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.     

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.     

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.     

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.     

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.     

Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET

The bacterial cell division machinery is organized in the so‐called divisome composed of highly dynamic but low abundant interacting (membrane‐bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background‐insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ‐ring and the so‐called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan‐synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto‐ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto‐ring elements.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Spatial separation of FtsZ and FtsN during cell division

The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as ‘the divisome’. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super‐complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.     

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.     

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.     

The order of the ring: assembly of Escherichia coli cell division components

Topological cues appear to override temporal events in the assembly of the Escherichia coli cell division ring. When a procedure that allows the recruitment of ring components based on their topological properties is used, a concerted mode of assembly of several components of the divisome, rather than a strict linear mode, is revealed. Three multimolecular complexes, the proto-ring, the periplasmic connector and the peptidoglycan factory, show some degree of concertation for their assembly. In addition, back-recruitment of all late proteins except FtsN into the division ring occurs even in the absence of proteins incorporated at earlier stages, i.e. FtsA or FtsQ.     

Maturation of the Escherichia coli divisome occurs in two steps

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14-21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.     

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.