ZipA Binds to FtsZ with High Affinity and Enhances the Stability of FtsZ Protofilaments

A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0) pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them

Cobalt hexamine trichloride induced toroidal condensation of FtsZ

491-497The bacterial cell division protein, FtsZ, polymerizes to form a cytokinetic Z-ring at the midcell, which engineers bacterial cell division. Herein, we have examined the effects of an inorganic polyamine, cobalt hexamine trichloride, on the assembly of FtsZ in vitro. Cobalt hexamine trichloride strongly enhances FtsZ assembly, suppresses its GTPase activity and stabilizes FtsZ polymers. However, CoCl2 and MnCl2 have no detectable effect on the assembly of FtsZ in vitro. Interestingly, FtsZ is found to assemble into toroidal structures in the presence of low concentrations of cobalt hexamine trichloride. The Z-ring in bacterial cells appears to be a toroidal-like structure, suggesting that the use of cobalt hexamine trichloride may help to understand the assembly of FtsZ into toroidal structure. The toroidal structures may also serve as templates to build toroidal resonators for electrical and thermal applications

Bis-ANS inhibited the interaction of FtsZ and ZipA.

<p>Bis-ANS inhibited the binding of FITC-ZipA to FtsZ (Panel A). FITC-ZipA (0.5 µM) was incubated without or with different concentrations of bis-ANS for 5 min at 25°C. Then, 0.5 µM FtsZ was added to the reaction mixtures and incubated for an additional 15 min at 25°C and the fluorescence spectra were recorded. Bis-ANS inhibited the effects of ZipA on the assembly of FtsZ (Panel B). FtsZ (6 µM) was polymerized in the presence of 4 µM ZipA without or with different concentrations (5 and 10 µM) of bis-ANS. The polymeric FtsZ was collected by sedimentation and the amount of FtsZ in the pellets was estimated by coomassie-blue staining of the SDS-PAGE. The experiment was performed five times. Panel C shows electron micrographs of ZipA-induced FtsZ polymers in the absence (i) and presence of 5 µM (ii) and 10 µM (iii) bis-ANS, respectively. Scale bar is 1000 nm.</p

ZipA prevented dilution-induced disassembly of FtsZ polymers.

<p>FtsZ (25 µM) was polymerized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028262#s2" target="_blank">materials and methods</a> section. The preformed FtsZ polymers were diluted 20 times in warm 25 mM PIPES buffer, pH 6.8 without and with different concentrations of ZipA and incubated for an additional 5 min at 37°C. The polymers were collected through centrifugation and the amount of FtsZ in the pellet was estimated using Coomassie blue stained SDS-PAGE. Lanes 1–5 denote FtsZ polymers pelleted in the absence and presence of 1, 2, 3 and 4 µM ZipA, respectively (Panel A). The relative amount of FtsZ in the pellets with respect to control was plotted against ZipA concentration (Panel B).</p

Characterization of equilibrium binding of FITC-ZipA to FtsZ.

<p>The fluorescence emission spectra of FITC-ZipA (0.5 µM) at pH 6.8 in the absence (▪) and presence of 10 (▴), 25 (▵), 50 (•), 100 (○), 200 (♦), 300 (◊), 500 (x), 600 (*), 1000 (+), 1500 (−) nM FtsZ (Panel A). Excitation wavelength used was 495 nm. The changes in the fluorescence intensities of FITC-ZipA in the presence of different concentrations of FtsZ at pHs of 6.0 (i), 6.8 (ii) and 8.0 (iii) (Panel B) and in the presence of 500 mM NaCl at pH 6.8 (Panel C) are shown.</p

Effects of ZipA on the assembly and bundling of FtsZ at different pHs.

<p>FtsZ (6 µM) was polymerized in 50 mM PIPES buffer containing 1 mM MgCl₂ and 1 mM GTP at 37°C in the absence (i, ii, iii) and presence (iv, v, vi) of ZipA (2 µM) at pHs of 6.0 (Panel A), 6.8 (Panel B) and 8.0 (Panel C). ZipA formed aggregates at different pHs used in this study (vii, viii, ix). Scale bar is 1000 nm.</p

Effects of ZipA on the assembly kinetics of FtsZ.

<p>FtsZ (6 µM) was polymerized in the absence (○) and presence of 2 µM (•) and 4 µM (▪) of ZipA (Panel A). FtsZ (6 µM) was polymerized in the absence of ZipA for 5 min. Then, 4 µM ZipA was added to the reaction milieu (indicated by an arrow) and the assembly kinetics of FtsZ was monitored for an additional 10 min (Panel B).</p

ZipA induced bundling of FtsZ and co-polymerized with FtsZ.

<p>FtsZ (2 µM) was polymerized in 25 mM PIPES buffer, pH 6.8 containing 1 mM MgCl₂ and 1 mM GTP at 37°C in the absence and presence of ZipA. FtsZ polymers were observed using a fluorescence microscope and a differential interference contrast microscope. FtsZ was polymerized in the absence (i), and presence of 0.5 (ii), 1.0 (iii) and 2.0 (iv) µM FITC-ZipA (Panel A). 0.5 (i), 1.0 (ii) and 2.0 (iii) µM of FITC-ZipA in the absence of FtsZ are shown (Panel B). FITC-FtsZ was polymerized in the absence (i), and presence of 0.5 (ii), 1.0 (iii) and 2.0 (iv) µM ZipA (Panel C), respectively. Scale bar is 10 µm.</p

An analysis of FtsZ assembly using small angle X-ray scattering and electron microscopy

Small angle X-ray scattering (SAXS) was used for the first time to study the self-assembly of the bacterial cell division protein, FtsZ, with three different additives: calcium chloride, monosodium glutamate and DEAE-dextran hydrochloride in solution. The SAXS data were analyzed assuming a model form factor and also by a model-independent analysis using the pair distance distribution function. Transmission electron microscopy (TEM) was used for direct observation of the FtsZ filaments. By sectioning and negative staining with glow discharged grids, very high bundling as well as low bundling polymers were observed under different assembly conditions. FtsZ polymers formed different structures in the presence of different additives and these additives were found to increase the bundling of FtsZ protofilaments by different mechanisms. The combined use of SAXS and TEM provided us a significant insight of the assembly of FtsZ and microstructures of the assembled FtsZ polymers