PBP1 is a component of the Bacillus subtilis cell division machinery

Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.     

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis.

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.     

The membrane-bound cell division protein DivIB is localized to the division site in Bacillus subtilis

The cell division gene divIB of Bacillus subtilis is essential for the normal rate of growth and division. The gene product, DivIB, is a membrane-bound protein in which the bulk of the protein (at the C-terminal end) is on the exterior surface of the cell membrane. DivIB is involved in the early stages of septum formation, but its exact role in cell division is unknown. To gain more information about the mode of action of DivIB in septum formation, we determined the location of DivIB within the cell membrane using immunofluorescence. This immunolocalization approach established that DivIB becomes localized to the division site before visible septation and remains localized to this site throughout the division process. Various DivIB immunostaining patterns were observed in immunofluorescence experiments and, together with cell length and nucleoid distance measurements, have allowed us to propose two models to describe DivIB localization during the cell cycle.     

Septation and chromosome segregation during sporulation in Bacillus subtilis.

The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division. It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.     

SpoIIIE and a novel type of DNA translocase, SftA, couple chromosome segregation with cell division in Bacillus subtilis

Cell division must only occur once daughter chromosomes have been fully separated. However, the initiating event of bacterial cell division, assembly of the FtsZ ring, occurs while chromosome segregation is still ongoing. We show that a two-step DNA translocase system exists in Bacillus subtilis that couples chromosome segregation and cell division. The membrane-bound DNA translocase SpoIIIE assembled very late at the division septum, and only upon entrapment of DNA, while its orthologue, SftA (YtpST), assembled at each septum in B. subtilis soon after FtsZ. Lack of SftA resulted in a moderate segregation defect at a late stage in the cell cycle. Like the loss of SpoIIIE, the absence of SftA was deleterious for the cells during conditions of defective chromosome segregation, or after induction of DNA damage. Lack of both proteins exacerbated all phenotypes. SftA forms soluble hexamers in solution, binds to DNA and has DNA-dependent ATPase activity, which is essential for its function in vivo . Our data suggest that SftA aids in moving DNA away from the closing septum, while SpoIIIE translocates septum-entrapped DNA only when septum closure precedes complete segregation of chromosomes.     

Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis.

The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B. subtilis 168. Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores. The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation. The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth. Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined. During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites. The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site. Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis. It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself.     

MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer

During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The direction of DNA translocation is controlled by the specific assembly of SpoIIIE in the mother cell and subsequent export of DNA into the forespore. We present evidence that the MinCD heterodimer, which spatially regulates cell division during vegetative growth, serves as a forespore-specific inhibitor of SpoIIIE assembly. The deletion of minCD increases the ability of forespore-expressed SpoIIIE to assemble and translocate DNA, and causes otherwise wild-type cells to reverse the direction of DNA transfer, producing anucleate forespores. We propose that two distinct mechanisms ensure the specific assembly of SpoIIIE in the mother cell, the partitioning of more SpoIIIE molecules into the larger mother cell by asymmetric cell division and the MinCD-dependent repression of SpoIIIE assembly in the forespore. Our results suggest that the ability of MinCD to sense positional information is utilized during sporulation to regulate protein assembly differentially on the two faces of the sporulation septum.     

Cytokinesis in bacteria.

Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.     

Balancing self-renewal and differentiation by asymmetric division: insights from brain tumor suppressors in Drosophila neural stem cells

Balancing self-renewal and differentiation of stem cells is an important issue in stem cell and cancer biology. Recently, the Drosophila neuroblast (NB), neural stem cell has emerged as an excellent model for stem cell self-renewal and tumorigenesis. It is of great interest to understand how defects in the asymmetric division of neural stem cells lead to tumor formation. Here, we review recent advances in asymmetric division and the self-renewal control of Drosophila NBs. We summarize molecular mechanisms of asymmetric cell division and discuss how the defects in asymmetric division lead to tumor formation. Gain-of-function or loss-of-function of various proteins in the asymmetric machinery can drive NB overgrowth and tumor formation. These proteins control either the asymmetric protein localization or mitotic spindle orientation of NBs. We also discuss other mechanisms of brain tumor suppression that are beyond the control of asymmetric division.     

SepF, a novel FtsZ-interacting protein required for a late step in cell division

Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.     

Evolution of cell division in bacteria

Molecular evolution in bacteria is examined with an emphasis on cell division. For a bacterial cell to assemble and then divide required an immense amount of integrated cell and molecular biology structures/functions to be present, such as a stable cellular structure, enzyme catalysis, minimal genome, septum formation at mid-cell and mechanisms to take up nutrients and produce and use energy, as well as store it. The first bacterial cell(s) capable of division must have had complex cell and molecular biology functions. At this stage of evolution, they would not have been primitive cells but would have reached a threshold in evolution where cell division occurred in a regulated manner.     

Cortical domains and the mechanisms of asymmetric cell division

Asymmetric cell divisions are central to the generation of cell-fate diversity because factors that are present in a mother cell and distributed unequally at cell division can generate distinct daughters. The process o f asymmetric cell division can be described as consisting of three steps: setting up an asymmetric cue in the mother cell, localizing factors with respect to this cue, and positioning the plane o f cell division so that localized factors are partitioned asymmetrically between daughters. This review describes how specialized cortical domains play a key role in each of these steps and discusses our current understanding of the molecular nature o f cortical domains and the mechanisms by which they may orchestrate asymmetric cell divisions.     

The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent manner

The process of bacterial cell division involves the assembly of a complex of proteins at the site of septation that probably provides both the structural and the cytokinetic functions required for elaboration and closure of the septal annulus. During sporulation in Bacillus subtilis , this complex of proteins is modified by the inclusion of a sporulation-specific protein, SpoIIE, which plays a direct role in gene regulation and also has a genetically separable role in determining the gross structural properties of the specialized sporulation septum. We demonstrate by both green fluorescent protein (GFP) fusions and indirect immunofluorescence microscopy that SpoIIGA, a protein required for proteolytic cleavage of pro-σ^E, is also targeted to the sporulation septum. Septal localization of SpoIIGA–GFP occurred even in the structurally abnormal septum formed by a SpoIIE null mutant. We also report the isolation of a spoIIGA homologue from Bacillus megaterium , a species in which the cells are significantly larger than those of B . subtilis . We have exploited the physical dimensions of the B . megaterium sporangium, in conjunction with wide-field deconvolution microscopy, to construct three-dimensional projections of sporulating cells. These projections indicate that SpoIIGA–GFP is initially localized in an annulus at the septal periphery and is only later localized uniformly throughout the septa. Localization was also detected in a B . subtilis spo0H null strain that fails to construct a spore septum. We propose that SpoIIGA is sequestered in the septum by an interaction with components of the septation machinery and that this interaction begins before the construction of the asymmetric septum.     

Cell-cycle-specific inhibition by chloramphenicol of septum fromation and cell division in synchronized cells of Bacillus subtilis.

The relationship between protein synthesis and processes of cell division was studied by using synchronized cells of Bacillus subtilis 168. The addition of chloramphenicol at the beginning of synchronous growth prevented septum formation and cell division, suggesting the requirement of protein synthesis for the processes of cell division. Experiments in which the drug was added to the cells at different cell ages showed that the protein synthesis required for the initiation of septum formation was completed at about 15 min and that the protein synthesis required for cell division was completed at about 45 min. By interpreting the result from the concept of the transition point for protein synthesis, it was suggested that the processes of cell division in B. subtilis require at least two kinds of protein molecules which are synthesized at distinct stages in the cell cycle. This was supported by the result of an experiment in which starvation and the readdition of a required amino acid to exponentially growing cells induced two steps of synchronous cell division. Further, the two transition points are in agreement with the estimations obtained by residual division after the inhibition of protein synthesis in asynchronous cells. The relationship of the timing between the completion of chromosome replication and the two transition points was also studied.     

The Bacillus subtilis cell division protein FtsL localizes to sites of septation and interacts with DivIC

FtsL is a small bitopic membrane protein required for vegetative cell division and sporulation in Bacillus subtilis. We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion. GFP-FtsL was localized at mid-cell in vegetative cells and at the asymmetric septum in sporulating cells. We also show that FtsL forms a ring-like structure at the division site and that it remains localized at mid-cell during the whole septation process. By yeast two-hybrid analysis and non-denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane-bound division protein, the FtsL-like DivIC protein.     

Taking shape: control of bacterial cell wall biosynthesis

The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape.