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.     

Crystal Structure and Site-directed Mutational Analysis Reveals Key Residues Involved in Escherichia coli ZapA Function

FtsZ is an essential cell division protein in Escherichia coli, and its localization, filamentation, and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. Zaps (FtsZ-associated proteins) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The x-ray crystallographic structure of E. coli ZapA was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in E. coli ZapA were modified using site-directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared with WT ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ΔzapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling and that ultimately affect Z-ring formation in dividing cells.     

Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.     

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.     

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.     

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.

     

MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli

The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K_d) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K_d for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k_on). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.     

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.     

Identification and Characterization of a Gene Cluster Required for Proper Rod Shape,Cell Division,and Pathogenesis in Clostridium difficile

Little is known about cell division in Clostridium difficile, a strict anaerobe that causes serious diarrheal diseases in people whose normal intestinal microbiome has been perturbed by treatment with broad-spectrum antibiotics. Here we identify and characterize a gene cluster encoding three cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, but MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of cyan fluorescent protein (CFP) to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. This application of CFP was possible because we discovered that O₂-dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they might be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.     

The β-Lactam Resistance Protein Blr,a Small Membrane Polypeptide,Is a Component of the Escherichia coli Cell Division Machinery

In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli β-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to β-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions.     

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.     

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.Bacterial cytokinesis is a highly coordinated process that is carried out by a multiprotein complex known as the divisome (9, 11, 37, 39). In Escherichia coli, there are at least 10 essential divisomal proteins that carry out the division process. Divisome formation is initiated at the incipient division site by the recruitment of the FtsZ ring (1) which provides a molecular scaffold onto which the other divisional proteins are subsequently loaded (24, 33) (Fig. ​(Fig.1).1). In E. coli, the first proteins to load after FtsZ are a group of predominantly cytoplasmic proteins (FtsA, ZapA, and ZipA) that stabilize nascent FtsZ protofilaments and tether them to the membrane. The stabilized Z-ring then acts as a platform for recruitment of the remaining essential divisomal proteins, which are all single- or multipass membrane proteins (i.e., FtsE/FtsX, FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, and FtsN). With the exception of FtsI, a transpeptidase that cross-links septal murein, the biochemical function of these proteins is unknown.Open in a separate windowFIG. 1.Schema showing the hierarchical pathway of divisome assembly in E. coli and B. subtilis (adapted from reference 30). For a protein to be recruited to the divisome, all of the proteins upstream from it in the hierarchical recruitment pathway must already be present at the septum. Groups of proteins that form a subcomplex independent of other divisomal proteins, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL, are highlighted by gray boxes. Red lines denote pairwise protein-protein interactions that have been experimentally demonstrated using genetic and/or biochemical approaches. The question mark indicates that the precise location of FtsW in the divisome assembly pathway in B. subtilis is currently unknown. (C) Possible outcomes of a heterologous septal targeting experiment in E. coli in which ZapA-DivIB is employed as the bait and GFP-PBP 2B is the prey. A direct interaction between DivIB and PBP 2B should result in a fluorescent ring at midcell (or a pair of dots when viewed in cross-section) due the recruitment of GFP-PBP 2B to the divisome (left panel). In contrast, a halo of fluorescence should be visible around the cell periphery due to the membrane-bound GFP-PBP 2B if there is no interaction between these two proteins (right panel).Divisomal protein recruitment in both Bacillus subtilis and E. coli occurs in a stepwise manner. For example, for FtsQ to be recruited to the E. coli divisome, all of the proteins upstream from it in the hierarchical recruitment pathway shown in Fig. ​Fig.1A1A must already be present at the septum. However, this pathway is not completely linear; some proteins appear to form subcomplexes prior to their recruitment to the divisome, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL (2, 12, 14, 15). The situation in B. subtilis is more complex and less well understood. For example, B. subtilis DivIB, DivIC, FtsL, and PBP 2B appear to be recruited to the septum as an interdependent group late in the cell cycle (10) (Fig. ​(Fig.1B).1B). To further complicate matters, once these individual proteins or subcomplexes have been recruited to the divisome, they engage in a complex network of protein-protein interactions with other divisomal proteins (7, 8, 18, 23).The plethora of protein-protein interactions at the bacterial divisome makes it difficult to decipher which molecular epitopes on individual proteins mediate their interaction with other divisomal proteins. Thus, we recently introduced an artificial septal targeting (AST) technique that allowed us to examine interactions between pairs of interacting B. subtilis divisomal proteins in E. coli (30). This technique involves artificially targeting one of the B. subtilis proteins (the “bait”) to the E. coli divisome by fusing it to E. coli ZapA and then using fluorescence microscopy to determine whether it can recruit to the septum a green fluorescent protein (GFP) fusion to a putative interacting partner (the “prey”) (Fig. ​(Fig.1C).1C). The primary advantage of the AST technique is that it allows direct assessment of the interaction between two B. subtilis divisomal proteins without interference from other members of the divisome.We previously used AST to demonstrate a direct interaction between B. subtilis FtsL and DivIC and between DivIB and PBP 2B (30). The latter finding is consistent with the observation from bacterial two-hybrid studies that B. subtilis DivIB interacts directly with both PBP 2B and FtsL (5) and that the E. coli orthologs of these proteins (FtsI and FtsQ, respectively) also interact strongly (18). The extracellular domain of DivIB is divided into three subdomains, termed α, β, and γ (31). It was recently shown using a combination of nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) that the concave face of the DivIB β domain makes direct contact with the C-terminal head of the FtsL-DivIC heterodimeric coiled coil (25), forming a stabilizing “cap” for these two intrinsically unstable proteins (32). In contrast, the α and γ regions of DivIB are not critical for formation of the DivIB/FtsL/DivIC ternary complex (25).The FtsQ/DivIB-FtsI/PBP 2B interaction appears to be widely conserved in both Gram-negative and Gram-positive bacteria, and therefore we decided to investigate the molecular details of this evolutionarily conserved interaction. By using a combination of AST and site-directed mutagenesis, we show that DivIB and PBP 2B interact exclusively through their extracytoplasmic regions and that this interaction is mediated by residues near the C terminus of DivIB. In combination with the results of previous studies, these new data have allowed us to construct a working model of the DivIB/PBP 2B/FtsL/DivIC complex.     

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.     

Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.     

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr⁸⁰⁵, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.     

Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsK_N) is essential for division, and the C terminus (FtsK_C) is a well characterized DNA translocase. Although the function of FtsK_N is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsK_N and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsK_N is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsK_N was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsK_N also provides an important platform for future studies on essential interactions required for this process.     

Structural and enzymatic characterization of the streptococcal ATP/diadenosine polyphosphate and phosphodiester hydrolase Spr1479/SapH

Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe³⁺ and Mn²⁺ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap_nA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap_nA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap_nA and phosphodiester hydrolase.     

The bacterial divisome: ready for its close-up

Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the correct time in the cell cycle. The structure and dynamics of this machine (divisome) in bacterial model systems are coming more clearly into focus, thanks to incisive cell biology methods in combination with biochemical and genetic approaches. The main conserved structural element of the machine is the tubulin homologue FtsZ, which assembles into a circumferential ring at the division site that is stabilized and anchored to the inner surface of the cytoplasmic membrane by FtsZ-binding proteins. Once this ring is in place, it recruits a series of transmembrane proteins that ultimately trigger cytokinesis. This review will survey the methods used to characterize the structure of the bacterial divisome, focusing mainly on the Escherichia coli model system, as well as the challenges that remain. These methods include recent super-resolution microscopy, cryo-electron tomography and synthetic reconstitution.