Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ

In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coli FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.     

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.     

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 a MAP-Tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division

The first visible event in prokaryotic cell division is the assembly of the soluble, tubulin-like FtsZ GTPase into a membrane-associated cytokinetic ring that defines the division plane in bacterial and archaeal cells. In the temperature-sensitive ftsZ84 mutant of Escherichia coli, this ring assembly is impaired at the restrictive temperature causing lethal cell filamentation. Here I present genetic and morphological evidence that a 2-fold higher dosage of the division gene zipA suppresses thermosensitivity of the ftsZ84 mutant by stabilizing the labile FtsZ84 ring structure in vivo. I demonstrate that purified ZipA promotes and stabilizes protofilament assembly of both FtsZ and FtsZ84 in vitro and cosediments with the protofilaments. Furthermore, ZipA organizes FtsZ protofilaments into arrays of long bundles or sheets that probably represent the physiological organization of the FtsZ ring in bacterial cells. The N-terminal cytoplasmic domain of membrane-anchored ZipA contains sequence elements that resemble the microtubule-binding signature motifs in eukaryotic Tau, MAP2 and MAP4 proteins. It is postulated that the MAP-Tau-homologous motifs in ZipA mediate its binding to FtsZ, and that FtsZ-ZipA interaction represents an ancient prototype of the protein-protein interaction that enables MAPs to suppress microtubule catastrophe and/or to promote rescue.     

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.     

Oligomerization of FtsZ converts the FtsZ tail motif (conserved carboxy‐terminal peptide) into a multivalent ligand with high avidity for partners ZipA and SlmA

A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (c onserved c arboxy‐t erminal p eptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z‐ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro.     

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.     

ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments

A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0) pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.     

The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ

ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.     

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.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

Concentration and assembly of the division ring proteins FtsZ,FtsA, and ZipA during the Escherichia coli cell cycle

The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.     

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.     

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.     

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.     

Analysis of the interaction of FtsZ with itself, GTP, and FtsA.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.     

Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring.

FtsZ and FtsA are essential for cell division in Escherichia coli and colocalize to the septal ring. One approach to determine what regions of FtsA and FtsZ are important for their interaction is to identify in vivo interactions between FtsA and FtsZ from different species. As a first step, the ftsA genes of Rhizobium meliloti and Agrobacterium tumefaciens were isolated and characterized. In addition, an FtsZ homolog that shared the unusual C-terminal extension of R. meliloti FtsZ1 was found in A. tumefaciens. In order to visualize their localization in cells, we tagged these proteins with green fluorescent protein (GFP). When R. meliloti FtsZ1-GFP or A. tumefaciens FtsZ-GFP was expressed at low levels in E. coli, they specifically localized only to the E. coli FtsZ ring, possibly by coassembly. When A. tumefaciens FtsA-GFP or R. meliloti FtsA-GFP was expressed in E. coli, they failed to localize detectably to the E. coli FtsZ ring. However, when R. meliloti FtsZ1 was coexpressed with them, fluorescence localized to a band at the midcell division site, strongly suggesting that FtsA from either A. tumefaciens or R. meliloti can bind directly to its cognate FtsZ. As expected, GFP-tagged FtsZ1 and FtsA from either R. meliloti or A. tumefaciens localized to the division site in A. tumefaciens cells. Therefore, the 61 amino acid changes between A. tumefaciens FtsA and R. meliloti FtsA do not prevent their direct interaction with FtsZ1 from either species, suggesting that those residues are not essential for protein-protein contacts. Moreover, the failure of the two non-E. coli FtsA derivatives to interact strongly with E. coli FtsZ in this in vivo system unless their cognate FtsZ was also present suggests that FtsA-FtsZ interactions have coevolved and that the residues which differ between the E. coli proteins and those of the two other species may be important for specific interactions.     

Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction

A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide.     

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.     

The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography

In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded beta-sheet packed against three alpha-helices and contains the split beta-alpha-beta motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended beta-strand followed by alpha-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.