Localized Dimerization and Nucleoid Binding Drive Gradient Formation by the Bacterial Cell Division Inhibitor MipZ

Summary Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells.

. Steady-state and transient kinetic analysis of nucleotide binding to MipZ, Related to Table 2. (A) Fluorescence emission spectra of mant-ATP. MipZ (1 µM) and/or mant-ATP (60 µM) were mixed in buffer BC containing no (+ Mg 2+ ) or 10 mM (-Mg 2+ ) EDTA. Emission spectra were recorded in a PC1 photon-counting spectrofluorimeter (ISS) using fluorescence resonance energy transfer (FRET) and an excitation wavelength of 290 nm. (B) Association of MipZ with mant nucleotides. Nucleotide binding did not change the intrinsic tryptophan fluorescence of MipZ (data not shown), necessitating the use of ADP and ATP analogues that carried a fluorescent methylanthraniloyl (mant) group attached to the 2'-or 3'-hydroxy group of the ribose moiety. Due to the bulkiness of the mant group and a critical role of these hydroxy groups in MipZ dimerization, the modified nucleotides are predicted to block dimer formation, consistent with their failure to be hydrolyzed by MipZ or mediate MipZ self-association in the SPR assay (data not shown). This circumstance allowed us to focus the measurements on the initial nucleotide binding step without interference from subsequent reactions. Interaction of the mant group with the protein surface led to a marked increase in fluorescence (panel A). Consistent with the easy accessibility of the nucleotide-binding site and the lack of major conformational changes, the transients recorded after rapid mixing of MipZ with mant nucleotides could be fitted to single-exponential equations, indicating single-step binding reactions. Shown is a typical curve representing the change in mant fluorescence upon binding of mant-ATP (30 µM) to MipZ (1 µM). Fitting of the data yielded a pseudo-first order rate constant (k obs ) of 4.7 s -1 . (C) Determination of the second-order association rate constants (k on ). The pseudo-first order rate constants for the association of MipZ with mant nucleotides showed a linear dependence on the nucleotide concentration used, with the slope of the fitted line determining the second-order rate constant of the association reaction. Shown is a representative graph in which the k obs values measured for mant-ATP were plotted against the corresponding mant-ATP concentrations. Fitting of the data to the linear function k obs =k on [mant-ATP]+k off yielded a value of 6.8 x 10 4 M -1 s -1 for k on . (D) Dissociation of mant nucleotides from MipZ. In order to determine the dissociation rate constants (k off ), we monitored the displacement of mant-labeled nucleotides from MipZ by an excess of unlabeled nucleotide. All transients obtained could be fitted to single-exponential functions. Shown is a typical curve representing the change in mant fluorescence upon dissociation of mant-ATP (30 µM) from MipZ (1 µM) in the presence of 2 mM ATP. Fitting of the data yielded a dissociation rate constant (k off ) of 2.1 s -1 . (A) Influence of DNA on the stimulation of FtsZ GTPase activity by MipZ. The GTPase activity of FtsZ (3 µM) was followed in the presence of 2 mM [α-32 P]-GTP and 3 mM ATP. The reactions were supplemented with 6 µM MipZ-D42A and/or 0.2 µM plasmid DNA (pMT428; 3.6 kb) as indicated. Data represent the mean of three independent measurements (± SD). (B) Inhibitory effect of DNA on the interaction between MipZ and ParB. ParB (200 RU) was immobilized on an SPR sensor chip and allowed to react with wild-type MipZ protein (0.5 µM) that had been preincubated with 2 mM ATP and the indicated concentrations of a 26-bp double-stranded oligonucleotide (ran2). Note that injection of the DNA fragment alone did not produce any response (data not shown). (A) Effect of Mg 2+ on the DNA-binding activity of MipZ. A double-stranded oligonucleotide (ran2, 700 RU) was immobilized on an SPR sensor chip and probed with MipZ (1 µM). The binding reactions were performed in buffer BC containing the indicated nucleotide (2 mM) and no (ATP + Mg 2+ ) or 10 mM (no nucleotide, ADP, ATP) EDTA, respectively. (B) DNA-binding specificity of MipZ. A double-stranded oligonucleotide (ran2, 500 RU) was immobilized on an SPR sensor chip and allowed to react with MipZ protein (1 µM) that had been preincubated with 2 mM ATP and a double-stranded oligonucleotide (ran2, 6 µM) (dsDNA), a single-stranded oligonucleotide (top strand of ran2, 6 µM) (ssDNA), an oligoribonucleotide (with a sequence analogous to the top strand of ran2, 6 µM) (RNA) or polyphosphate (25 µg/ml) (PP), respectively. A reaction performed in the absence of any competitor served as a control. Note that consistent with the results of this analysis, only dsDNA and ssDNA can stimulate the ATPase activity of MipZ (compare Fig. 3G; data not shown). (C) Inhibitory effect of ParB on the interaction between MipZ and DNA. A double-stranded oligonucleotide (ran2, 500 RU), immobilized on an SPR sensor chip, was probed with MipZ protein (0.5 µM) that had been preincubated with 2 mM ATP and the indicated concentrations of ParB. Note that ParB alone did not interact with the immobilized DNA fragment under the experimental conditions used (data not shown). The dissociation rate constant determined for the reaction without ParB was 1.5 min -1 . (D) As for (C), but using MipZ-D42A. , which express wild-type mipZ under the control of the ammonium-repressible P nrtX promoter and the indicated mipZ-eyfp allele under the control of the xylose-inducible P xyl promoter, were shifted from M2G N to M2G medium containing 0.3% xylose and cultivated for another 4 h to deplete wild-type MipZ and synthesize the fusion protein instead. Single polar regions were photo-bleached, and recovery was followed by fluorescence microscopy (bars: 2 µm). The bleached regions are highlighted by circles. (B) Cells of strain MT177 (ΔmipZ P nrtX ::mipZ P xyl ::mipZ-eyfp) were grown as described in (A). Chloramphenicol (20 µg/ml) was added to the growing culture 25 min prior to harvest as well as to the agarose pads used for the FRAP experiments. Fitting of the data to a single exponential equation yielded a recovery half-time of 12.4 s (± 0.5 s). (C) Cells of strain MT97 (mipZ-eyfp) were grown in M2G medium and subjected to FRAP analysis. The fitted curve corresponds to a recovery half-time of 12.0 s (± 1.2 s). (D and E) Cells of strains (D) DK2 (ΔmipZ P nrtX ::mipZ P xyl ::mipZ K13A -eyfp) and (E) MT179 (ΔmipZ P nrtX ::mipZ P xyl ::mipZ K18Qeyfp) were grown as described in (A) and subjected to FRAP analysis. Fitting of the data to a single-exponential equation yielded recovery half-times of 1.5 s (± 0.3 s) (K13A) and 1.2 s (± 0.2 s) (K18Q), respectively. Details on the experimental procedures and data analysis are described in the legend to   CB15N ΔmipZ P nrtX ::P nrtX -mipZ P xylX ::P xylX -mipZ K13A -eyfp P iol ::P iol -cfp-parB This study DK5 CB15N ΔmipZ P nrtX ::P nrtX -mipZ P xylX ::P xylX -mipZ G14V -eyfp P iol ::P iol -cfp-parB This study DK6 CB15N ΔmipZ P nrtX ::P nrtX -mipZ P xylX ::P xylX -mipZ K18Q -eyfp P iol ::P iol -cfp-parB This study DK7 CB15N ΔmipZ P nrtX ::P nrtX -mipZ P xylX ::P xylX -mipZ D42A -eyfp P iol ::P iol -cfp-parB This study

Media and growth conditions
All C. crescentus strains used in this study were derived from the synchronizable wild-type strain CB15N (NA1000) (Evinger and Agabian 1977). They were grown at 28°C in M2G (Ely, 1991)  -repressible P nrtX promoter and the indicated mipZeyfp allele under the control of the xylose-inducible P xyl promoter, were shifted from M2G N medium (-NH 4 + ) to M2G medium (+ NH 4 + ) supplemented with 0.3% xylose and incubated for 4-8 h. Strains carrying cerulean-parB under the control of the inositol-inducible P iol promoter (DK3, DK5, DK6 and DK7) were induced for 2 h with 0.3% myo-inositol before microscopy. To analyze the unspecific DNA-binding activity of MipZ, E. coli TOP10 was transformed with plasmid pDK2 (P BAD -mipZ-eyfp) or pDK3 (P BAD -mipZ D42A -eyfp) and grown for 2 h in the presence of cephalexin and arabinose to block cell division and activate synthesis of the fusion proteins, respectively. Thirty minutes prior to analysis, chloramphenicol (100 µg/ml) was added to induce nucleoid condensation. DNA was stained with DAPI, and the cells were visualized by microscopy.

Plasmid construction
Plasmids encoding fluorescent protein fusions. To generate pMT174, mipZ was PCR-amplified with the mutagenic primers CC2165-G14V and CC2165-rev2. The reaction product containing the mutant mipZ G14V allele was cut with NdeI and SacI and ligated into plasmid pMT151, an integrative plasmid carrying a mipZ-eyfp fusion, whose wild-type mipZ gene had been released by restriction with the same enzymes. Plasmid pMT209 was constructed as follows: Inverse PCR was performed on plasmid pMT151 using primers CC2165-K18Q-1 and CC2165-K18Q-2 to generate the mutant mipZ K18Q allele. After phosphorylation and selfligation of the PCR product, the resulting plasmid was cut with NdeI and SacI. The fragment containing mipZ K18Q was then ligated into plasmid pMT151 whose wild-type mipZ gene had been released by restriction with NdeI and SacI. To generate plasmid pMT977, the cerulean gene was released from pVCERN-3 by restriction with NdeI and KpnI. In parallel, the parB gene was isolated from pMT329 by restriction with KpnI and XbaI. The two fragments were combined and ligated into pAMIOL-4 cut with AseI and NheI. Plasmid pDK4 was constructed by site-directed mutagenesis of pMT151 using primers CC2165-K13A-QCM1 and CC2165-K13A-QCM2. The mipZ K13A gene was released by restriction of the PCR product with NdeI and SacI and ligated into pMT151 cut with the same enzymes. To generate plasmids pDK2 and pDK3, the mipZ-yfp and mipZ D42A -yfp fusions were released from plasmids pMT151 and pMT362, respectively, by restriction with XhoI, subsequent blunting with T4 DNA polymerase, and restriction with NdeI. The fragments were then ligated into pBAD24-CB that had been cut with HindIII, blunted with T4 DNA polymerase, and then cut with NdeI.

Plasmids for protein overproduction in E. coli.
To construct plasmid pDK10, a fragment bearing mipZ K13A was PCR-amplified from pDK4 using primers Pxyl-1 and CC2165-revHindIII. The product was cut with NdeI and HindIII and ligated into equally treated pET21a(+). For the construction of pMT413 and pMT414, the mipZ G14V and mipZ K18Q alleles were PCRamplified from suitable templates using primers CC2165-uni2 and CC2165-rev. The PCR products were cut with NdeI and NcoI and the 5' fragments released were ligated into equally treated pMT183, respectively. To generate pHis17mipZ, the mipZ gene from C. crescentus (ATCC 19089D) was cloned into pHis17, generating a hybrid that encodes a C-terminally hexahistidine-tagged protein. For the construction of plasmid pHis17mipZD42A, overlap extension PCR was used to generate the mutant mipZ D42A allele, and the resulting PCR product was cloned into pHis17.

Plasmids for bacterial two-hybrid analysis.
To construct pDK34, mipZ was PCR-amplified from pMT183 using primers mipZ-BACTH-for and mipZ-KT25-rev. The reaction product was cut with BamHI and EcoRI and ligated into equally treated pKT25. Plasmids pDK35 and pDK36 were generated by PCR amplification of mipZ from pMT183 with primers mipZ-BACTH-for and mipZ-KNT25-rev, restriction of the reaction product with BamHI and EcoRI, and subsequent ligation of the fragments into equally cut pKNT25 and pUT18, respectively. To construct pDK37, mipZ was PCR-amplified from pMT183 using primers mipZ-BACTH-for and mipZ-UT18C-rev. The products were digested with BamHI and EcoRI and ligated into pUT18C treated with the same enzymes.
Plasmids for yeast one-hybrid analysis. To generate plasmid pDK14, mipZ was PCR-amplified from genomic DNA with primers CC2165-uni2 and CC2165rev. The product was cut with NdeI and EcoRI and ligated into equally digested pGADT7. Plasmids pDK22, pDK23, pDK24 and pDK25 were constructed in the same way using plasmids bearing mutant alleles of mipZ as PCR templates.
Other plasmids. To construct plasmid pMT428, P gidA was PCR-amplified from genomic DNA using primers PgidA-1 and PgidA-2. The product was cut with EcoRI and BamHI and ligated into equally digested pBluescript II-KS(+).

Protein expression and purification
For biochemical studies, hexahistidine-tagged forms of MipZ or its mutant derivatives as well as native ParB and FtsZ were overproduced in E. coli Rosetta2 (DE3)pLysS (Invitrogen) and purified as described previously (Thanbichler and Shapiro 2006). The protein used for crystallizing apo-MipZ in crystal form 1 and the MipZ dimer was prepared as follows: E. coli strain BL21 AI (Invitrogen) was transformed with pHis17mipZ or pHis17mipZD42A and grown at 37˚C in 2xTY medium or, in the case for selenomethionine (SeMet)-labelled protein, in M9 minimal medium (Miller 1972) that was supplemented with amino acids and selenomethionine ten minutes prior to induction as described previously (van den Ent et al. 1999). At an OD 600 of 0.8, protein synthesis was induced with the addition of 0.2% arabinose. After another 4 h of incubation, the cells were harvested by centrifugation, frozen, and stored at -70˚C. To purify wild-type MipZ, the cells were lysed in 50 mM Tris/HCl (pH 8.0). The lysate was cleared by ultracentrifugation at 40,000 rpm in Beckman Ti45 rotor (4°C) and loaded on a HisTrap column (GE Healthcare). After a wash with 50 mM Tris/HCl/300 mM NaCl (pH 7.0), the protein was eluted with 50 mM Tris/HCl/ 300 mM imidazole (pH 7.0). Further purification involved ion-exchange chromatography on a HiTrap Q column (GE Healthcare), on which MipZ was not retained. The flow-through was concentrated in Vivaspin protein concentrators (10,000 MWCO) at 4˚C. Subsequently, the protein was applied to a Sephacryl S-200 gel filtration column (GE Healthcare) equilibrated in TEN buffer (20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM sodium azide) containing 100 mM NaCl. The final sample was concentrated to 2.6 mg/mL and stored at -70˚C. In the case of the SeMet-labeled protein, a reducing agent (either DTT or TCEP) was added to all buffers throughout the purification. MipZ D42A was purified in a similar manner, leaving out the ion-exchange chromatography step and using TEN buffer with 300 mM salt for gel filtration. Mass spectral analysis revealed a mass of 31598 Da for native MipZ, and 31918 Da for selenomethionine-labeled MipZ, indicating that both proteins lacked the first methionine. All seven remaining methionine residues were successfully labelled with selenium. For the crystallization of apo-MipZ in crystal form 2, hexahistidine-tagged MipZ was purified from Rosetta2 (DE3)/pLysS transformed with pMT183 as described previously (Thanbichler and Shapiro, 2006).

Crystallization and structural analysis
For apo-MipZ in crystal form 1, crystallization conditions were screened using MipZ at 2.6 mg/mL plated into 200 nl sitting-well drops in 1440 standard conditions using a robotics facility (Stock et al. 2005). Only one condition (100 mM MES, 30% PEG 400, pH 6.5) was identified. Crystals of SeMet-substituted MipZ were obtained in the same condition with streak-seeding from the native MipZ crystals. Data sets for both proteins were obtained from crystals flash frozen in mother liquor. Data were collected at ID23-1, ESRF (Grenoble, France). MipZ D42A was screened under the same 1440 standard conditions in the presence of 1 mM ATP or 1 mM ATPγS. Crystallization was observed in three buffers, which all contained ATPγS and 100 mM Tris (pH 8.5) with 18% -20% ethanol. Data sets for MipZ D42A were obtained from crystals flash frozen in a solution of 95% ethanol/50 mM Tris (pH 8.5), and collected at ID23-2, ESRF (Grenoble, France). For apo-MipZ in crystal form 2, 768 standard conditions were screened in 200 nl sitting drops using a Microsys 4004 Cartesian robotics system and MipZ at a concentration of 10 mg/ml. Crystals obtained in a buffer containing 100 mM Hepes (pH 7.5), 10% PEG 10000, 4% ethylene glycol and 30% glycerol were flash-frozen, and data were collected at ID23-1, ESRF. All crystals were indexed and integrated using the MOSFLM package and further processed using the CCP4 package (Collaborative Computational Project, 1994). Refinement was performed using PHENIX (Adams et al., 2002;Adams et al., 2004) for apo-MipZ in crystal form 1 and MipZ D42A and REFMAC5 (Murshudov et al. 1997) for apo-MipZ in crystal form 2. The structure of apo-MipZ in crystal form 1 was solved by multiple-wavelength anomalous diffraction (MAD), while the structures of apo-MipZ in crystal form 2 and MipZ D42A were solved by molecular replacement. For downstream analysis, sequences were aligned with ClustalW (Larkin et al., 2007) and secondary structural elements were identified using DSSP (Kabsch and Sander, 1983). 3D structural alignments and stereo superpositions were generated with Superpose V1.0 (Maiti et al., 2004), and surface electrostatic potentials were calculated using CCP4mg (Potterton et al., 2004).

Yeast one-hybrid analysis
Yeast reporter strain AH109 was transformed with plasmids encoding fusions of MipZ or its mutant variants to the GAL4 transactivation domain. Liquid βgalactosidase activity assays were performed following the protocol provided with the Matchmaker TM GAL4 Two Hybrid System 3 (Clontech).

Nucleotide hydrolysis assays
For ATPase activity assays, MipZ or its derivatives (6 µM) were preincubated for 10 min at 30°C in buffer P (50 mM Hepes/NaOH, pH 7.2, 50 mM KCl, 10 mM MgCl 2 , 1 mM β-mercaptoethanol) supplemented with the indicated components. The reaction was started by addition of [α-32 P]-ATP (25 Ci/mmol) to a final concentration of 1 mM. At 10-min intervals, 2 µl samples were taken and immediately spotted onto PEIcellulose F thin-layer chromatography plates (Merck). The plates were developed in a solvent system containing 0.5 M LiCl and 1 M formic acid, air-dried, and exposed to a storage phosphor screen (GE Healthcare).
After scanning of the screen with a Storm 840 PhosphorImager (GE Healthcare), the amount of [α-32 P]-ADP present in the samples was quantified using ImageQuant 5.2 (Molecular Dynamics). For GTPase assays, FtsZ (3 µM) was preincubated with MipZ or BSA (6 µM) and 1 mM ATP for 10 min at 30°C in buffer P. After starting the reaction by addition of [α-32 P]-GTP (25 Ci/mmol) to a final concentration of 2 mM, samples were taken and analyzed for the amount of [α-32 P]-GDP produced in the reaction as described above.
To analyze the concentration dependence of MipZ's ATPase activity, varying amounts of MipZ were incubated in buffer P containing 2 mM [α-32 P]-ATP and 0.3 mg/ml bovine serum albumin and assayed for hydrolytic activity as described above.
To determine the effect of ParB on MipZ's ATPase activity, varying amounts of MipZ were incubated with ParB (4.5 µM) and/or plasmid pMT428 (3.6 kb, 0.2 µM), which bears a chromosomal fragment containing all predicted C. crescentus parS sites in buffer P containing 2 mM [α-32 P]-ATP and assayed for hydrolytic activity as described above.

Transient kinetic analysis
Transient kinetic experiments were conducted at 30°C in a Hi-Tech Scientific SF-61MX stopped-flow spectrofluorimeter operated in FRET mode, using an excitation wavelength of 297 nm and a 360-nm emission cut-off filter. All solutions were prepared in buffer P (50 mM Hepes/NaOH, pH 7.2, 50 mM KCl, 10 mM MgCl 2 , 1 mM β-mercaptoethanol). In general, 10-20 traces were averaged for each condition. The observed rate constants (k obs ) were determined by fitting of the data to a single-exponential equation using Kinetic Studio 2.00 (TgK Scientific). Secondary analysis was performed using Excel (Microsoft) or Origin 6.1 (OriginLab).

Surface plasmon resonance analysis
Biosensor experiments were performed on a Biacore T100 system at 25°C and a flow rate of 30 µl/min using buffer P2 (10 mM Hepes/NaOH, pH 7.2, 150 mM NaCl, 10 mM MgCl 2 , 0.05 % Tween 20) or buffer BC (50 mM Hepes/NaOH, pH 7.2, 50 mM KCl, 5 mM MgCl 2 ). Proteins were dialyzed against the respective assay buffer before use. To analyze the binding of MipZ to dsDNA (in buffer P2), biotinylated doublestranded oligonucleotides (top strands: parS WT , GAG-GCTTGTTTCACGTGAAACATCGG; parS mt , GAGG-CTTGTTTGAGCTCAAACATCGG; ran1, GAGGCA-GACTAGATCTTCTAGTTCGG; ran2, GTCGACAG-AGTTCGCTAACATCGAGC) were immobilized on a streptavidin-coated Sensor Chip SA (GE Healthcare) according to the manufacturer`s recommendations. Self-association of MipZ and interaction of MipZ with ParB (in buffer BC) was examined using a Sensor Chip CM5 (GE Healthcare) to which MipZ or ParB was covalently attached via amine coupling as described by the manufacturer. For self-association of MipZ, the reactions contained 0.5 µM protein and 1 mM ATP or ADP when appropriate. For the analysis of the MipZ-ParB or MipZ-DNA interactions, proteins were used at a concentration of 6 µM in the presence of 1 mM ADP or ATP, if not indicated otherwise. After the binding phase, the chip was washed with buffer lacking protein and nucleotides to follow the dissociation reactions. The chip surface was regenerated by washing for 20 s with 7 mM NaOH (for protein-DNA interactions) or buffer BC containing 2 M NaCl (for protein-protein interactions). In all experiments, a mock-treated reference cell lacking immobilized ligand was used to correct for non-specific binding to the sensor chip surface. Primary data analysis was performed with the BIAevaluation software (GE Healthcare). Dissociation rate constants were determined by fitting of the response curves to a single-exponential equation using the Origin 6.1 software (OriginLab).

Size exclusion chromatography
Wild-type MipZ or MipZ-D42A (both at 2 mg/ml) were incubated for 2 min in a buffer (20 mM CHES, pH 10, 100 mM NaCl, 1 mM MgCl 2 , 1 mM azide) containing either 1 mM ADP, ATP or ATPγS. The samples (25 µl) were spun in a microfuge for 1 min. Subsequently, the soluble fraction was injected onto a Superdex 200 Precision Column (2.4 ml; GE Healthcare) that was equilibrated in CHES buffer (20 mM CHES, 100 mM NaCl, 1 mM MgCl and 1 mM azide, pH 10) containing 0.1 mM ADP (for ADP-bound protein) or ATP (for ATP-and ATPγS-bound protein). Protein was eluted at a flow rate of 0.05 ml/min.

Electrophoretic mobility shift assays
The ability of MipZ to bind double-stranded DNA was assayed in the presence of 1.25 mM nucleotide (ADP, ATP or ATPγS) and 2.5 mM Mg 2+ . Binding reactions were performed in 15 µl volumes containing 25 mM Tris/HCl (pH 8.0), 100 mM KCl, 10% glycerol and 50 µg/mL BSA. Each reaction contained 60 ng (23 nM) of a 282-bp PCR product, amplified from Thermotoga maritima chromosomal DNA using the primers CCGGAATTCCATATGCAGCTCAAGGCCGCTAT-TGAG and CGGGATCCTCAAGACTCGCCACCA-TCGC and protein at concentrations ranging from 270 nM to 5.6 µM. Reactions were incubated at room temperature for 20 min and then loaded on a 4-20% gradient polyacrylamide gel (Criterion TBE, Bio-Rad), which was run for 90 min at 4˚C and a constant voltage of 100 V in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). DNA was stained with ethidium bromide before visualisation with UV light.

FRAP analysis
For FRAP analyses were performed on the same microscope setup equipped with a 488 nm-solid state laser and a 2D-VisiFRAP Galvo System multi-point FRAP module (Visitron Systems, Germany), using single 30-ms pulses at a laser power of 1%. Cells were transferred onto pads made of 1.5% agarose in M2G medium. After acquisition of a pre-bleach image and application of the laser pulse, fluorescence recovery was monitored at regular intervals over a period of at least two minutes. For each timepoint, the integrated fluorescence intensities of the whole cell, the bleached region and an equally sized unbleached region were measured using Metamorph 7.1.2. After background correction, the fluorescence intensities of the bleached and unbleached regions were divided by the whole cell intensity to correct for general photobleaching during the imaging process, and the average values of 17-29 cells were plotted. Recovery rates were determined with the SOLVER function of Microsoft Excel by fitting the data obtained for the bleached region to the single exponential function F(t) = A(1-e-k*t)+F0, where F(t) is the fluorescence at time t, A the maximum intensity, k the rate constant and F0 the relative fluorescence intensity at t=0 min. In all cases, fits with R2 ≥ 0.95 were obtained. Recovery half-times were calculated according to the equation t1/2 = ln(0.5)/-k.

Rotary shadowing electron microscopy
Phage ΦX174 RFII DNA (NEB) was digested with XhoI (NEB), extracted with phenol/ chloroform, and used at a final concentration of 100 pg µl -1 . MipZ D42A (at concentrations ranging from 150 nM to 5 µM) was incubated with ATPγS and/or DNA for 10 min at room temperature. The samples were then dialyzed for 1 h at 4°C against 150 mM ammonium bicarbonate, 40% glycerol, 100 µM ATP, using 10000 MWCO micro dialysis units (Pierce). 5-µl samples were spread onto freshly cleaved mica sheets and rotary-shadowed at an oblique angle of 8˚ and maximum rotation speed for 20 sec in a freeze fracture and etching system (Cressington Scientific, PA), using a platinum electron-beam metal evaporation gun operated at 80 mA/2.2 kV. Immediately following platinum coating, two sequential 16-sec depositions of carbon (100 mA/2.5 kV) were made at an angle of 90˚ to the rotating sample for increased structural support. After removal from the vacuum chamber, samples were floated off on water and transferred onto copper grids. Images were taken with a Philips EM 208 transmission electron microscope at 80 kV and magnifications between 79,000 and 180,000 x.