In long bacterial cells,the Min system can act off‐center

In many rod‐shaped bacteria, the Min system is well‐known for generating a cell‐pole to cell‐pole standing wave oscillation with a single node at mid‐cell to align cell division. In filamentous E. coli cells, the single‐node standing wave transitions into a multi‐nodal oscillation. These multi‐nodal dynamics have largely been treated simply as an interesting byproduct of artificially elongated cells. However, a recent in vivo study by Muraleedharan et al. shows how multi‐nodal Min dynamics are used to align non‐mid‐cell divisions in the elongated swarmer cells of Vibrio parahaemolyticus. The authors propose a model where the combined actions of cell‐length dependent Min dynamics, in concert with nucleoid occlusion along the cell length and regulation of FtsZ levels ensures Z ring formation and complete chromosome segregation at a single off‐center position. By limiting the number of cell division events to one per cell at an off‐center position, long swarmer cells are preserved within a multiplying population. The findings unveil an elegant mechanism of cell‐division regulation by the Min system that allows long swarmer cells to divide without the need to ‘dedifferentiate’.     

Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non‐medial septa

Mycobacterium spp., rod‐shaped cells belonging to the phylum Actinomycetes, lack the Min‐ and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid‐cell. We show that the position for establishment of the FtsZ‐ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non‐medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane‐specific dye FM4‐64 and fluorescent antibiotic vancomycin (FL‐Vanco), respectively, showed that many division sites were off‐centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time‐lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post‐septal DNA transport and unequal polar growth may compensate for the non‐medial division site placement in Mycobacterium spp.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.     

Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.     

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

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

Specific amino acid substitutions in β strand S2 of FtsZ cause spiraling septation and impair assembly cooperativity in Streptomyces spp.

Bacterial cell division is orchestrated by the Z ring, which is formed by single‐stranded treadmilling protofilaments of FtsZ. In Streptomyces, during sporulation, multiple Z rings are assembled and lead to formation of septa that divide a filamentous hyphal cell into tens of prespore compartments. We describe here mutant alleles of ftsZ in Streptomyces coelicolor and Streptomyces venezuelae that perturb cell division in such a way that constriction is initiated along irregular spiral‐shaped paths rather than as regular septa perpendicular to the cell length axis. This conspicuous phenotype is caused by amino acid substitutions F37I and F37R in β strand S2 of FtsZ. The F37I mutation leads, instead of regular Z rings, to formation of relatively stable spiral‐shaped FtsZ structures that are capable of initiating cell constriction. Further, we show that the F37 mutations affect the polymerization properties and impair the cooperativity of FtsZ assembly in vitro. The results suggest that specific residues in β strand S2 of FtsZ affect the conformational switch in FtsZ that underlies assembly cooperativity and enable treadmilling of protofilaments, and that these features are required for formation of regular Z rings. However, the data also indicate FtsZ‐directed cell constriction is not dependent on assembly cooperativity.     

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

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

In vivo organization of the FtsZ‐ring by ZapA and ZapB revealed by quantitative super‐resolution microscopy

In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring‐like structure (Z‐ring) at the midcell. Despite its essential role, the molecular architecture of the Z‐ring remains elusive. In this work we examine the roles of two FtsZ‐associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z‐ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single‐molecule‐based super‐resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z‐ring. Furthermore, we find these clusters are not due to the loss of ZapB–MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.     

A conserved coiled‐coil protein pair focuses the cytokinetic Z‐ring in Caulobacter crescentus

The cytoskeletal GTPase FtsZ assembles at midcell, recruits the division machinery and directs envelope invagination for bacterial cytokinesis. ZapA, a conserved FtsZ‐binding protein, promotes Z‐ring stability and efficient division through a mechanism that is not fully understood. Here, we investigated the function of ZapA in Caulobacter crescentus. We found that ZapA is encoded in an operon with a small coiled‐coil protein we named ZauP. ZapA and ZauP co‐localized at the division site and were each required for efficient division. ZapA interacted directly with both FtsZ and ZauP. Neither ZapA nor ZauP influenced FtsZ dynamics or bundling, in vitro, however. Z‐rings were diffuse in cells lacking zapA or zauP and, conversely, FtsZ was enriched at midcell in cells overproducing ZapA and ZauP. Additionally, FtsZ persisted at the poles longer when ZapA and ZauP were overproduced, and frequently colocalized with MipZ, a negative regulator of FtsZ polymerization. We propose that ZapA and ZauP promote efficient cytokinesis by stabilizing the midcell Z‐ring through a bundling‐independent mechanism. The zauPzapA operon is present in diverse Gram‐negative bacteria, indicating a common mechanism for Z‐ring assembly.     

Robust Min‐system oscillation in the presence of internal photosynthetic membranes in cyanobacteria

The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ‐ring formation and represents one of the best‐understood examples of emergent protein self‐organization in nature. The oscillatory patterns of the Min‐system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self‐organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, it was shown that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole‐to‐pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction‐diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Interestingly, in addition to oscillating, MinC exhibits a midcell localization dependent on MinD and the DivIVA‐like protein Cdv3, indicating that two distinct pools of MinC are coordinated in S. elongatus. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min‐system function in bacteria and organelles with internal membrane systems.     

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re‐direct peptidoglycan biosynthesis to mid‐cell during cell division in polar‐growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid‐cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid‐cell, exhibit GTPase activity and form co‐polymers, only one, FtsZ_AT, is required for cell division. We find that FtsZ_AT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid‐cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ_AT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid‐cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.     

Divisome‐dependent subcellular localization of cell–cell joining protein SepJ in the filamentous cyanobacterium Anabaena

Heterocyst‐forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA‐dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two‐hybrid system. We found SepJ self‐interaction and a specific interaction with FtsQ, confirmed by co‐purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.     

FtsA reshapes membrane architecture and remodels the Z‐ring in Escherichia coli

Cell division in prokaryotes initiates with assembly of the Z‐ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z‐ring is comprised of polymers of tubulin‐like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C‐terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide‐dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C‐terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z‐ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.     

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

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

Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritinlabeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.In memory: R. Y. Stanier     

A gradient‐forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillum gryphiswaldense

Cell division needs to be tightly regulated and closely coordinated with other cellular processes to ensure the generation of fully viable offspring. Here, we investigate division site placement by the cell division regulator MipZ in the alphaproteobacterium Magnetospirillum gryphiswaldense, a species that forms linear chains of magnetosomes to navigate within the geomagnetic field. We show that M. gryphiswaldense contains two MipZ homologs, termed MipZ1 and MipZ2. MipZ2 localizes to the division site, but its absence does not cause any obvious phenotype. MipZ1, by contrast, forms a dynamic bipolar gradient, and its deletion or overproduction cause cell filamentation, suggesting an important role in cell division. The monomeric form of MipZ1 interacts with the chromosome partitioning protein ParB, whereas its ATP‐dependent dimeric form shows non‐specific DNA‐binding activity. Notably, both the dimeric and, to a lesser extent, the monomeric form inhibit FtsZ polymerization in vitro. MipZ1 thus represents a canonical gradient‐forming MipZ homolog that critically contributes to the spatiotemporal control of FtsZ ring formation. Collectively, our findings add to the view that the regulatory role of MipZ proteins in cell division is conserved among many alphaproteobacteria. However, their number and biochemical properties may have adapted to the specific needs of the host organism.     

A novel component of the division‐site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD

Cell division in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod‐shaped bacteria divide precisely at mid‐cell between segregated sister chromosomes. Selection of the correct site for cell division is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents division in the vicinity of the chromosomes, and the Min system, which prevents inappropriate division at the cell poles. In Bacillus subtilis recruitment of the division inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the division‐site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in division because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and divIVA mutants showed that their block in division occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of division per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate division.     

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

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

Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z‐ring

FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z‐ring prior to division. Two small coiled‐coil proteins, ZapA and ZapB, are both recruited early to the Z‐ring. We show here that ZapB is recruited to the Z‐ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two‐hybrid and in vitro interaction assays. Using high‐resolution 3‐D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z‐ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto‐filaments and ZapB mediates further stabilization of this interaction by cross‐linking ZapA molecules bound to adjacent FtsZ proto‐filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z‐ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z‐ring assembly by two different mechanisms.     

The essential role of SepF in mycobacterial division

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