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.     

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.     

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 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.     

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.     

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

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

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.     

Roles of pneumococcal DivIB in cell division

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of a divIB deletion mutant of Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the Delta divIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the Delta divIB mutant in defined medium. Functional mapping of the three distinct alpha, beta, and gamma domains of the extracellular region of DivIB revealed that a complete beta domain is required to fully rescue the deletion mutant. DivIB with a truncated beta domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.     

The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components

The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the 'septasome') that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two-hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdependence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.     

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.     

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.     

Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength

In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.     

Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization

In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its Haemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.     

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.     

Localization of FtsL to the Escherichia coli septal ring

In Escherichia coli, nine gene products are known to be essential for assembly of the division septum. One of these, FtsL, is a bitopic membrane protein whose precise function is not understood. Here we use fluorescence microscopy to study the subcellular localization of FtsL, both in a wild-type strain and in a merodiploid strain that expresses a GFP-FtsL fusion protein. We show that FtsL localizes to the cell septum where it forms a ring analogous to the cytoplasmic FtsZ ring. FtsL localization is dependent upon the function of FtsZ, FtsA and FtsQ, but not FtsI. In a reverse approach, we use fusions of green fluorescent protein (GFP) to FtsZ, FtsA and ZipA to show that these proteins localize to the division site in an FtsL-independent fashion. We propose that FtsL is a relatively late recruit to the ring structure that mediates septation.     

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.     

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.     

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.     

Lipoprotein FtsB in Streptococcus pyogenes Binds Ferrichrome in Two Steps with Residues Tyr137 and Trp204 as Critical Ligands

Lipoprotein FtsB is a component of the FtsABCD transporter that is responsible for ferrichrome binding and uptake in the Gram-positive pathogen Streptococcus pyogenes. In the present study, FtsB was cloned and purified from the bacteria and its Fch binding characteristics were investigated in detail by using various biophysical and biochemical methods. Based on the crystal structures of homogeneous proteins, FtsB was simulated to have bi-lobal structure forming a deep cleft with four residues in the cleft as potential ligands for Fch binding. With the assistance of site-directed mutagenesis, residue Trp204 was confirmed as a key ligand and Tyr137 was identified to be another essential residue for Fch binding. Kinetics experiments demonstrated that Fch binding in FtsB occurred in two steps, corresponding to the bindings to Tyr137 at N-lobe and Trp204 from C-lobe, respectively, and so that closing the protein conformation. Without either residue Tyr137 or Trp204, Fch binding in the protein as mutants Fch-Y137A and Fch-W204A may have a loose conformation, resembling the apo-proteins in proteolysis resistance and migration behaviors in native gel. This study revealed the inconsistence in the key amino acids among Fch-binding proteins from Gram-positive and –negative bacteria, providing interesting findings for understanding the differences between Gram-positive and –negative bacteria in the mechanism of iron uptake via siderophore (Fch) binding and transport.     

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.