Intrinsic instability of the essential cell division protein FtsL of Bacillus subtilis and a role for DivIB protein in FtsL turnover

Cell division in most eubacteria is driven by an assembly of about eight conserved division proteins. These proteins form a ring structure that constricts in parallel with the formation of the division septum. Here, we show that one of the division proteins, FtsL, is highly unstable. We also show that the protein is targeted to the ring structure and that targeting occurs in concert with the recruitment of several other membrane-associated division proteins. FtsL stability is further reduced in the absence of DivIB protein (probably homologous to E. coli FtsQ) at high temperature, suggesting that DivIB is involved in the control of FtsL turnover. The reduced stability of FtsL may explain the temperature dependence of divIB mutants, because their phenotype can be suppressed by overexpression of FtsL. The results provide new insights into the roles of the FtsL and DivIB proteins in bacterial cell division.     

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.     

A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region.     

In vitro reconstitution of a trimeric complex of DivIB, DivIC and FtsL, and their transient co-localization at the division site in Streptococcus pneumoniae

DivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer.     

Central Domain of DivIB Caps the C-terminal Regions of the FtsL/DivIC Coiled-coil Rod

DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the “bean”-shaped central β-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.Cell division is one of the defining features of life. Understanding the division of bacteria is also required to find novel antibiotic strategies. Numerous studies, carried out mostly with the model organisms Escherichia coli and Bacillus subtilis have uncovered several components of the divisome, which can be defined as the ensemble of proteins localized at the division site and participating in the process. Comparison of genomes and deletion studies indicate that the core of the divisome comprises eight conserved, mostly essential proteins: FtsZ, FtsA, FtsK, FtsQ(DivIB), FtsL, FtsB(DivIC), FtsW, and FtsI. Fts nomenclature applies to Gram-negative organisms, whereas Div nomenclature applies to Gram-positive bacteria. These proteins are listed here in the conditional order of their recruitment to the division site of E. coli (1–4).Processes in which they participate have been attributed to several division proteins. FtsZ forms polymers with an annular distribution on the cytoplasmic side of the membrane and governs the recruitment of the other proteins. FtsA may mediate the interaction of FtsZ with the membrane. FtsK participates to the resolution of chromosome dimers, and possibly to the membrane fission. FtsI, and likely FtsW, participate to septal cell wall formation (1–4). In contrast, the roles of FtsQ(DivIB), FtsL, and FtsB(DivIC) have not been firmly linked to any particular process.FtsQ(DivIB), FtsL, and FtsB(DivIC) are positioned in the middle of the conditional order of recruitment in E. coli and B. subtilis. When the temporality of the recruitment was examined, FtsQ(DivIB) was found to belong to the late recruits, together with the proteins involved in cell wall assembly (5). In E. coli, the presence of FtsL and FtsB at the division site is mutually dependent, and their localization depends on that of FtsQ (6, 7). In B. subtilis, the presence of FtsL and DivIC at mid-cell depends on that of DivIB, at the temperature at which DivIB is essential, and reciprocally (8, 9). A complex comprising FtsQ, FtsL, and FtsB was isolated from E. coli by co-immunoprecipitation (10), and reconstituted in vitro with recombinant soluble forms of pneumococcal DivIB, FtsL, and DivIC (11). The interaction of the three proteins was also confirmed by yeast and bacterial triple hybrid (12, 13).The genes ftsL and ftsB(divIC) are essential in E. coli and B. subtilis (6, 14–16) and presumably in Streptococcus pneumoniae (17). The essentiality of ftsQ(divIB) in laboratory conditions varies between species. The gene ftsQ is essential in E. coli (18), but divIB is essential only at high temperatures in B. subtilis (9, 19), or in a chemically defined medium in S. pneumoniae (17). Under these conditions, the essentiality of DivIB appears to be a consequence of the protection from proteolysis that it provides to FtsL (8, 17).FtsQ(DivIB), FtsL, and FtsB(DivIC) are bitopic membrane proteins with an N-terminal cytoplasmic region, a single transmembrane segment, and an extracytoplasmic region. The extracellular part is necessary and sufficient for the localization and function of FtsQ(DivIB), provided that it is anchored to the membrane (e.g. Refs. 20 and 21)), although the transmembrane segment also contributes to the localization (22, 23). The extracellular part is organized in three regions termed α, β, and γ. The crystal structure of a region consisting of the α- and β-domains was solved for FtsQ from E. coli and Yersinia enterocolitica (24). The α-domain, comprising about 70 amino acids proximal to the cytoplasmic membrane, corresponds to the POTRA (for polypeptide transport-associated) domain first identified by sequence analysis and proposed to function as a molecular chaperone (25). The α- and β-domains form the conserved region of the FtsQ(DivIB) protein. The γ-region constitutes a C-terminal tail. It is highly variable in length and sequence and predicted to be unfolded. The γ-region was not observed in the structures from E. coli and Y. entercolitica, thus confirming its flexible nature (24).The α-domain in the recombinant soluble form of the extracellular part of DivIB from Geobacillus stearothermophilus was digested by trypsin and therefore considered to be largely unfolded (26). The γ-region was also removed by trypsin digestion, together with a C-terminal fragment of the β-domain. The structure of the resulting shorter β-domain from G. stearothermophilus was solved by NMR (26) and lacks the two C-terminal β-strands.Localization epitopes have been identified in the transmembrane segment, the α-domain, and a region encompassing the C-terminal part of the β-domain and γ-tail of DivIB from B. subtilis (23). Likewise in E. coli, a region in the α-domain is required for localization of FtsQ, whereas the C-terminal region of the β-domain and the last α-helix are required for recruitment of FtsL and FtsB (24). In S. pneumoniae, the essentiality of DivIB in defined medium was found to reside in the C-terminal region of the β-domain (17).No experimental structure is known for FtsL or FtsB(DivIC). Both are small proteins comprising between 90 and 140 amino acids. The number of residues is sometimes larger, as in Mycobacterium tuberculosis (384 for FtsL and 228 for FtsB), due to N- and/or C-terminal extensions consisting of mostly charged and polar amino acids or proline-rich sequences. The major part of FtsL or FtsB(DivIC) is extracellular and contains a region proximal to the transmembrane segment, predicted to form a coiled-coil of about five heptads. Coiled-coil helices associate longitudinally to mediate protein association. It is possible that the coiled-coil helices are continuations of the transmembrane helices, although a proline (known to break helices) is present in some species between the two segments. Following the coiled-coil region is a 25–35-residue long C-terminal region in both FtsL and DivIC(FtsB). This region was recently shown in FtsB to be required for interaction with FtsQ in E. coli (27).We report here the results of structural studies in solution of a ternary complex consisting of the β- and γ-segments of DivIB, and a constrained dimer of the extracellular parts of FtsL and DivIC from S. pneumoniae. Despite the coiled-coil predictions, the recombinant extracellular domains of FtsL and DivIC did not interact in vitro (11, 28). Forced dimerization was obtained by fusion with artificial coiled-coil peptides k5 and e5 (35 residues long), which are known to form a heterodimer due to their complementarity of charge, with a nanomolar dissociation constant (29). The k5- and e5-coils were fused to the extracellular domain of FtsL and DivIC, to give rise to KL and EC fusion proteins, respectively. The constrained dimer (KL/EC) was shown to interact with the extracellular part of DivIB (DivIB_ext), yielding a soluble complex amenable to structural studies (11).The overall shape of the complex and its constituents was probed using small-angle x-ray scattering (SAXS)² and small-angle neutron scattering (SANS). NMR was used to investigate the interface between the proteins by chemical shift mapping. The interaction was further investigated using surface plasmon resonance with truncated forms of the proteins. The complex of DivIB, FtsL, and DivIC is formed by the interaction of one face of the β-domain of DivIB with the C-terminal regions of FtsL and DivIC, at the tip of an elongated rod formed by the coiled-coil segments. The α-domain of DivIB and the coiled-coil regions of FtsL and DivIC remain free to interact with other proteins of the division apparatus.     

Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly

Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQ's interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wrinkled-colony morphology, which yielded several new point mutations in ftsQ. Two of these mutations affect localization of FtsQ to midcell and together define a targeting role for FtsQ's alpha domain. Further characterization of one localization-defective mutant protein [FtsQ(V92D)] revealed an unexpected role in localization for the first 49 amino acids of FtsQ. Finally, we found a suppressor of FtsQ(V92D) that was due to a point mutation in domain 1C of FtsA, a domain previously implicated in the recruitment of divisome proteins. However, despite reports of a potential interaction between FtsA and FtsQ, suppression by FtsA(I143L) is not mediated via direct contact with FtsQ. Rather, this mutation acts as a general suppressor of division defects, which include deletions of the normally essential genes zipA and ftsK and mutations in FtsQ that affect both localization and recruitment. Together, these results reveal increasingly complex connections within the bacterial divisome.     

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.     

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.     

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.     

Membrane-bound division proteins DivIB and DivIC of Bacillus subtilis function solely through their external domains in both vegetative and sporulation division

The Bacillus subtilis membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both divIB and divIC was performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of the Escherichia coli TolR protein. B. subtilis strains expressing the divIB and divIC hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed.     

Characterization of the essential cell division gene ftsL (yllD ) of Bacillus subtilis and its role in the assembly of the division apparatus

We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL , of Escherichia coli . Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, σ^F. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.     

Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.     

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.     

Structural and mutational analysis of the cell division protein FtsQ

Bacterial cytokinesis requires the divisome, a complex of proteins that co-ordinates the invagination of the cytoplasmic membrane, inward growth of the peptidoglycan layer and the outer membrane. Assembly of the cell division proteins is tightly regulated and the order of appearance at the future division site is well organized. FtsQ is a highly conserved component of the divisome among bacteria that have a cell wall, where it plays a central role in the assembly of early and late cell division proteins. Here, we describe the crystal structure of the major, periplasmic domain of FtsQ from Escherichia coli and Yersinia enterocolitica . The crystal structure reveals two domains; the α-domain has a striking similarity to polypeptide transport-associated (POTRA) domains and the C-terminal β-domain forms an extended β-sheet overlaid by two, slightly curved α-helices. Mutagenesis experiments demonstrate that two functions of FtsQ, localization and recruitment, occur in two separate domains. Proteins that localize FtsQ need the second β-strand of the POTRA domain and those that are recruited by FtsQ, like FtsL/FtsB, require the surface formed by the tip of the last α-helix and the two C-terminal β-strands. Both domains act together to accomplish the role of FtsQ in linking upstream and downstream cell division proteins within the divisome.     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.     

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.     

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.     

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.     

Localization of the Bacillus subtilis murB gene within the dcw cluster is important for growth and sporulation

The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for "division and cell wall") cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivIB levels were normal in a second mutant containing an in-frame deletion of murB (DeltamurB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the DeltamurB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a murB(+)PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.