FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

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.     

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.     

New data on structures formed by the FtsZ protein during the cell division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> obtained using the localization microscopy method

The FtsZ protein, a bacterial tubulin homolog, is one of the key proteins in bacterial cell division, forming a contractile Z-ring in the middle of the dividing cell. In the present study, immunofluorescence staining, in combination with the localization microscopy method, was used for visualization of the structures formed by unlabelled FtsZ in Escherrichia coli cells. The techniques employed allowed reconstruction of the multistep mechanism of formation of FtsZ structures during the cytokinesis process. New data were obtained confirming the hypothesis that FtsZ is a helixlike structure that constricts during the division, producing constriction between the daughter cells.     

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.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.     

Influence of FtsZ proteins from some mycoplasma species on the division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

FtsZ is a highly conserved protein that performs one of the key roles in cell division of most prokaryotes. At the present time it is believed that the main role of FtsZ in the cells of the microorganisms studied (Escherichia coli, Bacillus subtilis, etc.) is to control the process of cell wall synthesis at the site of future septum. In this regard, the presence of FtsZ in the cells of mycoplasmas – bacteria that lack the cell wall, looks intriguing. In the present work we investigated the influence of FtsZ proteins of three mycoplasmas – Mycoplasma hominis, Mycoplasma gallisepticum and Acholeplasma laidlawii – on the cytokinesis of E. coli. FtsZ proteins were able to interact with the E. coli division machinery, including inhibiting the cytokinesis process, while demonstrating various patterns of distribution in the cell. The data obtained support the hypothesis that FtsZ plays a significant role in the division of mycoplasma cells.     

Large ring polymers align FtsZ polymers for normal septum formation

Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria. Mutation of sepF results in deformed division septa. It is unclear how SepF contributes to the synthesis of normal septa. We have studied SepF by electron microscopy (EM) and found that the protein assembles into very large (～50 nm diameter) rings. These rings were able to bundle FtsZ protofilaments into strikingly long and regular tubular structures reminiscent of eukaryotic microtubules. SepF mutants that disturb interaction with FtsZ or that impair ring formation are no longer able to align FtsZ filaments in vitro, and fail to support normal cell division in vivo. We propose that SepF rings are required for the regular arrangement of FtsZ filaments. Absence of this ordered state could explain the grossly distorted septal morphologies seen in sepF mutants.     

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.     

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.     

SepF, a novel FtsZ-interacting protein required for a late step in cell division

Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.     

Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.     

FtsZ and the division of bacterial cell

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.     

Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ

With the emergence of multidrug-resistant bacterial strains, there is a dire need for new drug targets for antibacterial drug discovery and development. Filamentous temperature sensitive protein Z (FtsZ), is a GTP-dependent prokaryotic cell division protein, sharing less than 10% sequence identity with the eukaryotic cell division protein, tubulin. FtsZ forms a dynamic Z-ring in the middle of the cell, leading to septation and subsequent cell division. Inhibition of the Z-ring blocks cell division, thus making FtsZ a highly attractive target. Various groups have been working on natural products and synthetic small molecules as inhibitors of FtsZ. This review summarizes the recent advances in the development of FtsZ inhibitors, focusing on those in the last 5 years, but also includes significant findings in previous years.     

钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻（Spirulina platensis）FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis

The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.     

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.     

FtsZ may have dual roles in the filamentous cyanobacterium Nostoc/Anabaena sp. strain PCC 7120

The cellular and subcellular localization of FtsZ, a bacterial cell division protein, were investigated in vegetative cells of the filamentous cyanobacterium Nostoc/Anabaena sp. strain PCC 7120. We show by using immunogold-transmission electron microscopy that FtsZ forms a ring structure in a filamentous cyanobacterium, similar to observations in unicellular bacteria and chloroplasts. This finding, that the FtsZ in a filamentous cyanobacterium accumulates at the growing edge of the division septa leading to the formation of the typical prokaryotic Z-ring arrangement, is novel. Moreover, an apparent cytoplasmic distribution of FtsZ occurred in vegetative cells. During the transition of vegetative cells into terminally differentiated heterocysts the cytoplasmic FtsZ levels decreased substantially. These findings suggest a conserved function of FtsZ among prokaryotes, including filamentous cyanobacteria with cell differentiation capacity, and possibly a role of FtsZ as a cytoskeletal component in the cytoplasm.     

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.