Fine-mapping the Contact Sites of the Escherichia coli Cell Division Proteins FtsB and FtsL on the FtsQ Protein

Escherichia coli cell division is effected by a large assembly of proteins called the divisome, of which a subcomplex consisting of three bitopic inner membrane proteins, FtsQ, FtsB, and FtsL, is an essential part. These three proteins, hypothesized to link cytoplasmic to periplasmic events during cell division, contain large periplasmic domains that are of major importance for function and complex formation. The essential nature of this subcomplex, its low abundance, and its multiple interactions with key divisome components in the relatively accessible periplasm make it an attractive target for the development of protein-protein interaction inhibitors. Although the crystal structure of the periplasmic domain of FtsQ has been solved, the structure of the FtsQBL complex is unknown, with only very crude indications of the interactions in this complex. In this study, we used in vivo site-specific photo cross-linking to probe the surface of the FtsQ periplasmic domain for its interaction interfaces with FtsB and FtsL. An interaction hot spot for FtsB was identified around residue Ser-250 in the C-terminal region of FtsQ and a membrane-proximal interaction region for both proteins around residue Lys-59. Sequence alignment revealed a consensus motif overlapping with the C-terminal interaction hot spot, underlining the importance of this region in FtsQ. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division.     

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.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.     

The essential role of SepF in mycobacterial division

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

The Escherichia coli FtsK functional domains involved in its interaction with its divisome protein partners

FtsK is a multifunctional protein involved in both cell division and chromosome segregation. As far as its role in cell division is concerned, FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components. The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by the two-hybrid assay was already shown by our group. In this work, we describe the identification of the protein domain(s) involved in the interaction with the cell division partner proteins. The biological role of some interactions is also discussed.     

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.     

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.     

Septal Localization of FtsQ, an Essential Cell Division Protein in Escherichia coli

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.     

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.     

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.     

Staphylococcus aureus DivIB is a peptidoglycan‐binding protein that is required for a morphological checkpoint in cell division

Bacterial cell division is a fundamental process that requires the coordinated actions of a number of proteins which form a complex macromolecular machine known as the divisome. The membrane‐spanning proteins DivIB and its orthologue FtsQ are crucial divisome components in Gram‐positive and Gram‐negative bacteria respectively. However, the role of almost all of the integral division proteins, including DivIB, still remains largely unknown. Here we show that the extracellular domain of DivIB is able to bind peptidoglycan and have mapped the binding to its β subdomain. Conditional mutational studies show that divIB is essential for Staphylococcus aureus growth, while phenotypic analyses following depletion of DivIB results in a block in the completion, but not initiation, of septum formation. Localisation studies suggest that DivIB only transiently localises to the division site and may mark previous sites of septation. We propose that DivIB is required for a molecular checkpoint during division to ensure the correct assembly of the divisome at midcell and to prevent hydrolytic growth of the cell in the absence of a completed septum.     

The structural dynamics of full-length divisome transmembrane proteins FtsQ,FtsB, and FtsL in FtsQBL complex formation

FtsQBL is a transmembrane protein complex in the divisome of Escherichia coli that plays a critical role in regulating cell division. Although extensive efforts have been made to investigate the interactions between the three involved proteins, FtsQ, FtsB, and FtsL, the detailed interaction mechanism is still poorly understood. In this study, we used hydrogen-deuterium exchange mass spectrometry to investigate these full-length proteins and their complexes. We also dissected the structural dynamic changes and the related binding interfaces within the complexes. Our data revealed that FtsB and FtsL interact at both the periplasmic and transmembrane regions to form a stable complex. Furthermore, the periplasmic region of FtsB underwent significant conformational changes. With the help of computational modeling, our results suggest that FtsBL complexation may bring the respective constriction control domains (CCDs) in close proximity. We show that when FtsBL adopts a coiled-coil structure, the CCDs are fixed at a vertical position relative to the membrane surface; thus, this conformational change may be essential for FtsBL’s interaction with other divisome proteins. In the FtsQBL complex, intriguingly, we show only FtsB interacts with FtsQ at its C-terminal region, which stiffens a large area of the β-domain of FtsQ. Consistent with this, we found the connection between the α- and β-domains in FtsQ is also strengthened in the complex. Overall, the present study provides important experimental evidence detailing the local interactions between the full-length FtsB, FtsL, and FtsQ protein, as well as valuable insights into the roles of FtsQBL complexation in regulating divisome activity.     

The distinctive cell division interactome of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis>

Background

Bacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW.

Results

Using a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsA_Ng were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW.

Conclusions

Results from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.

     

The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites

Bacterial cytokinesis is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. DivIB and its orthologue FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. A molecular dissection approach was used to determine which of these domains are essential for recruitment of DivIB to incipient division sites and for its cell division functions. We show that DivIB has three molecular epitopes that mediate its localization to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and we have used this information to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localization epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein–protein interactions, many of which are redundant and likely to be individually non-essential.     

Division‐site localization of RodZ is required for efficient Z ring formation in Escherichia coli

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ‐dependent manner. We also found that division‐site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division‐site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.     

ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division

Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C‐terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non‐replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.     

The N‐succinyl‐l,l‐diaminopimelic acid desuccinylase DapE acts through ZapB to promote septum formation in Escherichia coli

Spatial regulation of cell division in Escherichia coli occurs at the stage of Z ring formation. It consists of negative (the Min and NO systems) and positive (Ter signal mediated by MatP/ZapA/ZapB) regulators. Here, we find that N‐succinyl‐L,L‐diaminopimelic acid desuccinylase (DapE) facilitates functional Z ring formation by strengthening the Ter signal via ZapB. DapE depends on ZapB to localize to the Z ring and its overproduction suppresses the division defect caused by loss of both the Min and NO systems. DapE shows a strong interaction with ZapB and requires the presence of ZapB to exert its function in division. Consistent with the idea that DapE strengthens the Ter signal, overproduction of DapE supports cell division with reduced FtsZ levels and provides some resistance to the FtsZ inhibitors MinCD and SulA, while deletion of dapE, like deletion of zapB, exacerbates the phenotypes of cells impaired in Z ring formation such as ftsZ84 or a min mutant. Taken together, our results report DapE as a new component of the divisome that promotes the integrity of the Z ring by acting through ZapB and raises the possibility of the existence of additional divisome proteins that also function in other cellular processes.     

ZipN, an FtsA-like orchestrator of divisome assembly in the model cyanobacterium Synechocystis PCC6803

We pursued the characterization of the divisome of the spherical-celled cyanobacterium Synechocystis PCC6803, through deletion, site-directed mutagenesis, GFP tagging, two-hybrid and co-immunoprecipitation assays. We presently report that the DivIVA-like protein Cdv3 is essential to both cell growth and division, whereas the AmiC, AmpH, FtsE, FtsN, SpoIID, YlmD, YlmE and YlmG proteins are dispensable. With the exception of the self-interacting protein YlmD, none of these dispensable factors appeared to interact with ZipN, the crucial cytokinetic factor we previously characterized. By contrast, we found that ZipN interacts with itself and the self-interacting protein Cdv3, as well as with all other crucial cytokinetic factors we previously characterized, namely: FtsZ, FtsI, FtsQ, SepF and ZipS. We also identified ZipN amino acids selectively involved in ZipN interaction with one of its following partners, Cdv3, FtsQ or SepF. Finally, we found no direct interaction between Cdv3, SepF and ZipS. Collectively, these results indicate that ZipN is a central player of divisome assembly in cyanobacteria, similarly to the FtsA protein of E. coli that is absent in cyanobacteria and chloroplast.     

Contribution of the FtsQ transmembrane segment to localization to the cell division site

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.