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Wagner-Herman JK Bernard R Dunne R Bisson-Filho AW Kumar K Nguyen T Mulcahy L Koullias J Gueiros-Filho FJ Rudner DZ 《Journal of bacteriology》2012,194(17):4608-4618
During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed. 相似文献
3.
In Escherichia coli the Z ring has the potential to assemble anywhere along the cell length but is restricted to midcell by the action of negative regulatory systems, including Min. In the current model for the Min system, the MinC/MinD division inhibitory complex is evenly distributed on the membrane and can disrupt Z rings anywhere in the cell; however, MinE spatially regulates MinC/MinD by restricting it to the cell poles, thus allowing Z ring formation at midcell. This model assumes that Z rings formed at different cellular locations have equal sensitivity to MinC/MinD in the absence of MinE. However, here we report evidence that differences in MinC/MinD sensitivity between polar and nonpolar Z rings exists even when there is no MinE. MinC/MinD at proper levels is able to block minicell production in Δmin strains without increasing the cell length, indicating that polar Z rings are preferentially blocked. In the FtsZ-I374V strain (which is resistant to MinC(C)/MinD), wild-type morphology can be easily achieved with MinC/MinD in the absence of MinE. We also show that MinC/MinD at proper levels can rescue the lethal phenotype of a min slmA double deletion mutant, which we think is due to the elimination of polar Z rings (or FtsZ structures), which frees up FtsZ molecules for assembly of Z rings at internal sites to rescue division and growth. Taken together, these data indicate that polar Z rings are more susceptible to MinC/MinD than internal Z rings, even when MinE is absent. 相似文献
4.
Deletion of the min operon results in increased thermosensitivity of an ftsZ84 mutant and abnormal FtsZ ring assembly, placement, and disassembly
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To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C. Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes. 相似文献
5.
Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ
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In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the min system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts min function, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly. 相似文献
6.
MinC spatially controls bacterial cytokinesis by antagonizing the scaffolding function of FtsZ 总被引:1,自引:0,他引:1
BACKGROUND: Cytokinesis in bacteria is mediated by a cytokinetic ring, termed the Z ring, which forms a scaffold for recruitment of other cell-division proteins. The Z ring is composed of FtsZ filaments, but their organization in the Z ring is poorly understood. In Escherichia coli, the Min system contributes to the spatial regulation of cytokinesis by preventing the assembly of the Z ring away from midcell. The effector of the Min system, MinC, inhibits Z ring assembly by a mechanism that is not clear. RESULTS: Here, we report that MinC controls the scaffolding function of FtsZ by antagonizing the mechanical integrity of FtsZ structures. Specifically, MinC antagonizes the ability of FtsZ filaments to be in a solid-like gel state. MinC is a modular protein whose two domains (MinC(C) and MinC(N)) synergize to inhibit FtsZ function. MinC(C) interacts directly with FtsZ polymers to target MinC to Z rings. MinC(C) also prevents lateral interactions between FtsZ filaments, an activity that seems to be unique among cytoskeletal proteins. Because MinC(C) is inhibitory in vivo, it suggests that lateral interactions between FtsZ filaments are important for the structural integrity of the Z ring. MinC(N) contributes to MinC activity by weakening the longitudinal bonds between FtsZ molecules in a filament leading to a loss of polymer rigidity and consequent polymer shortening. On the basis of our results, we develop the first computational model of the Z ring and study the effects of MinC. CONCLUSIONS: Control over the scaffolding activity of FtsZ probably represents a universal regulatory mechanism of bacterial cytokinesis. 相似文献
7.
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. 相似文献
8.
Sheng‐Wen Chiu Mark A. J. Roberts Mark C. Leake Judith P. Armitage 《Molecular microbiology》2013,90(2):322-337
Bacterial chemotaxis depends on signalling through large protein complexes. Each cell must inherit a complex on division, suggesting some co‐ordination with cell division. In Escherichia coli the membrane‐spanning chemosensory complexes are polar and new static complexes form at pre‐cytokinetic sites, ensuring positioning at the new pole after division and suggesting a role for the bacterial cytoskeleton. Rhodobacter sphaeroides has both membrane‐associated and cytoplasmic, chromosome‐associated chemosensory complexes. We followed the relative positions of the two chemosensory complexes, FtsZ and MreB in aerobic and in photoheterotrophic R. sphaeroides cells using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which FtsZ extends circumferentially to form the Z‐ring. Membrane‐associated chemosensory proteins form a number of dynamic unit‐clusters with mature clusters containing about 1000 CheW3 proteins. Individual clusters diffuse randomly within the membrane, accumulating at new poles after division but not colocalizing with either FtsZ or MreB. The cytoplasmic complex colocalizes with FtsZ at midcells in new‐born cells. Before cytokinesis one complex moves to a daughter cell, followed by the second moving to the other cell. These data indicate that two homologous complexes use different mechanisms to ensure partitioning, and neither complex utilizes FtsZ or MreB for positioning. 相似文献
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Cell division in bacteria is facilitated by a polymeric ring structure, the Z ring, composed of tubulin-like FtsZ protofilaments. Recently it has been shown that in Bacillus subtilis , the Z ring forms through the cell cycle-mediated remodelling of a helical FtsZ polymer. To investigate how this occurs in vivo , we have exploited a unique temperature-sensitive strain of B. subtilis expressing the mutant protein FtsZ(Ts1). FtsZ(Ts1) is unable to complete Z ring assembly at 49°C, becoming trapped at an intermediate stage in the helix-to-ring progression. To determine why this is the case, we used a combination of methods to identify the specific defect of the FtsZ(Ts1) protein in vivo . Our results indicate that while FtsZ(Ts1) is able to polymerize normally into protofilaments, it is defective in the ability to support lateral associations between these filaments at high temperatures. This strongly suggests that lateral FtsZ association plays a crucial role in the polymer transitions that lead to the formation of the Z ring in the cell. In addition, we show that the FtsZ-binding protein ZapA, when overproduced, can rescue the FtsZ(Ts1) defect in vivo . This suggests that ZapA functions to promote the helix-to-ring transition of FtsZ by stimulating lateral FtsZ association. 相似文献
11.
The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division. 相似文献
12.
A key event in bacterial cytokinesis is the formation of the Z ring, which serves as a mechanical scaffold that recruits other cytokinetic proteins to establish functional divisomes. This scaffolding function of Z rings is essential throughout cytokinesis, but the underlying molecular interactions are poorly understood. Here we report that a widely conserved FtsZ binding protein, ZapA, has cytological, biochemical and biophysical properties that argue for the importance of cross-linking interactions between FtsZ polymers in the coherence of Z rings. Escherichia coli zapA null mutant cells have Z rings that are structurally looser and many helical precursors of Z rings fail to coalesce into coherent rings. Biophysical behaviour of FtsZ in the presence of ZapA reveals that ZapA not only bundles, but also cross-links FtsZ polymers, which makes it the first cross-linking protein of the bacterial cytoskeleton. Cross-linking in vitro occurs at the stoichiometry of FtsZ-ZapA interaction at the Z rings in vivo, where nearly all intracellular ZapA is dynamically associated. ZapA also stabilizes longitudinal bonds between FtsZ monomers since it promotes the polymerization of FtsZ mutants with lesions at the polymerization interface and since it reverses the inhibitory effects of SulA, a known antagonist of FtsZ longitudinal interactions. 相似文献
13.
A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis 总被引:1,自引:1,他引:0
The earliest event in bacterial cell division is the formation of a Z ring, composed of the tubulin-like FtsZ protein, at the division site at midcell. This ring then recruits several other division proteins and together they drive the formation of a division septum between two replicated chromosomes. Here we show that, in addition to forming a cytokinetic ring, FtsZ localizes in a helical-like pattern in vegetatively growing cells of Bacillus subtilis. FtsZ moves rapidly within this helix-like structure. Examination of FtsZ localization in individual live cells undergoing a single cell cycle suggests a new assembly mechanism for Z ring formation that involves a cell cycle-mediated multistep remodelling of FtsZ polymers. Our observations suggest that initially FtsZ localizes in a helical pattern, with movement of FtsZ within this structure occurring along the entire length of the cell. Next, movement of FtsZ in a helical-like pattern is restricted to a central region of the cell. Finally the FtsZ ring forms precisely at midcell. We further show that another division protein, FtsA, shown to interact with FtsZ prior to Z ring formation in B. subtilis, also localizes to similar helical patterns in vegetatively growing cells. 相似文献
14.
Harold P. Erickson David E. Anderson Masaki Osawa 《Microbiology and molecular biology reviews》2010,74(4):504-528
Summary: FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation. 相似文献
15.
Influence of FtsZ GTPase activity and concentration on nanoscale Z‐ring structure in vivo revealed by three‐dimensional Superresolution imaging
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FtsZ is an essential bacterial cytoskeletal protein that assembles into a ring‐like structure (Z‐ring) at midcell to carry out cytokinesis. In vitro, FtsZ exhibits polymorphism in polymerizing into different forms of filaments based on its GTPase activity, concentration, and buffer condition. In vivo, the Z‐ring appeared to be punctate and heterogeneously organized, although continuous, homogenous Z‐ring structures have also been observed. Understanding how the Z‐ring is organized in vivo is important because it provides a structural basis for the functional role of the Z‐ring in cytokinesis. Here, we assess the effects of both GTPase activity and FtsZ concentration on the organization of the Z‐ring in vivo using three‐dimensional (3D) superresolution microscopy. We found that the Z‐ring became more homogenous when assembled in the presence of a GTPase‐deficient mutant, and upon overexpression of either wt or mutant FtsZ. These results suggest that the in vivo organization of the Z‐ring is largely dependent on the intrinsic polymerization properties of FtsZ, which are significantly influenced by the GTPase activity and concentration of FtsZ. Our work provides a unifying theme to reconcile previous observations of different Z‐ring structures, and supports a model in which the wt Z‐ring comprises loosely associated, heterogeneously distributed FtsZ clusters. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 725–734, 2016. 相似文献
16.
FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments. 相似文献
17.
Jackson A. Buss Nick T. Peters Jie Xiao Thomas G. Bernhardt 《Molecular microbiology》2017,104(4):652-663
Cell division in Escherichia coli begins with the polymerization of FtsZ into a ring‐like structure, the Z‐ring, at midcell. All other division proteins are thought to require the Z‐ring for recruitment to the future division site. Here, it is reported that the Z‐ring associated proteins ZapA and ZapB form FtsZ‐independent structures at midcell. Upon Z‐ring disruption by the FtsZ polymerization antagonist SulA, ZapA remained at midcell as a cloud‐like accumulation. Using ZapA(N60Y), a variant defective for interaction with FtsZ, it was established that these ZapA structures form without a connection to the Z‐ring. Furthermore, midcell accumulations of GFP‐ZapA(N60Y) often preceded Z‐rings at midcell and required ZapB to assemble, suggesting that ZapB polymers form the foundation of these structures. In the absence of MatP, a DNA‐binding protein that links ZapB to the chromosomal terminus region, cloud‐like ZapA structures still formed but failed to track with the chromosome terminus and did not consistently precede FtsZ at midcell. Taken together, the results suggest that FtsZ‐independent structures of ZapA–ZapB provide additional positional cues for Z‐ring formation and may help coordinate its assembly with chromosome replication and segregation. 相似文献
18.
Jackson Buss Carla Coltharp Tao Huang Chris Pohlmeyer Shih‐Chin Wang Christine Hatem Jie Xiao 《Molecular microbiology》2013,89(6):1099-1120
In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring‐like structure (Z‐ring) at the midcell. Despite its essential role, the molecular architecture of the Z‐ring remains elusive. In this work we examine the roles of two FtsZ‐associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z‐ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single‐molecule‐based super‐resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z‐ring. Furthermore, we find these clusters are not due to the loss of ZapB–MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments. 相似文献
19.
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. 相似文献
20.
Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings
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Chauhan A Madiraju MV Fol M Lofton H Maloney E Reynolds R Rajagopalan M 《Journal of bacteriology》2006,188(5):1856-1865
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. 相似文献