Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.     

FtsA reshapes membrane architecture and remodels the Z‐ring in Escherichia coli

Cell division in prokaryotes initiates with assembly of the Z‐ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z‐ring is comprised of polymers of tubulin‐like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C‐terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide‐dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C‐terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z‐ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.     

FtsZ Polymers Tethered to the Membrane by ZipA Are Susceptible to Spatial Regulation by Min Waves

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.     

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.     

A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis

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.     

Identification of a region of FtsA required for interaction with FtsZ

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.     

Targeting of (D)MinC/MinD and (D)MinC/DicB complexes to septal rings in Escherichia coli suggests a multistep mechanism for MinC-mediated destruction of nascent FtsZ rings

The MinC protein is an important determinant of septal ring positioning in Escherichia coli. The N-terminal domain ((Z)MinC) suppresses septal ring formation by interfering with FtsZ polymerization, whereas the C-terminal domain ((D)MinC) is required for dimerization as well as for interaction with the MinD protein. MinD oscillates between the membrane of both cell halves in a MinE-dependent fashion. MinC oscillates along with MinD such that the time-integrated concentration of (Z)MinC at the membrane is minimal, and hence the stability of FtsZ polymers is maximal, at the cell center. MinC is cytoplasmic and fails to block FtsZ assembly in the absence of MinD, indicating that recruitment of MinC by MinD to the membrane enhances (Z)MinC function. Here, we present evidence that the binding of (D)MinC to MinD endows the MinC/MinD complex with a more specific affinity for a septal ring-associated target in vivo. Thus, MinD does not merely attract MinC to the membrane but also aids MinC in specifically binding to, or in close proximity to, the substrate of its (Z)MinC domain. MinC-mediated division inhibition can also be activated in a MinD-independent fashion by the DicB protein of cryptic prophage Kim. DicB shows little homology to MinD, and how it stimulates MinC function has been unclear. Similar to the results obtained with MinD, we find that DicB interacts directly with (D)MinC, that the (D)MinC/DicB complex has a high affinity for some septal ring target(s), and that MinC/DicB interferes with the assembly and/or integrity of FtsZ rings in vivo. The results suggest a multistep mechanism for the activation of MinC-mediated division inhibition by either MinD or DicB and further expand the number of properties that can be ascribed to the Min proteins.     

Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins

Bacterial cell division typically requires assembly of the cytoskeletal protein FtsZ into a ring (Z-ring) at the nascent division site that serves as a foundation for assembly of the division apparatus. High resolution imaging suggests that the Z-ring consists of short, single-stranded polymers held together by lateral interactions. Several proteins implicated in stabilizing the Z-ring enhance lateral interactions between FtsZ polymers in vitro. Here we report that residues at the C terminus of Bacillus subtilis FtsZ (C-terminal variable region (CTV)) are both necessary and sufficient for stimulating lateral interactions in vitro in the absence of modulatory proteins. Swapping the 6-residue CTV from B. subtilis FtsZ with the 4-residue CTV from Escherichia coli FtsZ completely abolished lateral interactions between chimeric B. subtilis FtsZ polymers. The E. coli FtsZ chimera readily formed higher order structures normally seen only in the presence of molecular crowding agents. CTV-mediated lateral interactions are important for the integrity of the Z-ring because B. subtilis cells expressing the B. subtilis FtsZ chimera had a low frequency of FtsZ ring formation and a high degree of filamentation relative to wild-type cells. Site-directed mutagenesis of the B. subtilis CTV suggests that electrostatic forces are an important determinant of lateral interaction potential.     

FtsZ ring in bacterial cytokinesis

FtsZ is localized to a cytokinetic ring at the cell division site in bacteria. In this review a model is discussed that suggests that FtsZ self assembles into a ring at a nucleation site formed on the cytoplasmic membrane under cell-cycle control. This model suggests that formation of the cytokinetic FtsZ ring initiates and coordinates the circumferential invagination of the cytoplasmic membrane and cell wall, leading to formation of the septum. It is also suggested that this process may be conserved among the peptidoglycan-containing eubacteria. In addition, similarities between FtsZ and tubulin are discussed.     

The essential role of SepF in mycobacterial division

Mycobacteria lack several of the components that are essential in model systems as Escherichia coli or Bacillus subtilis for the formation of the divisome, a ring‐like structure assembling at the division site to initiate bacterial cytokinesis. Divisome assembly depends on the correct placement of the FtsZ protein into a structure called the Z ring. Notably, early division proteins that assist in the localisation of the Z ring to the cytoplasmic membrane and modulate its structure are missing in the so far known mycobacterial cell division machinery. To find mycobacterium‐relevant components of the divisome that might act at the level of FtsZ, a yeast two‐hybrid screening was performed with FtsZ from Mycobacterium tuberculosis. We identified the SepF homolog as a new interaction partner of mycobacterial FtsZ. Depending on the presence of FtsZ, SepF‐GFP fusions localised in ring‐like structures at potential division sites. Alteration of SepF levels in Mycobacterium smegmatis led to filamentous cells, indicating a division defect. Depletion of SepF resulted in a complete block of division. The sepF gene is highly conserved in the M. tuberculosis complex members. We therefore propose that SepF is an essential part of the core division machinery in the genus Mycobacterium.     

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

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

The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ

Summary Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure establishes the location of the nascent division site in prokaryotes. Factors that modulate FtsZ assembly are essential for ensuring the precise spatial and temporal regulation of cytokinesis. We have identified ClpX, the substrate recognition subunit of the ClpXP protease, as an inhibitor of FtsZ assembly in Bacillus subtilis. Genetic data indicate that ClpX but not ClpP inhibits FtsZ-ring formation in vivo. In vitro, ClpX inhibits FtsZ assembly in a ClpP-independent manner through a mechanism that does not require ATP hydrolysis. Together our data support a model in which ClpX helps maintain the cytoplasmic pool of unassembled FtsZ that is required for the dynamic nature of the cytokinetic ring. ClpX is conserved throughout bacteria and has been shown to interact directly with FtsZ in Escherichia coli. Thus, we speculate that ClpX functions as a general regulator of FtsZ assembly and cell division in a wide variety of bacteria.     

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.     

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.     

Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z‐ring

FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z‐ring prior to division. Two small coiled‐coil proteins, ZapA and ZapB, are both recruited early to the Z‐ring. We show here that ZapB is recruited to the Z‐ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two‐hybrid and in vitro interaction assays. Using high‐resolution 3‐D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z‐ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto‐filaments and ZapB mediates further stabilization of this interaction by cross‐linking ZapA molecules bound to adjacent FtsZ proto‐filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z‐ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z‐ring assembly by two different mechanisms.     

MinC spatially controls bacterial cytokinesis by antagonizing the scaffolding function of FtsZ

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.     

Identification and characterization of ZapC, a stabilizer of the FtsZ ring in Escherichia coli

In Escherichia coli, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with E. coli. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division.     

ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains

FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.     

Kinetic modeling of the assembly, dynamic steady state, and contraction of the FtsZ ring in prokaryotic cytokinesis

Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling.