Role of Escherichia coli FtsN protein in the assembly and stability of the cell division ring

Deprivation of FtsN, the last protein in the hierarchy of divisome assembly, causes the disassembly of other elements from the division ring, even extending to already assembled proto‐ring proteins. Therefore the stability and function of the divisome to produce rings active in septation is not guaranteed until FtsN is recruited. Disassembly follows an inverse sequential pathway relative to assembly. In the absence of FtsN, the frequencies of FtsN and FtsQ rings are affected similarly. Among the proto‐ring components, ZipA are more sensitive than FtsZ or FtsA rings. In contrast, removal of FtsZ leads to an almost simultaneous disappearance of the other elements from rings. Although restoration of FtsN allows for a quick reincorporation of ZipA into proto‐rings, the de novo joint assembly of the three components when FtsZ levels are restored to FtsZ‐deprived filaments is even faster. This suggests that the recruitment of ZipA into FtsZ‐FtsA incomplete proto‐rings may require first a period for the reversal of these partial assemblies.     

A role for the FtsQLB complex in cytokinetic ring activation revealed by an ftsL allele that accelerates division

The cytokinetic apparatus of bacteria is initially formed by the polymerization of the tubulin‐like FtsZ protein into a ring structure at midcell. This so‐called Z‐ring facilitates the recruitment of many additional proteins to the division site to form the mature divisome machine. Although the assembly pathway leading to divisome formation has been well characterized, the mechanisms that trigger cell constriction remain unclear. In this report, we study a ‘forgotten’ allele of ftsL from Escherichia coli, which encodes a conserved division gene of unknown function. We discovered that this allele promotes the premature initiation of cell division. Further analysis also revealed that the mutant bypasses the requirement for the essential division proteins ZipA, FtsK and FtsN, and partially bypasses the need for FtsA. These findings suggest that rather than serving simply as a protein scaffold within the divisome, FtsL may play a more active role in the activation of the machine. Our results support a model in which FtsL, along with its partners FtsB and FtsQ, function as part of a sensing mechanism that promotes the onset of cell wall remodeling processes needed for the initiation of cell constriction once assembly of the divisome complex is deemed complete.     

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 bypass of ZipA by overexpression of FtsN requires a previously unknown conserved FtsN motif essential for FtsA–FtsN interaction supporting a model in which FtsA monomers recruit late cell division proteins to the Z ring

Assembly of the divisome in Escherichia coli occurs in two temporally distinct steps. First, FtsZ filaments attached to the membrane through interaction with FtsA and ZipA coalesce into a Z ring at midcell. Then, additional proteins are recruited to the Z ring in a hierarchical manner to form a complete divisome, activated by the arrival of FtsN. Recently, we proposed that the interaction of FtsA with itself competes with its ability to recruit downstream division proteins (both require the IC domain of FtsA) and ZipA's essential function is to promote the formation of FtsA monomers. Here, we tested whether overexpression of a downstream division protein could make ZipA dispensable, presumably by shifting the FtsA equilibrium to monomers. Only overexpression of FtsN bypassed ZipA and a conserved motif in the cytoplasmic domain of FtsN was required for both the bypass and interaction with FtsA. Also, this cytoplasmic motif had to be linked to the periplasmic E domain of FtsN to bypass ZipA, indicating that linkage of FtsA to periplasmic components of the divisome through FtsN was essential under these conditions. These results are used to further elaborate our model for the role of FtsA in recruiting downstream division proteins.     

Recruitment of ZipA to the Septal Ring of Escherichia coli Is Dependent on FtsZ and Independent of FtsA

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.     

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.     

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N‐terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.     

Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB–GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84 ^ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro , ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self-assemble into larger structures.     

A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface

In Escherichia coli, initial assembly of the Z ring for cell division requires FtsZ plus the essential Z ring‐associated proteins FtsA and ZipA. Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP binding pocket and result in cell division arrest at non‐permissive temperatures. We found that purified wild‐type FtsA bound and hydrolysed ATP, whereas FtsA27 was defective in both activities. FtsA27 was also less able to localize to the Z ring in vivo. To investigate the role of ATP transactions in FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably, suppressors outside of the ATP binding site, including some mapping to the FtsA‐FtsA subunit interface, also enhanced ATP transactions and exhibited gain of function phenotypes in vivo. These results suggest that allosteric effects, including changes in oligomeric state, may influence the ability of FtsA to bind and/or hydrolyse ATP.     

The C‐terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an ～ 50‐amino‐acid (aa) linker between the protofilament‐forming globular domain and the C‐terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct‐linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct‐linkers from three bacterial species behaved as IDPs in vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct‐linkers from other bacteria, and even for an unrelated IDP from human α‐adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95 aa had compromised or no function. We conclude that the Ct‐linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct‐linker as a worm‐like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane.     

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re‐direct peptidoglycan biosynthesis to mid‐cell during cell division in polar‐growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid‐cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid‐cell, exhibit GTPase activity and form co‐polymers, only one, FtsZ_AT, is required for cell division. We find that FtsZ_AT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid‐cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ_AT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid‐cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.     

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.     

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.     

Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.     

The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil⁺ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.     

E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。     

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.     

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.     

Adenine Nucleotide-dependent Regulation of Assembly of Bacterial Tubulin-like FtsZ by a Hypermorph of Bacterial Actin-like FtsA

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.Bacterial cell division requires a large number of proteins that colocalize to form a putative protein machine at the cell membrane (1). This machine, sometimes called the divisome, recruits enzymes to synthesize the septum cell wall and to initiate and coordinate the invagination of the cytoplasmic membrane (and in Gram-negative bacteria, the outer membrane). The most widely conserved and key protein for this process is FtsZ, a homolog of tubulin that forms a ring structure called the Z ring, which marks the site of septum formation (2, 3). Like tubulin, FtsZ assembles into filaments with GTP but does not form microtubules (4). The precise assembly state and conformation of these FtsZ filaments at the division ring is not clear, although recent electron tomography work suggests that the FtsZ ring consists of multiple short filaments tethered to the membrane at discrete junctures (5), which may represent points along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One of these, ZipA, is not well conserved but is an essential protein in E. coli. ZipA binds to the C-terminal tail of FtsZ (6–8), and purified ZipA promotes bundling of FtsZ filaments in vitro (9, 10). The other, FtsA, is also essential in E. coli and is more widely conserved among bacterial species. FtsA is a member of the HSP70/actin superfamily (11, 12), and like ZipA, it interacts with the C-terminal tail of FtsZ (7, 13–15). FtsA can self-associate (16, 17) and bind ATP (12, 18), but reports of ATPase activity vary, with Bacillus subtilis FtsA having high activity (19) and Streptococcus pneumoniae FtsA exhibiting no detectable activity (20). There are no reports of any other in vitro activities of FtsA, including effects on FtsZ assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has a number of key activities in the cell. It is required for recruitment of a number of divisome proteins (21, 22) and helps to tether the Z ring to the membrane via a C-terminal membrane-targeting sequence (23). FtsA, like ZipA and other divisome proteins, is necessary to activate the contraction of the Z ring (24, 25). In E. coli, the FtsA:FtsZ ratio is crucial for proper cell division, with either too high or too low a ratio inhibiting septum formation (26, 27). This ratio is roughly 1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell (28), which works out to concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in the protein can result in significant enhancement of divisome activity. For example, the R286W mutation of FtsA, also called FtsA*, can substitute for the native FtsA and divide the cell. However, this mutant FtsA causes E. coli cells to divide at less than 80% of their normal length (29) and allows efficient division of E. coli cells in the absence of ZipA (30), indicating that it has gain-of-function activity. FtsA* and other hypermorphic mutations such as E124A and I143L can also increase division activity in cells lacking other essential divisome components (31–33). The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule, allowing cell division to occur at higher ratios than with WT² FtsA. This may be because the altered FtsA proteins self-associate more readily than WT FtsA, which may cause different changes in FtsZ assembly state as compared with WT FtsA (17, 34).In this study, we use an in vitro system with purified FtsZ and a purified tagged version of FtsA* to elucidate the role of FtsA in activating constriction of the Z ring in vivo. We show that FtsA*, at physiological concentrations in the presence of ATP or ADP, has significant effects on the assembly of FtsZ filaments.     

3D-SIM Super-resolution of FtsZ and Its Membrane Tethers in Escherichia coli Cells

FtsZ, a bacterial homolog of eukaryotic tubulin, assembles into the Z ring required for cytokinesis. In Escherichia coli, FtsZ interacts directly with FtsA and ZipA, which tether the Z ring to the membrane. We used three-dimensional structured illumination microscopy to compare the localization patterns of FtsZ, FtsA, and ZipA at high resolution in Escherichia coli cells. We found that FtsZ localizes in patches within a ring structure, similar to the pattern observed in other species, and discovered that FtsA and ZipA mostly colocalize in similar patches. Finally, we observed similar punctate and short polymeric structures of FtsZ distributed throughout the cell after Z rings were disassembled, either as a consequence of normal cytokinesis or upon induction of an endogenous cell division inhibitor.