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.     

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 Rhodanine Derivative CCR-11 Inhibits Bacterial Proliferation by Inhibiting the Assembly and GTPase Activity of FtsZ

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (～15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.     

Berberine targets assembly of Escherichia coli cell division protein FtsZ

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.     

Effects of pH and ionic strength on the assembly and bundling of FtsZ protofilaments: a possible role of electrostatic interactions in the bundling of protofilaments

Assembly, bundling and stability of FtsZ protofilaments are important for the formation and functioning of the cytokinetic Z-ring during bacterial division. We found that the bundling of FtsZ protofilaments decreased strongly with increasing pH from 6.0 to 7.9, while the assembly of FtsZ monomers did not decrease considerably. In addition, the disassembly of FtsZ protofilaments was strongly suppressed at pH 6.0 as compared to the elevated pHs. The far-UV circular dichroism spectra of the native FtsZ and the tryptophan emission spectra of mutated FtsZ (Y371W) did not change by increasing pH from 6 to 7.9 indicating that the structure of FtsZ was not altered significantly. Further, the inhibition of bundling of FtsZ protofilaments predominantly, and the inhibition of assembly to a lesser extent by salt indicated that electrostatic interactions are important for the assembly and bundling of FtsZ protofilaments. These observations are supported by the results of computational docking of Escherichia coli dimer structure. The results suggest that the basic intracellular pH (7.4-7.8) of E. coli may play a role in regulating the assembly dynamics of FtsZ in the Z-ring by reducing protofilament stability and bundling in bacterial cytoplasm.     

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.     

Cell division genes and proteins in bacterial cells

In this review, genes and proteins involved in cytokinesis and cell proliferation of cell-wall bacteria and mycoplasms are considered. We hope that this comparative analysis of genes and proteins of phylogenetically distant bacteria, including the minimal cells of mycoplasmas, can be useful for understanding the basic principles of prokaryotic cell division. The ftsZ gene was found among representatives of all bacterial groups. The recent data indicate that FtsZ protein plays the central role in the process of bacterial cell division. FtsZ protein was revealed in all Eubacterial groups (including mycoplasmas), in Archaebacteria and chloroplasts, All FtsZ proteins are able to form protofilaments as a result of polymerization in vitro and demonstrate GTF-ase activity. On the base of these properties and some similarities in amino acid sequences with tubulins, it has been suggested that FtsZ protein is an evolutionary ancestor of Eukaryotic tubulins. On the earliest stage of bacterial cytokinesis FtsZ protein assembles into a submembranous Z-ring which encircles bacterial cell in the predivisional site. Some other bacterial proteins take part in stabilization and contraction of the Z-ring, which is considered as a cytoskeleton-like bacterial structure.     

Acid-induced loss of functional properties of bacterial cell division protein FtsZ: evidence for an alternative conformation at acidic pH

Several types of bacteria live in highly acidic environments. Since the assembly of FtsZ is important for bacterial cytokinesis, the effects of pH on the assembly and structural properties of FtsZ were examined. FtsZ retained GTP binding ability but lost GTPase activity at pH 2.5. In the presence of GTP, FtsZ formed protofilaments at pH 7 while it formed aggregates instead of protofilaments at pH 2.5, indicating that GTP hydrolysis is important for the assembly of FtsZ into protofilaments. Further, the acid-inactivated state of FtsZ recovered its structural and functional properties upon refolding at pH 7, indicating that the cellular functions of FtsZ may be restored after removal of the external stress. In addition, the affinity of 1-anilinonaphthalene-8-sulfonic acid (ANS) binding to FtsZ was found to be higher at pH 2.5 than at pH 7. FtsZ-ANS complex had a higher quantum yield and lifetime at pH 2.5 than at pH 7. However, the secondary structures of FtsZ were similar at pH 7 and 2.5, indicating that FtsZ attained an alternatively folded state (A) at pH 2.5, which has some characteristics of a molten-globule-like state. The A state was more stable than the native state (N) against urea-induced unfolding. The transition from N to A state involves the formation of aggregates of FtsZ (I). The association of FtsZ monomers occurred in the narrow pH range (3.2-2.8) and it was found to be a fully reversible process. The results suggest that a productive intermediate (I) forms in the acid-induced unfolding pathway of FtsZ and that the unfolding pathway may be minimally described as N <==> I <==> A.     

The Nucleoid Occlusion SlmA Protein Accelerates the Disassembly of the FtsZ Protein Polymers without Affecting Their GTPase Activity

Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ.     

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.     

A 4-aminofurazan derivative-A189-inhibits assembly of bacterial cell division protein FtsZ in vitro and in vivo

Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4-aminofurazan derivative was found to inhibit FtsZ GTPase with an IC(50) of 80 mug/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189-treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA-deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.     

Substituted 1,6-diphenylnaphthalenes as FtsZ-targeting antibacterial agents

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multidrug-resistant strains of infectious bacteria. Several 1-phenylbenzo[c]phenanthridines exhibit notable antibacterial activity. Based upon their structural similarity to these compounds, a distinct series of substituted 1,6-diphenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In addition, the effect of select 1,6-diphenylnaphthalenes on the polymerization dynamics of S. aureus FtsZ and mammalian β-tubulin was also assessed. The presence of a basic functional group or a quaternary ammonium substituent on the 6-phenylnaphthalene was required for significant antibacterial activity. Diphenylnaphthalene derivatives that were active as antibiotics, did exert a pronounced effect on bacterial FtsZ polymerization and do not appear to cross-react with mammalian tubulin to any significant degree.     

FtsZ inhibitors as a new genera of antibacterial agents

The continuous emergence and rapid spread of a multidrug-resistant strain of bacterial pathogens have demanded the discovery and development of new antibacterial agents. A highly conserved prokaryotic cell division protein FtsZ is considered as a promising target by inhibiting bacterial cytokinesis. Inhibition of FtsZ assembly restrains the cell-division complex known as divisome, which results in filamentation, leading to lysis of the cell. This review focuses on details relating to the structure, function, and influence of FtsZ in bacterial cytokinesis. It also summarizes on the recent perspective of the known natural and synthetic inhibitors directly acting on FtsZ protein, with prominent antibacterial activities. A series of benzamides, trisubstituted benzimidazoles, isoquinolene, guanine nucleotides, zantrins, carbonylpyridine, 4 and 5-Substituted 1-phenyl naphthalenes, sulindac, vanillin analogues were studied here and recognized as FtsZ inhibitors that act either by disturbing FtsZ polymerization and/or GTPase activity. Doxorubicin, from a U.S. FDA, approved drug library displayed strong interaction with FtsZ. Several of the molecules discussed, include the prodrugs of benzamide based compound PC190723 (TXA-709 and TXA707). These molecules have exhibited the most prominent antibacterial activity against several strains of Staphylococcus aureus with minimal toxicity and good pharmacokinetics properties. The evidence of research reports and patent documentations on FtsZ protein has disclosed distinct support in the field of antibacterial drug discovery. The pressing need and interest shall facilitate the discovery of novel clinical molecules targeting FtsZ in the upcoming days.     

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.     

Essential cell division protein FtsZ assembles into one monomer-thick ribbons under conditions resembling the crowded intracellular environment

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked.     

Rational Design of Berberine-Based FtsZ Inhibitors with Broad-Spectrum Antibacterial Activity

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.     

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.     

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.     

Ruthenium red-induced bundling of bacterial cell division protein, FtsZ

The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.