The C-terminal domain of MinC inhibits assembly of the Z ring in Escherichia coli

In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(122-231), the C terminus of full-length MinC, or the C terminus of MinC(122-231) perturbed MinC function, which may explain why cell division inhibition by MinC(122-231) was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.     

Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in Escherichia coli

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.     

Morphogenesis of rod-shaped sacculi

For growth and division of rod-shaped bacteria, the cylindrical part of the sacculus has to be elongated and two new cell poles have to be synthesized. The elongation is performed by a protein complex, the elongase that inserts disaccharidepentapeptide units at a limited number of discrete sites while using the cytoskeletal MreB helix as a tracking device. Upon initiation of cell division by positioning of the cytoskeletal Z-ring at mid cell, a switch from dispersed to concentrated local peptidoglycan-synthesis occurs. From this point on, peptidoglycan synthesis is for a large part redirected from elongating activity to synthesis of new cell poles by the divisome. The divisome might be envisioned as an extended elongase because apart from its basic peptidoglycan synthesizing activity, specific functions have to be added. These are conversion from a cylinder to a sphere, invagination of the outer membrane and addition of hydrolases that allow separation of the daughter cells. The elongase and the divisome are dynamic hyperstructures that probably share part of their proteins. Although this multifunctionality and flexibility form a barrier to the functional elucidation of its individual subunits, it helps the cells to survive a variety of emergency situations and to proliferate securely.     

Dynamic proteins in bacteria

Growth of the bacterial cell involves proteins that assemble into dynamic localized structures that are required for cellular morphogenesis and division. During the past year, the continued application of fluorescence microscopy has led to the discovery of novel actin-like filaments involved in cell shape and plasmid DNA segregation, and to new insights into the regulation and dynamics of the Z-ring. Studies on the Min proteins, which rapidly oscillate between the cell poles to spatially regulate Z-ring assembly, has led to a biochemical basis for the oscillation and a suggestion that MinD assembles into dynamic filaments. These studies further demonstrate that the eukaryotic cytoskeleton had its origins in bacteria.     

Maturation of the Escherichia coli divisome occurs in two steps

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14-21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.     

Timing of Z-ring localization in Escherichia coli

Bacterial cell division takes place in three phases: Z-ring formation at midcell, followed by divisome assembly and building of the septum per se. Using time-lapse microscopy of live bacteria and a high-precision cell edge detection method, we have previously found the true time for the onset of septation, τ(c), and the time between consecutive divisions, τ(g). Here, we combine the above method with measuring the dynamics of the FtsZ-GFP distribution in individual Escherichia coli cells to determine the Z-ring positioning time, τ(z). To analyze the FtsZ-GFP distribution along the cell, we used the integral fluorescence profile (IFP), which was obtained by integrating the fluorescence intensity across the cell width. We showed that the IFP may be approximated by an exponential peak and followed the peak evolution throughout the cell cycle, to find a quantitative criterion for the positioning of the Z-ring and hence the value of τ(z). We defined τ(z) as the transition from oscillatory to stable behavior of the mean IFP position. This criterion was corroborated by comparison of the experimental results to a theoretical model for the FtsZ dynamics, driven by Min oscillations. We found that τ(z) < τ(c) for all the cells that were analyzed. Moreover, our data suggested that τ(z) is independent of τ(c), τ(g) and the cell length at birth, L(0). These results are consistent with the current understanding of the Z-ring positioning and cell septation processes.     

DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus

Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.     

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.A unique lipid composition and lipid-protein interactions appear to exist at the transient membrane domain that defines the division site in bacterial cells (1). Using the cardiolipin (CL)⁴-specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2). Subsequently others reported similar CL domains in Bacillus subtilis (3) and Pseudomonas putida (4). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ΔminCDE mutant (5). We suggested that formation of CL domains at cell pole/division sites plays an important role in selection and recognition of the division site by amphitropic cell cycle and cell division proteins, such as DnaA (initiation of DNA replication at oriC), MinD (a part of MinCDE system preventing positioning of the divisome at cell poles in E. coli), and FtsA (bacterial actin, which is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin), responsible for targeting the Z-ring to the mid-cell membrane domain). They interact directly with membrane phospholipids through specific amphipathic motifs enriched in basic amino acids, which confers the preference for anionic lipids (for references see Ref. 1). In E. coli the ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP bound to MinD resulting in release of MinD, and consequently MinC, from the membrane into the cytoplasm. As a result, all three proteins oscillate between the cell poles maintaining the maximum concentration of the inhibitor MinC at the cell poles and its minimum concentration at the cell center. Pole-to-pole oscillation of Min proteins occurs by dynamic redistribution of the proteins within a helical oligomeric structure that winds around the cell (for recent review and references see Ref. 6).Our previous study of a mutant lacking phosphatidylethanolamine (PE) and containing highly elevated levels of phosphatidylglycerol (PG) and CL demonstrated a strong inhibition of cell division and aggregation of MinD and FtsZ/FtsA proteins at domains enriched in CL (7, 8). To further investigate the role of lipids in the process of cell division, we chose an E. coli mutant with an opposite extreme in phospholipid composition to PE-lacking mutants, namely a ΔpgsA mutant (pgsA encodes phosphatidylglycerol phosphate synthase, which catalyzes the committed step to PG and CL synthesis (9)). This mutant is devoid of PG and CL (contribute ∼20 mole % of phospholipids in wild type) and contains higher levels of PE (∼90 mole % versus 80 mole % in wild type) (10, 11). Interestingly, the ΔpgsA null mutant accumulates elevated amounts of the phospholipid precursors, phosphatidic acid (PA) (∼4 mole %) and CDP-diacylglycerol (∼3 mole %), which are also anionic lipids that were proposed to fulfill the structural and functional roles of PG and CL (10, 11). These results suggest that a minimum of 5–10% anionic lipid is required to support viability. Another minor anionic phospholipid, N-acylphosphatidylethanolamine was suggested to be present in wild-type E. coli (12), which, if proven true, might be also elevated in this mutant. Finally, if anionic lipids are essential for cell division, then we would expect these normally minor lipids to segregate into similar anionic lipid domains, as does CL.In this report we identified N-acyl-PE in E. coli and, along with PA, its enrichment in polar/septal membrane domains of the ΔpgsA mutant UE54 (11) lacking PG and CL. Thus E. coli has a mechanism for preferential segregation of anionic phospholipids to the polar/septal regions where several amphitropic proteins, which show preference for interaction with anionic phospholipids in vitro, are functionally located.     

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.     

Deletion of the min operon results in increased thermosensitivity of an ftsZ84 mutant and abnormal FtsZ ring assembly, placement, and disassembly

To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C. Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.     

Cytological characterization of YpsB, a novel component of the Bacillus subtilis divisome

Cell division in bacteria is carried out by an elaborate molecular machine composed of more than a dozen proteins and known as the divisome. Here we describe the characterization of a new divisome protein in Bacillus subtilis called YpsB. Sequence comparisons and phylogentic analysis demonstrated that YpsB is a paralog of the division site selection protein DivIVA. YpsB is present in several gram-positive bacteria and likely originated from the duplication of a DivIVA-like gene in the last common ancestor of bacteria of the orders Bacillales and Lactobacillales. We used green fluorescent protein microscopy to determine that YpsB localizes to the divisome. Similarly to that for DivIVA, the recruitment of YpsB to the divisome requires late division proteins and occurs significantly after Z-ring formation. In contrast to DivIVA, however, YpsB is not retained at the newly formed cell poles after septation. Deletion analysis suggests that the N terminus of YpsB is required to target the protein to the divisome. The high similarity between the N termini of YpsB and DivIVA suggests that the same region is involved in the targeting of DivIVA. YpsB is not essential for septum formation and does not appear to play a role in septum positioning. However, a ypsB deletion has a synthetic effect when combined with a mutation in the cell division gene ftsA. Thus, we conclude that YpsB is a novel B. subtilis cell division protein whose function has diverged from that of its paralog DivIVA.     

Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA

At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.     

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.     

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.     

Chloroplast Division Protein ARC3 Regulates Chloroplast FtsZ-Ring Assembly and Positioning in Arabidopsis through Interaction with FtsZ2

Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid.     

MinC mutants deficient in MinD- and DicB-mediated cell division inhibition due to loss of interaction with MinD, DicB, or a septal component

The min locus encodes a negative regulatory system that limits formation of the cytokinetic Z ring to midcell by preventing its formation near the poles. Of the three Min proteins, MinC is the inhibitor and prevents Z-ring formation by interacting directly with FtsZ. MinD activates MinC by recruiting it to the membrane and conferring a higher affinity on the MinCD complex for a septal component. MinE regulates the cellular location of MinCD by inducing MinD, and thereby MinC, to oscillate between the poles of the cell, resulting in a time-averaged concentration of MinCD on the membrane that is lowest at midcell. MinC can also be activated by the prophage-encoded protein DicB, which targets MinC to the septum without recruiting it first to the membrane. Previous studies have shown that the C-terminal domain of MinC is responsible for the interaction with MinD, DicB, and the septal component. In the present study, we isolated mutations in the C-terminal domain of MinC that affected its interaction with MinD, DicB, and the septal component. Among the mutations isolated, R133A and S134A are specifically deficient in the interaction with MinD, E156A is primarily affected in the interaction with DicB, and R172A is primarily deficient in the interaction with the septum. These mutations differentiate the interactions of MinC with its partners and further support the model of MinCD- and MinC-DicB-mediated cell division inhibition.     

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod‐shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re‐assemble, and MreB‐free zones were subsequently observed in the cytoplasmic membrane. These MreB‐free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y‐shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.     

MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis

In Bacillus subtilis, FtsZ ring formation and cell division is favoured at the midcell because the inhibitor proteins MinC and MinD are indirectly restricted to the cell poles by the protein DivIVA. Here we identify MinJ, a topological determinant of medial FtsZ positioning that acts as an intermediary between DivIVA and MinD. Due to unrestricted MinD activity, cells mutated for minJ exhibited pleiotropic defects in homologous recombination, swarming motility and cell division. MinJ restricted MinD activity by localizing MinD to the cell poles through direct protein-protein interaction. MinJ itself localized to cell poles in a manner that was dependent on DivIVA. MinJ is conserved in other low G+C Gram-positive bacteria and may be an important component of cell division site selection in these organisms.     

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.     

Topological regulation of cell division in E. coli. spatiotemporal oscillation of MinD requires stimulation of its ATPase by MinE and phospholipid.

Topological regulation of cell division in E. coli requires positioning a cell division inhibitor, MinC, at the poles of the cell, thus restricting the potential for division to midcell. This positioning is achieved through a rapid oscillation of MinC from pole to pole, a process requiring MinD and MinE. However, the mechanistic basis for this oscillation is not known. Here we report that MinE stimulates MinD ATPase activity, but only in the presence of phospholipid vesicles. Analysis of MinE mutants demonstrates that this stimulation is required for MinD oscillation and suggests that the level of stimulation determines the period of the oscillation. A model is presented in which the requirements for the MinD ATPase contribute spatial and temporal inputs that provide the mechanistic basis for the oscillation.