E93R substitution of Escherichia coli FtsZ induces bundling of protofilaments, reduces GTPase activity, and impairs bacterial cytokinesis

Abstract

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7-1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg(93)-Glu(138) salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.

FIGURE 1.

A, effect of E93R mutation on FtsZ assembly. WT and mutant FtsZ (14.4 μm) were polymerized at 37 °C. Shown are the light scattering traces of WT-FtsZ (○), E93R-FtsZ (●), D84A-FtsZ (♦), and S231R-FtsZ (△) in the presence of 1 mm GTP and E93R-FtsZ in the absence of 1 mm GTP (□). B, WT-FtsZ and E93R-FtsZ existed in the same oligomeric state in solution. E93R-FtsZ or WT-FtsZ was eluted through a size exclusion column (Superdex G-200). The elution profiles of WT- and E93R-FtsZs are similar. C, E93R mutant induced bundling of FtsZ protofilaments. E93R- and WT-FtsZ were polymerized at 37 °C in the assembly buffer containing 25 mm Pipes (pH 6.8), 50 mm KCl, 10 mm MgCl₂, and 1 mm GTP. Shown are the electron micrographs of WT-FtsZ and E93R-FtsZ polymerized for 5 min (i and ii) and WT-FtsZ and E93R-FtsZ polymerized for 30 min (iii and iv). Scale bar, 1000 nm. Fluorescence images of FITC-labeled WT (v) and E93R (vi) FtsZ polymers (polymerized for 5 min) are also shown. Scale bar, 10 μm. HSA, human serum albumin.

FIGURE 2.

E93R mutation decreased the critical concentration of EcFtsZ. Different concentrations of WT-FtsZ (●) and E93R-FtsZ (○) were polymerized at 37 °C for 10 min. Polymers were pelleted down by high speed centrifugation. The experiment was performed three times.

FIGURE 3.

A, E93R mutation suppressed the GTPase activity of FtsZ. E93R- and WT-FtsZ were polymerized at 37 °C in the assembly buffer containing 25 mm Pipes (pH 6.8), 50 mm KCl, 10 mm MgCl₂, and 1 mm GTP. P_i released was monitored by a standard malachite green ammonium molybdate assay. Shown are the mol of P_i released/mol of WT-FtsZ (●) and E93R-FtsZ (○). B, E93R mutation did not inhibit the TNPGTP binding to FtsZ. WT-FtsZ or E93R-FtsZ (6 μm) was incubated with 50 μm TNPGTP on ice for 4 h. Fluorescence spectra were taken using 410 nm as the excitation wavelength. The emission spectra of free TNPGTP (▴) and TNPGTP bound with WT (○) and E93R (●) FtsZ have been shown in the figure. C, E93R mutation increased the steady state duration of FtsZ assembly. WT and mutant FtsZ (18 μm) were polymerized in the limited amount of GTP (250 μm) at 37 °C. Shown are the light scattering traces of WT-FtsZ (○) and E93R-FtsZ (●). D, E93R mutation prevented the disassembly of preformed FtsZ polymers. WT- and E93R-FtsZ (36 μm) were polymerized in the presence of 1 m glutamate at 37 °C. Polymers were diluted 30-fold in warm 25 mm Pipes buffer and incubated for an additional 5 min at 37 °C as described under “Experimental Procedures.” Polymers were collected by centrifugation and loaded on SDS-PAGE. Shown in the figure are WT-FtsZ (lane 1) and E93R-FtsZ (lane 2) collected after centrifugation. The experiment was repeated three times. Error bars, S.D.

FIGURE 4.

Secondary structure analysis of WT and mutant FtsZ. Far-UV CD spectra of WT-FtsZ, E93R-FtsZ, E93V-FtsZ, and E93A-FtsZ (4.8 μm) were monitored over the wavelength range 190–260 nm using a 0.1-cm path length cuvette in a JASCO J810 spectropolarimeter. Shown are the far-UV CD spectra of WT-FtsZ (●), E93R-FtsZ (▴), E93V-FtsZ (■), and E93A-FtsZ (♦).

FIGURE 5.

E93R mutant showed a dominating effect on assembly of WT-FtsZ. Increasing concentrations of E93R-FtsZ were mixed with decreasing concentrations of WT-FtsZ while keeping the total protein concentration in the reaction mixture constant (7.2 μm). Polymers were collected by centrifugation, and protein concentration in the supernatant was estimated by the Bradford method (26). Error bars, S.D.

FIGURE 6.

Effect of E93R mutation on the Z-ring assembly and nucleoid segregation in JKD7–1/pKD3 cells. WT-FtsZ and E93R-FtsZ genes were inserted in complementation vector pJSB2. JKD7–1/pKD3 cells were transformed with the recombinant plasmids. A, distribution of JKD7–1/pKD3 cell lengths in cells expressing WT-FtsZ and E93R-FtsZ in the presence of glucose (0.5%) at 30 °C and different concentrations (0.1, 0.2, and 0.3%) of arabinose at 42 °C. A length of 200 cells was measured in each case, and the percentage of cells was plotted as a function of cell length. WT-FtsZ (B) and E93R-FtsZ (C) were expressed in the presence of different concentrations of arabinose (0.1, 0.2, and 0.3%) at 42 °C in JKD7–1/pKD3 cells. FtsZ and DNA were stained with anti-FtsZ antibody and DAPI, respectively. Scale bar, 5 μm.

FIGURE 7.

Homology model of E93R-FtsZ built using MtbFtsZ (Protein Data Bank code 1RQ7; top) and MjFtsZ (Protein Data Bank code 1W59; bottom) as templates. Homology modeling was carried out using the automated 3D-Jigsaw server. The alignment reported by the server did not require any tweaking because MtbFtsZ, EcFtsZ, and MjFtsZ share high sequence similarity. A, the two chains of a dimer are shown as gray and black wire frames. Residues Arg⁹³, Met¹⁰⁴, and Glu¹³⁸ are shown as green or cyan sticks. The dimer was constructed by combining models built on both templates. The dimers were built to mimic the dimerization in their respective templates. B, the salt bridge between Arg⁹³:Nη and Glu¹³⁸:Oϵ in E93R-FtsZ is shown by a black dashed line (lower panel); the distance between these two atoms is 2.8 Å.