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.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

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.     

FtsZ-ZapA-ZapB interactome of Escherichia coli

Bacterial cell division relies on the formation and contraction of the Z ring, coordinated and regulated by a dynamic protein complex called the divisome. The cell division factor ZapA interacts directly with FtsZ and thereby increases FtsZ protofilament association and Z-ring stability. Here, we investigated ZapB interaction with ZapA and its effect on Z-ring formation and FtsZ protofilament bundling. The combination of the ftsZ84 allele that encodes an FtsZ variant that polymerizes inefficiently with a zapB null mutant resulted in a synthetic defective phenotype. Overproduction of ZapA led to the formation of aberrant FtsZ helical structures and delocalization of ZapB. The N-terminal end of ZapB was essential for ZapB-ZapA interaction, and amino acid changes close to the N terminus of ZapB exhibited reduced interaction with ZapA. Sedimentation assays showed that ZapB interacts strongly with ZapA and reduces ZapA's interaction with FtsZ in vitro. The morphology of the structures formed by ZapA and ZapB together was similar to the cables formed by ZapB in the presence of CaCl(2), a known ZapB bundling agent. The in vivo and in vitro data support a model in which ZapA interacts strongly with ZapB and the ZapA-ZapB interaction is favored over ZapA-FtsZ.     

Recruitment of ZipA to the division site by interaction with FtsZ

ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle. As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring. In this study, we have investigated the requirements for ZipA localization to the division site. ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ. Consistent with this, apparently normal Z rings are formed in the absence of ZipA. The interaction between FtsZ and ZipA occurs through their carboxy-terminal domains. Although a MalE-ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments. These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.     

Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery

SulA and MinCD are specific inhibitors of cell division in Escherichia coli. In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented the formation of high-molecular-weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo. However, MinCD did prevent the formation of FtsA rings. Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.     

衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E．coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。     

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.     

Inhibition of cell division suppresses heterocyst development in Anabaena sp. strain PCC 7120

When the filamentous cyanobacterium Anabaena PCC 7120 is exposed to combined nitrogen starvation, 5 to 10% of the cells along each filament at semiregular intervals differentiate into heterocysts specialized in nitrogen fixation. Heterocysts are terminally differentiated cells in which the major cell division protein FtsZ is undetectable. In this report, we provide molecular evidence indicating that cell division is necessary for heterocyst development. FtsZ, which is translationally fused to the green fluorescent protein (GFP) as a reporter, is found to form a ring structure at the mid-cell position. SulA from Escherichia coli inhibits the GTPase activity of FtsZ in vitro and prevents the formation of FtsZ rings when expressed in Anabaena PCC 7120. The expression of sulA arrests cell division and suppresses heterocyst differentiation completely. The antibiotic aztreonam, which is targeted to the FtsI protein necessary for septum formation, has similar effects on both cell division and heterocyst differentiation, although in this case, the FtsZ ring is still formed. Therefore, heterocyst differentiation is coupled to cell division but independent of the formation of the FtsZ ring. Consistently, once the inhibitory pressure of cell division is removed, cell division should take place first before heterocyst differentiation resumes at a normal frequency. The arrest of cell division does not affect the accumulation of 2-oxoglutarate, which triggers heterocyst differentiation. Consistently, a nonmetabolizable analogue of 2-oxoglutarate does not rescue the failure of heterocyst differentiation when cell division is blocked. These results suggest that the control of heterocyst differentiation by cell division is independent of the 2-oxoglutarate signal.     

Examination of the interaction between FtsZ and MinCN in E. coli suggests how MinC disrupts Z rings

In Escherichia coli the Min system prevents Z ring assembly at cell poles by topologically regulating the division inhibitor MinC. The MinC protein has two domains of equal size and both domains can target FtsZ and block cell division in the proper context. Recently, we have shown that, along with MinD, the C‐terminal domain of MinC (MinC^C) competes with FtsA, and to a lesser extent with ZipA, for interaction with the C‐terminal tail of FtsZ to block division. Here we explored the interaction between the N‐terminal domain of MinC (MinC^N) and FtsZ. A search for mutations in ftsZ that confer resistance to MinC^N identified an α‐helix at the interface of FtsZ subunits as being critical for the activity of MinC^N. Focusing on one such mutant FtsZ–N280D, we showed that it greatly reduced the FtsZ–MinC interaction and was resistant to MinC^N both in vivo and in vitro. With these results, an updated model for the action of MinC on FtsZ is proposed: MinC interacts with FtsZ to disrupt two interactions, FtsZ–FtsA/ZipA and FtsZ–FtsZ, both of which are essential for Z ring formation.     

The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

Overproduction of FtsZ induces minicell formation in E. coli

The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli.     

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.     

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.     

Division‐site localization of RodZ is required for efficient Z ring formation in Escherichia coli

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ‐dependent manner. We also found that division‐site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division‐site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.     

The conserved C-terminal tail of FtsZ is required for the septal localization and division inhibitory activity of MinCC/MinD

The Escherichia coli Min system contributes to spatial regulation of cytokinesis by preventing assembly of the Z ring away from midcell. MinC is a cell division inhibitor whose activity is spatially regulated by MinD and MinE. MinC has two functional domains of similar size, both of which have division inhibitory activity in the proper context. However, the molecular mechanism of the inhibitory action of either domain is not very clear. Here, we report that the septal localization and division inhibitory activity of MinC^C/MinD requires the conserved C-terminal tail of FtsZ. This tail also mediates interaction with two essential division proteins, ZipA and FtsA, to link FtsZ polymers to the membrane. Overproduction of MinC^C/MinD displaces FtsA from the Z ring and eventually disrupts the Z ring, probably because it also displaces ZipA. These results support a model for the division inhibitory action of MinC/MinD. MinC/MinD binds to ZipA and FtsA decorated FtsZ polymers located at the membrane through the MinC^C/MinD–FtsZ interaction. This binding displaces FtsA and/or ZipA, and more importantly, positions MinC^N near the FtsZ polymers making it a more effective inhibitor.     

The developmental regulator PatD modulates assembly of the cell-division protein FtsZ in the cyanobacterium Anabaena sp. PCC 7120

FtsZ is a tubulin-like GTPase that polymerizes to initiate the process of cell division in bacteria. Heterocysts are terminally differentiated cells of filamentous cyanobacteria that have lost the capacity for cell division and in which the ftsZ gene is downregulated. However, mechanisms of FtsZ regulation during heterocyst differentiation have been scarcely investigated. The patD gene is NtcA dependent and involved in the optimization of heterocyst frequency in Anabaena sp. PCC 7120. Here, we report that the inactivation of patD caused the formation of multiple FtsZ-rings in vegetative cells, cell enlargement, and the retention of peptidoglycan synthesis activity in heterocysts, whereas its ectopic expression resulted in aberrant FtsZ polymerization and cell division. PatD interacted with FtsZ, increased FtsZ precipitation in sedimentation assays, and promoted the formation of thick straight FtsZ bundles that differ from the toroidal aggregates formed by FtsZ alone. These results suggest that in the differentiating heterocysts, PatD interferes with the assembly of FtsZ. We propose that in Anabaena FtsZ is a bifunctional protein involved in both vegetative cell division and regulation of heterocyst differentiation. In the differentiating cells PatD-FtsZ interactions appear to set an FtsZ activity that is insufficient for cell division but optimal to foster differentiation.     

Interaction between cell division proteins FtsE and FtsZ

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.     

Polymerization and bundling kinetics of FtsZ filaments

FtsZ is a tubulin homolog essential for prokaryotic cell division. In living bacteria, FtsZ forms a ringlike structure (Z-ring) at the cell midpoint. Cell division coincides with a gradual contraction of the Z-ring, although the detailed molecular structure of the Z-ring is unknown. To reveal the structural properties of FtsZ, an understanding of FtsZ filament and bundle formation is needed. We develop a kinetic model that describes the polymerization and bundling mechanism of FtsZ filaments. The model reveals the energetics of the FtsZ filament formation and the bundling energy between filaments. A weak lateral interaction between filaments is predicted by the model. The model is able to fit the in vitro polymerization kinetics data of another researcher, and explains the cooperativity observed in FtsZ kinetics and the critical concentration in different buffer media. The developed model is also applicable for understanding the kinetics and energetics of other bundling biopolymer filaments.