Investigation of regulation of FtsZ assembly by SulA and development of a model for FtsZ polymerization

In Escherichia coli FtsZ organizes into a cytoskeletal ring structure, the Z ring, which effects cell division. FtsZ is a GTPase, but the free energy of GTP hydrolysis does not appear to be used for generation of the constriction force, leaving open the question of the function of the GTPase activity of FtsZ. Here we study the mechanism by which SulA, an inhibitor of FtsZ induced during the SOS response, inhibits FtsZ function. We studied the effects of SulA on the in vitro activities of FtsZ, on Z rings in vivo, and on a kinetic model for FtsZ polymerization in silico. We found that the binding of SulA to FtsZ is necessary but not sufficient for inhibition of polymerization, since the assembly of FtsZ polymers in the absence of the GTPase activity was not inhibited by SulA. We developed a new model for FtsZ polymerization that accounts for the cooperativity of FtsZ and could account for cooperativity observed in other linear polymers. When SulA was included in the kinetic scheme, simulations revealed that SulA with strong affinity for FtsZ delayed, but did not prevent, the assembly of polymers when they were not hydrolyzing GTP. Furthermore, the simulations indicated that SulA controls the assembly of FtsZ by binding to a polymerization-competent form of the FtsZ molecule and preventing it from participating in assembly. In vivo stoichiometry of the disruption of Z rings by SulA suggests that FtsZ may undergo two cooperative transitions in forming the Z ring.     

Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity

The assembly and stability of FtsZ protofilaments have been shown to play critical roles in bacterial cytokinesis. Recent evidence suggests that FtsZ may be considered as an important antibacterial drug target. Curcumin, a dietary polyphenolic compound, has been shown to have a potent antibacterial activity against a number of pathogenic bacteria including Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus. We found that curcumin induced filamentation in the Bacillus subtilis 168, suggesting that it inhibits bacterial cytokinesis. Further, curcumin strongly inhibited the formation of the cytokinetic Z-ring in B. subtilis 168 without detectably affecting the segregation and organization of the nucleoids. Since the assembly dynamics of FtsZ protofilaments plays a major role in the formation and functioning of the Z-ring, we analysed the effects of curcumin on the assembly of FtsZ protofilaments. Curcumin inhibited the assembly of FtsZ protofilaments and also increased the GTPase activity of FtsZ. Electron microscopic analysis showed that curcumin reduced the bundling of FtsZ protofilaments in vitro. Further, curcumin was found to bind to FtsZ in vitro with a dissociation constant of 7.3+/-1.8 microM and the agent also perturbed the secondary structure of FtsZ. The results indicate that the perturbation of the GTPase activity of FtsZ assembly is lethal to bacteria and suggest that curcumin inhibits bacterial cell proliferation by inhibiting the assembly dynamics of FtsZ in the Z-ring.     

Totarol inhibits bacterial cytokinesis by perturbing the assembly dynamics of FtsZ

Totarol, a diterpenoid phenol, has been shown to inhibit the proliferation of several pathogenic Gram-positive bacteria including Mycobacterium tuberculosis. In this study, totarol was found to inhibit the proliferation of Bacillus subtilis cells with a minimum inhibitory concentration of 2 microM. It did not detectably perturb the membrane structure of B. subtilis; it strongly induced the filamentation in B. subtilis cells, suggesting that it inhibits bacterial cytokinesis. Totarol (1.5 microM) reduced the frequency of the Z-ring occurrence per micrometer of the bacterial cell length but did not affect the nucleoid frequency, suggesting that it blocks cytokinesis by inhibiting the formation of the Z-ring. The assembly dynamics of FtsZ is thought to play an important role in the formation and functioning of the Z-ring, a machine that engineers cytokinesis in bacteria. Since totarol was shown to inhibit the proliferation of M. tuberculosis, we examined the effects of totarol on the assembly dynamics of M. tuberculosis FtsZ (MtbFtsZ) in vitro. Totarol decreased the assembly of MtbFtsZ protofilaments and potently suppressed the GTPase activity of MtbFtsZ. It bound to MtbFtsZ with a dissociation constant of 11 +/- 2.3 microM. It increased the fluorescence intensity of the MtbFtsZ-1-anilinonaphthalene-8-sulfonic acid complex and inhibited the fluorescence intensity of N-(1-pyrene)maleimide-labeled MtbFtsZ, suggesting that totarol induces conformational changes in MtbFtsZ. The results indicated that totarol can perturb the assembly dynamics of FtsZ protofilaments in the Z-ring. Totarol exhibited extremely weak inhibitory effects on HeLa cell proliferation. It did not affect microtubule organization in HeLa cells. The results suggest that totarol inhibits bacterial proliferation by targeting FtsZ and it may be useful as a lead compound to develop an effective antitubercular drug.     

Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.     

Bacterial SOS Checkpoint Protein SulA Inhibits Polymerization of Purified FtsZ Cell Division Protein

Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring.

Immunoelectron microscopy was used to assess the effects of inhibitors of cell division on formation of the FtsZ ring in Escherichia coli. Induction of the cell division inhibitor SulA, a component of the SOS response, or the inhibitor MinCD, a component of the min system, blocked formation of the FtsZ ring and led to filamentation. Reversal of SulA inhibition by blocking protein synthesis in SulA-induced filaments led to a resumption of FtsZ ring formation and division. These results suggested that these inhibitors block cell division by preventing FtsZ localization into the ring structure. In addition, analysis of min mutants demonstrated that FtsZ ring formation was also associated with minicell formation, indicating that all septation events in E. coli involve the FtsZ ring.     

Dynamic assembly of FtsZ regulated by GTP hydrolysis.

FtsZ forms a cytokinetic ring, designated the Z ring, that directs cytokinesis in prokaryotes. It has limited sequence similarity to eukaryotic tubulins and, like tubulin, it has GTPase activity and the ability to assemble into various structures including protofilaments, bundles and minirings. By using both electron microscopy and sedimentation, we demonstrate that FtsZ from Escherichia coli undergoes a strictly GTP-dependent polymerization and the polymers disappear as the GTP is consumed. Thus, FtsZ polymerization, like that of tubulin, is dynamic and regulated by GTP hydrolysis. These results provide the basis for the dynamics of the Z ring and favor a model in which the Z ring is formed by a nucleation event.     

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Cationic lipid enhances assembly of bacterial cell division protein FtsZ: a possible role of bacterial membrane in FtsZ assembly dynamics

The assembly of FtsZ plays an important role in bacterial cell division. Lipids in the bacterial cell membrane have been suggested to play a role in directing the site of FtsZ assembly. Using lipid monolayer and bilayer (liposome) systems, we directly examined the effects of cationic lipids on FtsZ assembly. We found that cationic lipids enhanced the assembly of FtsZ in association with an increase in the GTPase activity of FtsZ. The system consisting of lipid monolayer and bilayer (liposome) may mimic the bacterial membrane and therefore, the data might indicate the influence of bacterial membrane on the assembly of FtsZ protofilaments.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

A rapid fluorescence assay for FtsZ assembly indicates cooperative assembly with a dimer nucleus

FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?     

SepF increases the assembly and bundling of FtsZ polymers and stabilizes FtsZ protofilaments by binding along its length

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90 degrees light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZDelta16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.     

Sanguinarine blocks cytokinesis in bacteria by inhibiting FtsZ assembly and bundling

Bacterial diseases are among the leading causes of human death. The development of antibiotic resistance greatly contributes to the high mortality rate, and thus, the discovery of antibacterial drugs with novel mechanisms of action is needed. In this study, we found that sanguinarine, a benzophenanthridine alkaloid, strongly induced filamentation in both Gram-positive and Gram-negative bacteria and prevented bacterial cell division by inhibiting cytokinesis. Sanguinarine did not perturb the membrane structure in Escherichia coli. However, it perturbed the cytokinetic Z-ring formation in E. coli. In addition, sanguinarine strongly reduced the frequency of the occurrence of Z rings/micrometer of Bacillus subtilis length but did not alter the number of nucleoids/micrometer of cell length. The results suggested that sanguinarine inhibited cytokinesis in B. subtilis by inhibiting Z-ring formation without affecting nucleoid segregation. Sanguinarine inhibited the assembly of purified FtsZ and reduced the bundling of FtsZ protofilaments in vitro. Further, the interaction of sanguinarine to FtsZ was investigated using size-exclusion chromatography, an extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, and tryptophan fluorescence of mutated FtsZ (Y371W). Sanguinarine was found to bind to FtsZ with a dissociation constant of 18-30 microM. The results together show that sanguinarine inhibits bacterial division by perturbing FtsZ assembly dynamics in the Z ring and provide evidence in support of the hypothesis that the assembly and bundling of FtsZ play a critical role in bacterial cytokinesis. The results suggest that sanguinarine may be used as a lead compound to develop FtsZ-targeted antibacterial agents.     

A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ

FtsZ polymerizes to form a dynamic ring structure called the Z-ring at the midcell of bacteria. EzrA, a membrane protein, has been shown to prevent the formation of aberrant Z-rings in the low GC Gram-positive bacteria by inhibiting FtsZ assembly. In this study, we show that Bacillus subtilis (B. subtilis) EzrA inhibited the assembly and bundling of B. subtilis FtsZ. It increased the critical concentration of FtsZ assembly and depolymerized the preformed FtsZ polymers in vitro. We obtained evidence suggesting that B. subtilis EzrA forms complex with B. subtilis FtsZ in vitro. EzrA was found to bind to FtsZ at a single site with a dissociation constant of 4.3 +/- 0.6 microM. EzrA-FtsZ interaction has a significant electrostatic contribution as apparent from the effect of salt on their binding interactions. To elucidate the site of interaction between EzrA and FtsZ, we deleted 16 amino acid residues from the extreme C-terminal tail of B. subtilis FtsZ, which are conserved in FtsZ orthologues. EzrA did not inhibit the assembly of C-terminal truncated B. subtilis FtsZ. It also did not bind to the C-terminal truncated FtsZ detectably, suggesting that EzrA interacts with FtsZ through its conserved C-terminal tail residues. Further, a 17-residue synthetic peptide (365-382) of the C-terminal tail of FtsZ (CTP17) was used to probe the interaction of EzrA with the C-terminal tail of FtsZ. CTP17 bound to EzrA, inhibited the binding of EzrA to FtsZ, and surmounted the inhibitory effects of EzrA on the assembly of FtsZ in vitro. The data together showed that EzrA binds to the C-terminal tail of FtsZ. FtsA, a positive regulator of FtsZ assembly, is also known to interact with the C-terminal tail of FtsZ. The results indicated an interesting possibility that the assembly dynamics of FtsZ in the Z-ring is regulated by the competition between positive and negative regulators sharing the same binding site on FtsZ.     

Guanine nucleotide-dependent assembly of FtsZ into filaments.

FtsZ is an essential cell division protein that is localized to the leading edge of the bacterial septum in a cytokinetic ring. It contains the tubulin signature motif and is a GTP binding protein with a GTPase activity. Further comparison of FtsZ with eukaryotic tubulins revealed some additional sequence similarities, perhaps indicating a similar GTP binding site. Examination of FtsZ incubated in vitro by electron microscopy revealed a guanine nucleotide-dependent assembly into protein filaments, supporting the hypothesis that the FtsZ ring is formed through self-assembly. FtsZ3, which is unable to bind GTP, does not polymerize, whereas FtsZ2, which binds GTP but is deficient in GTP hydrolysis, is capable of polymerization.     

Glutamate-induced assembly of bacterial cell division protein FtsZ

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; approximately 90% of the protein was sedimented as polymer in the presence of 1 m glutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 m glutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.     

EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.