Volume 12, Issue 6, Pages 1477-1487 (December 2003) Bacterial Mitosis  Jakob Møller-Jensen, Jonas Borch, Mette Dam, Rasmus B Jensen, Peter Roepstorff, Kenn Gerdes  Molecular Cell  Volume 12, Issue 6, Pages 1477-1487 (December 2003) DOI: 10.1016/S1097-2765(03)00451-9

Figure 1 Overview of the R1 par System (A) The parM and parR genes are shown as bars and the location of the par promoter is shown as an arrow. Opposing arrows indicate the transcriptional terminator. The centromere-like parC site that is located upstream of the parM and parR genes is enlarged. The locations of the ten 11 bp direct repeats (iterons) and the core promoter region are indicated. The number of each iteron used here is shown below the boxes. (B) Sequence logo showing the conservation of the nucleotides in the different positions in the iterons. The relative sizes of the letters indicate the degree of conservation measured as number of bits. The ten iterons were aligned and the sequence logo was calculated as described by Schneider and Stephens (1990). Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)

Figure 2 ParR-parC Interaction In Vitro (A) Specific binding of ParR to the full-length parC region examined using gel shift analysis. A 32P-labeled 161 bp fragment (1.5 nM) containing the full-length parC region (an EcoRI-XbaI digest of pMD330) was incubated with increasing concentrations of purified ParR protein. The amounts of ParR used (in nM) are indicated below the lanes. The reactions were analyzed by electrophoresis in a 5% poly-acrylamide gel. (B) Binding of ParR to DNA fragments containing different numbers of iterons. The panels show the gel shift patterns when ParR binds to DNA fragments containing increasing numbers of iterons. The DNA concentration was 2 nM in all experiments and the concentrations of ParR used (in nM) are indicated below each lane. (a) Binding to a fragment that does not contain sequences with homology to the iterons (a 27 bp fragment from pUC19). (b) Binding to a fragment containing iteron 1 (a 36 bp fragment from pMD191). (c) Binding to a fragment containing iterons 1 and 2 (a 47 bp fragment from pMD192). (d) Binding to a fragment containing iterons 1–3 (a 58 bp fragment from pMD193). (e) Binding to a fragment containing iterons 1–4 (a 69 bp fragment from pMD194). (f) Binding to a fragment containing iterons 1–5 (a 95 bp fragment from pMD333). (C) DNase I foot-printing of protein-DNA complexes formed when parC DNA is incubated with purified ParR and ParM proteins. A 32P end-labeled 310 bp fragment containing the full-length parC region was incubated with ParR and ParM. The concentrations of the proteins used are indicated below the lanes (in nM). ATP was included in the reactions since ATP is required for ParM-mediated enhancement of plasmid pairing in vitro (Jensen et al., 1998). The locations of the ten iterons are indicated with arrows, and the locations of the −10 and −35 sequences of the parA promoter are marked to the left. Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)

Figure 3 Surface Plasmon Resonance Analysis of ParM-ParR-parC Interactions Biotinylated double-stranded parC or control DNA fragments were immobilized on a streptavidin sensor chip and assayed for ParR and ParM binding. (A) Binding of ParR and ParM to parC DNA in the presence of different nucleotides. Time windows of protein addition are indicated by double arrows. Black, ATP; green, ATPγS; red, ADP. (B) Binding of ParR and ParM to control DNA in the presence of different nucleotides. Black, ATP; green, ATPγS; red, ADP. (C) Binding of ParM to the ParR/parC complex at different ATP concentrations. Black, 500 μM; red, 125 μM; green, 32 μM; blue, 8 μM; purple, 2 μM. (D) ATP dependency of ParM binding to the ParR/parC complex. (E) Binding of ParM to the ParR/parC complex at different ParM concentrations. Black, 10 μM; red, 5 μM; green, 2.5 μM; blue, 1.25 μM; purple, 0.625 μM; cyan, 0 μM. (F) Cooperative binding of ParM to the ParR/parC complex. ParM binding responses are plotted against the respective ParM concentrations. Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)

Figure 4 Intracellular Localization of Plasmid DNA and ParM Protein Visualized by Combined Phase-Contrast and Immunofluorescence Microscopy Plasmid DNA was labeled indirectly by induction of a LacI-GFP fusion protein. Escherichia coli MC1000 cells harboring par+ plasmids (MC1000/pJMJ178/pRBJ460) divided into groups that contain: one plasmid focus (A), two nonpolar foci (B), two polar foci (C), three foci (D), or two polar foci separated by incomplete filaments (E). Size bar represents 2 μm. Cells were grown at 30°C with a generation time of 35 min, except for single-focus cells, which were grown at 25°C with a generation time of 65 min. Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)

Figure 5 Replication Arrest Experiment (A) Fraction of cells containing R1 plasmids (circles) and ParM filaments (triangles) after inhibition of plasmid replication. At time zero, synthesis of CopA-RNA was induced by addition of IPTG, and after 60 min, half of the culture was transferred to fresh medium to remove the inducer (open circles/triangles). Each data point represents the counting of at least 500 cells. (B) Western blot showing the intracellular amount of ParM during the experiment in cells exposed to IPTG for 210 min (+IPTG) and in cells washed after 60 min (−IPTG). (C) Typical examples of fixed cells immunostained for ParM (a) before and 120 min after replication arrest in the (b) unwashed fraction and the (c) washed fraction. −IPTG indicates that IPTG was removed at t = 60 min. Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)

Figure 6 Plasmid Segregation by the R1 par System (A) Mechanism of R1 plasmid transport by insertional polymerization of ParM. ParR protein (yellow) serves as a link between the centromere like parC DNA (red) and the ParM filaments. ParM-ATP (green) binds to ParR-parC and the filament end, whereby its ATPase activity is stimulated. Conversion of ParM-ATP into ParM-ADP (blue) leads to detachment of the filament, thus allowing the entry of another ParM-ATP monomer. The asynchronous growth of several parallel proto-filaments ensures that overall contact between plasmid and filament is conserved during transport. (B) Model for the mechanism of plasmid partitioning by the R1 par system. (1) A partitioning complex is formed via specific binding of ParR proteins (yellow) to parC regions of newly replicated plasmid molecules (red). (2) The partitioning complex serves as a nucleation point for ParM filament formation. Continuous insertion of ParM-ATP (green) onto the filament ends pushes the plasmid copies apart. (3) Nucleotide hydrolysis within the polymers leads to formation of ParM-ADP (blue) and filament destabilization. (4) At cell division, the partitioned plasmid copies are present near opposite cell poles and hence end up in future daughter cells. ParM nucleotide exchange is required to rejuvenate the par function. For simplicity, only two ParM protofilaments are shown in this cartoon. Molecular Cell 2003 12, 1477-1487DOI: (10.1016/S1097-2765(03)00451-9)