained by addition of 10 µg/ml cephalexin to the growth medium for 90 min prior to fixation. ParM and mutant proteins were expressed to wild-type levels from an inducible ptac promoter construction by the addition of 20 µM IPTG to the growth medium.

Fig. 4. ParM polymerization is dynamic and is stimulated by the ParR–parC complex. (A) Polymerization of the ParM protein was assayed in the presence of different nucleotides: ATP (squares), ATP-γ-S (circles), ADP (triangles). The protein (5 µM) was allowed to equilibrate in the cuvette for 60 s before addition of 500 µM nucleotide. Light scattering at a 90° angle was measured as arbitrary units every 5 s. (B) Polymerization of ParM (5 µM) after addition of increasing amounts of ATP: 0.1 mM (diamonds), 0.2 mM (inverted triangles), 0.5 mM (upright triangles), 1 mM (circles) and 2 mM (squares). (C) Recycling of ParM protein. Light scattering signal measured after two successive additions of ATP: 100 µM ATP was added after 60 s and another 200 µM was added after 240 s. (D) Induction of ParM polymerization by ParR and parC DNA. Polymerization of 1 µM ParM was measured in the presence of ParR and/or parC DNA: ParR + pMD330 (pUC19-parC⁺) (squares), ParR (circles), pMD330 (pUC19-parC⁺) (upright triangles), ParR + pUC19 (control DNA) (inverted triangles), no addition (diamonds). ATP (500 µM) was added after 60 s of pre-equilibration.

Fig. 6. Model for R1 par-mediated plasmid partitioning during the cell cycle. Plasmids (red) are replicated by the host cell replication machinery (yellow), which is located at mid-cell (Lemon and Grossman, 1998; Koppes et al., 1999). Replicated plasmid parC regions are paired through interactions with ParR protein (grey), thereby forming a partitioning complex. The partitioning complex forms a nucleation point for ParM filamentation. Continuous addition of ATP-ParM (green) to the filament poles provides the force for active movement of plasmid copies to opposite poles. Within the filaments, ATP is hydrolysed, leading to destabilization of the ParM polymer. Nucleotide exchange is required to recharge the ADP-ParM (blue) molecules for a subsequent round of partitioning.