The early divisome protein FtsA interacts directly through its 1c subdomain with the cytoplasmic domain of the late divisome protein FtsN

In Escherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsN(Cyto)), a single transmembrane domain (FtsN(TM)), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directly in vitro. FtsN(Cyto) is sufficient to bind to FtsA, but only when it is tethered to FtsN(TM) or to a leucine zipper. Mutation of a conserved patch of positive charges in FtsN(Cyto) to negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsN(Cyto-TM) is not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsN(Cyto) protein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.     

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 FtsA in SPOR‐independent localization of the essential Escherichia coli cell division protein FtsN

FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self‐enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR‐independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN_Cyto‐TM) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR‐independent localization preceded SPOR‐dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsN_Cyto‐TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA–FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR‐dependent localization.     

FtsN, a late recruit to the septum in Escherichia coli

The localization of FtsN in Escherichia coli was investigated by immunofluorescence microscopy. FtsN is an essential cell division protein with a simple bitopic topology, a short N-terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain. FtsN was found to localize to the septum in a ring pattern similar to that observed for FtsZ and FtsA, although the frequency of cells with rings was less. A MalG–FtsN fusion was also localized to the septum, indicating that the information for FtsN localization is supplied by its periplasmic domain. FtsN localization was dependent upon the prior localization of FtsZ and FtsA and required the function of FtsI and FtsQ. Consistent with FtsN functioning after FtsZ, Z rings were observed in a mutant depleted of FtsN.     

An altered FtsA can compensate for the loss of essential cell division protein FtsN in Escherichia coli

FtsN is the last known essential protein component to be recruited to the Escherichia coli divisome, and has several special properties. Here we report the isolation of suppressor mutants of ftsA that allow viability in the absence of ftsN. Cells producing the FtsA suppressors exhibited a mild cell division deficiency in the absence of FtsN, and no obvious phenotype in its presence. Remarkably, these altered FtsA proteins also could partially suppress a deletion of ftsK or zipA, were less toxic than wild-type FtsA when in excess, and conferred resistance to excess MinC, indicating that they share some properties with the previously isolated FtsA* suppressor mutant, and bypass the need for ftsN by increasing the integrity of the Z ring. TolA, which normally requires FtsN for its recruitment to the divisome, localized proficiently in the suppressed ftsN null strain, strongly suggesting that FtsN does not recruit the Tol-Pal complex directly. Therefore, despite its classification as a core divisome component, FtsN has no unique essential function but instead promotes overall Z ring integrity. The results strongly suggest that FtsA is conformationally flexible, and this flexibility is a key modulator of divisome function at all stages.     

Last but not least: new insights into how FtsN triggers constriction during Escherichia coli cell division

The arrival of FtsN at the division site triggers synthesis of septal peptidoglycan and constriction of the cell envelope. New findings are changing our view of how this happens. Binding of FtsN's cytoplasmic domain to a protein named FtsA recruits a small amount of FtsN to the division site earlier than previously recognized. The ability of FtsA to interact with FtsN is regulated by the ZipA protein. The FtsN–FtsA interaction pushes FtsA into an ‘on’ conformation that activates the machinery for peptidoglycan synthesis. In addition, a small region of FtsN's periplasmic domain appears to interact with the FtsQLB complex, pushing it into an ‘on’ state that also triggers synthesis of peptidoglycan. Thus, FtsN allosterically activates peptidoglycan synthesis by two pathways, one in the cytoplasm and involving FtsA, and the other in the periplasm and involving FtsQLB.     

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto‐ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto‐ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZ_L169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZ_L169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain‐of‐function mutants such as FtsA* or increased levels of the FtsA‐interacting protein FtsN. Similar to FtsA*, FtsZ_L169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZ_L169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.     

FtsA mutants impaired for self-interaction bypass ZipA suggesting a model in which FtsA's self-interaction competes with its ability to recruit downstream division proteins

Z-ring assembly requires polymers of the tubulin homologue FtsZ to be tethered to the membrane. Although either ZipA or FtsA is sufficient to do this, both of these are required for recruitment of downstream proteins to form a functional cytokinetic ring. Gain of function mutations in ftsA, such as ftsA* (ftsA-R286W), bypass the requirement for ZipA suggesting that this atypical, well-conserved, actin homologue has a more critical role in Z-ring function. FtsA forms multimers both in vitro and in vivo, but little is known about the role of FtsA polymerization. In this study we identify FtsA mutants impaired for self-interaction. Such mutants are able to support Z-ring assembly and are also able to bypass the requirement for ZipA. These mutants, including FtsA*, have reduced ability to self-interact but interact normally with FtsZ and are less toxic if overexpressed. These results do not support a model in which FtsA monomers antagonize FtsZ polymers. Instead, we propose a new model in which FtsA self-interaction competes with its ability to recruit downstream proteins. In this model FtsA self-interaction at the Z ring is antagonized by ZipA, allowing unpolymerized FtsA to recruit downstream proteins such as FtsN.     

ZipA is required for recruitment of FtsK, FtsQ, FtsL, and FtsN to the septal ring in Escherichia coli

The septal ring in Escherichia coli consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.     

Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli

ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ. Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA. We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins. In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized. Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings. These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.     

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.     

FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division

During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.     

Solution structure and domain architecture of the divisome protein FtsN

Prokaryotic cell division occurs through the formation of a septum, which in Escherichia coli requires coordination of the invagination of the inner membrane, biosynthesis of peptidoglycan and constriction of the outer membrane. FtsN is an essential cell division protein and forms part of the divisome, a putative complex of proteins located in the cytoplasmic membrane. Structural analyses of FtsN by nuclear magnetic resonance (NMR) reveals an RNP-like fold at the C-terminus (comprising residues 243-319), which has significant sequence homology to a peptidoglycan-binding domain. Sequential deletion mutagenesis in combination with NMR shows that the remaining of the periplasmic region of FtsN is unfolded, with the exception of three short, only partially formed helices following the trans-membrane helix. Based on these findings we propose a model in which FtsN, anchored in the inner membrane, bridges over to the peptidoglycan layer, thereby enabling the coordination of the divisome and the murein-shaping machinery in the periplasm.     

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.     

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.     

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division 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.     

Role for the nonessential N terminus of FtsN in divisome assembly

FtsN, the last essential protein in the cell division localization hierarchy in Escherichia coli, has several peculiar characteristics, suggesting that it has a unique role in the division process despite the fact that it is conserved in only a subset of bacteria. In addition to suppressing temperature-sensitive mutations in ftsA, ftsK, ftsQ, and ftsI, overexpression of FtsN can compensate for a complete lack of FtsK in the cell. We examined the requirements for this phenomenon. We found that the N-terminal terminal region (cytoplasmic and transmembrane domains) is critical for suppression, while the C-terminal murein-binding domain is dispensable. Our results further suggest that FtsN and FtsK act cooperatively to stabilize the divisome.     

Compensation for the loss of the conserved membrane targeting sequence of FtsA provides new insights into its function

The bacterial actin homologue FtsA has a conserved C-terminal membrane targeting sequence (MTS). Deletion or point mutations in the MTS, such as W408E, were shown previously to inactivate FtsA function and inhibit cell division. Because FtsA binds to the tubulin-like FtsZ protein that forms the Z ring, it is thought that the MTS of FtsA is required, along with the transmembrane protein ZipA, to assemble the Z ring and anchor it to the cytoplasmic membrane. Here, we show that despite its reduced membrane binding, FtsA-W408E could localize to the Z ring and recruit the late cell division protein FtsI, but was defective in self-interaction and recruitment of FtsN, another late cell division protein. These defects could be suppressed by a mutation that stimulates membrane association of FtsA-W408E, or by expressing a tandem FtsA-W408E. Remarkably, the FtsA MTS could be completely replaced with the transmembrane domain of MalF and remain functional for cell division. We propose that FtsA function in cell division depends on additive effects of membrane binding and self-interaction, and that the specific requirement of an amphipathic helix for tethering FtsA to the membrane can be bypassed.     

Roles for both FtsA and the FtsBLQ subcomplex in FtsN‐stimulated cell constriction in Escherichia coli

Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain (^EFtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic ^EFtsN*‐suppressing mutations that restore division in cells producing otherwise non‐functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without ^EFtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN (^NFtsN) and FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN⁺ cells, ^EFtsN*‐suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to de‐repress septal peptidoglycan synthesis and membrane invagination.