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.     

MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter

Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.     

Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.     

Assembly of the FtsZ ring at the central division site in the absence of the chromosome

The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli , we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.     

FtsZ ring: the eubacterial division apparatus conserved in archaebacteria

FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.     

Chloroplast division machinery as revealed by immunofluorescence and electron microscopy

The division of chloroplasts (plastids) is critical for the viability of photosynthetic eukaryotes. Previously we reported on the chloroplast division apparatus, which consists of inner and outer double or triple rings (PD rings). Chloroplasts are assumed to arise from bacterial endosymbionts, while bacterial division is instigated by a bacterial cytokinesis Z-ring protein (FtsZ). Here we present immunofluorescence and electron-microscopic evidence of chloroplast division via complex machinery involving the FtsZ and PD rings in the higher plant Pelargonium zonale Ait. Prior to invagination, the FtsZ protein was attached to a ring at the stromal division site. Following formation of the FtsZ ring, the inner stromal and outer cytosolic PD rings appeared, signifying the initiation of invagination. The FtsZ ring and the PD rings were found at the leading edge of chloroplast constriction throughout division. During chloroplast division, neither the FtsZ nor the inner rings changed width, but the volume of the outer ring gradually increased. We suggest that the FtsZ ring determines the division region, after which the inner and outer PD rings are formed as a lining for the FtsZ ring. With the outer ring providing the motivating force, the FtsZ and inner PD rings ultimately decompose to their base components.     

ZapA and ZapB form an FtsZ‐independent structure at midcell

Cell division in Escherichia coli begins with the polymerization of FtsZ into a ring‐like structure, the Z‐ring, at midcell. All other division proteins are thought to require the Z‐ring for recruitment to the future division site. Here, it is reported that the Z‐ring associated proteins ZapA and ZapB form FtsZ‐independent structures at midcell. Upon Z‐ring disruption by the FtsZ polymerization antagonist SulA, ZapA remained at midcell as a cloud‐like accumulation. Using ZapA(N60Y), a variant defective for interaction with FtsZ, it was established that these ZapA structures form without a connection to the Z‐ring. Furthermore, midcell accumulations of GFP‐ZapA(N60Y) often preceded Z‐rings at midcell and required ZapB to assemble, suggesting that ZapB polymers form the foundation of these structures. In the absence of MatP, a DNA‐binding protein that links ZapB to the chromosomal terminus region, cloud‐like ZapA structures still formed but failed to track with the chromosome terminus and did not consistently precede FtsZ at midcell. Taken together, the results suggest that FtsZ‐independent structures of ZapA–ZapB provide additional positional cues for Z‐ring formation and may help coordinate its assembly with chromosome replication and segregation.     

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.     

Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.     

The min locus, which confers topological specificity to cell division, is not involved in its coupling with nucleoid separation.

In Escherichia coli, nucleoid separation and cell constriction remain tightly linked when division is retarded by altering the level of synthesis of the protein FtsZ. In this study, we have examined the role of the min locus, which is responsible for the inactivation of polar division sites, in the partition-septation coupling mechanism. We conclude that the coupling persists in a delta min strain and that its timing relative to replication remains dependent on the level of FtsZ synthesis. We suggest that the retarded nucleoid segregation observed in min mutants is the result of this coupling in cells with a perturbed pattern of nonpolar divisions.     

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.     

Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain

In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favour of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.     

The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains

FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.     

Timing of FtsZ Assembly in Escherichia coli

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.     

Bacterial cell division and the septal ring

Cell division in bacteria is mediated by the septal ring, a collection of about a dozen (known) proteins that localize to the division site, where they direct assembly of the division septum. The foundation of the septal ring is a polymer of the tubulin-like protein FtsZ. Recently, experiments using fluorescence recovery after photobleaching have revealed that the Z ring is extremely dynamic. FtsZ subunits exchange in and out of the ring on a time scale of seconds even while the overall morphology of the ring appears static. These findings, together with in vitro studies of purified FtsZ, suggest that the rate-limiting step in turnover of FtsZ polymers is GTP hydrolysis. Another component of the septal ring, FtsK, is involved in coordinating chromosome segregation with cell division. Recent studies have revealed that FtsK is a DNA translocase that facilitates decatenation of sister chromosomes by TopIV and resolution of chromosome dimers by the XerCD recombinase. Finally, two murein hydrolases, AmiC and EnvC, have been shown to localize to the septal ring of Escherichia coli, where they play an important role in separation of daughter cells.     

EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.     

Spatial and temporal organization of replicating Escherichia coli chromosomes

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.