Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.     

Asticcacaulis biprosthecum sp.nov. Life cycle,morphology and cultural characteristics

Asticcacaulis biprosthecum sp. n. is a gram-negative aerobic heterotroph which undergoes a dimorphic life cycle typical of bacteria of the caulobacter group. Prosthecae of these cells are shown to be structurally homologous with prosthecae of two strains ofCaulobacter crescentus. The general physiological and cultural characteristics are recorded.     

Effect of cis-platinum(II)diamminodichloride on cell division of Hyphomicrobium and Caulobacter.

Low concentrations of the radiomimetic agent cis-platinum(II)diamminodichloride (PDD) inhibited cell division in Caulobacter crescentus (0.1 mug/ml) and Hyphomicrobium sp. strain B-522 (1.0 mug/ml) without altering the length of prosthecae. After exposure, cells of C. crescentus appeared as long filaments, whereas only the bud portion of Hyphomicrobium underwent elongation. PDD-treated cells of both species were multinucleated. After the removal of PDD by washing, filaments of C. crescentus fragmented unequally and then normal growth resumed. In Hyphomicrobium (where division involves release of swarmer cells that arise as buds on the distal ends of hyphae), potential septation sites formed in the presence of PDD remained inactive after washing. Reinitiation of cell division in this species was dependent upon the synthesis of new hyphae that could arise from either end of the elongated bud. This finding suggests that the PDD-induced lesion at a given septation site is irreversible and, upon removal of this compound, alternate sites must be synthesized for the subsequent occurrence of cell division.     

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus

Cell division in Gram‐negative bacteria involves the co‐ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin‐like protein FtsZ into a Z‐ring at mid‐cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z‐ring constriction, inner‐ and outer‐membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs‐Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid‐cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast‐growth conditions. State‐of‐the‐art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM‐depleted cells compared with wild‐type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.     

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.     

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.     

FzlA,an essential regulator of FtsZ filament curvature,controls constriction rate during Caulobacter division

During bacterial division, polymers of the tubulin‐like GTPase FtsZ assemble at midcell to form the cytokinetic Z‐ring, which coordinates peptidoglycan (PG) remodeling and envelope constriction. Curvature of FtsZ filaments promotes membrane deformation in vitro, but its role in division in vivo remains undefined. Inside cells, FtsZ directs PG insertion at the division plane, though it is unclear how FtsZ structure and dynamics are mechanistically coupled to PG metabolism. Here we study FzlA, a division protein that stabilizes highly curved FtsZ filaments, as a tool for assessing the contribution of FtsZ filament curvature to constriction. We show that in Caulobacter crescentus, FzlA must bind to FtsZ for division to occur and that FzlA‐mediated FtsZ curvature is correlated with efficient division. We observed that FzlA influences constriction rate, and that this activity is associated with its ability to bind and curve FtsZ polymers. Further, we found that a slowly constricting fzlA mutant strain develops ‘pointy’ poles, suggesting that FzlA influences the relative contributions of radial versus longitudinal PG insertion at the septum. These findings implicate FzlA as a critical coordinator of envelope constriction through its interaction with FtsZ and suggest a functional link between FtsZ curvature and efficient constriction in C. crescentus.     

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.     

Cell division and trichome breakage in Beggiatoa

The process ofBeggiatoa trichome division was elucidated through phase-contrast microscopy and transmission and scanning electron microscopy. Trichome breakage and dispersion is accomplished by the formation of sacrificial cells (necridia) at various points within the trichome. Upon dying, the sacrificial cells lyse, dividing the trichome into two daughter trichomes. This process is identical with that found in many oscillatorian blue-green bacteria, but differs from the mechanism of trichome division in most of the other flexuous gliding bacteria. Cellular division within the trichome occurs by septation, involving the cytoplasmic membrane and the electron-dense L2 (peptidoglycan) layer. The outer envelope layers do not take part in division.     

Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti.

Inhibition of cell division in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis results in elongation into long filaments many times the length of dividing cells. As a first step in characterizing the Rhizobium meliloti cell division machinery, we tested whether R. meliloti cells could also form long filaments after cell division was blocked. Unexpectedly, DNA-damaging agents, such as mitomycin C and nalidixic acid, caused only limited elongation. Instead, mitomycin C in particular induced a significant proportion of the cells to branch at the poles. Moreover, methods used to inhibit septation, such as FtsZ overproduction and cephalexin treatment, induced growing cells to swell, bud, or branch while increasing in mass, whereas filamentation was not observed. Overproduction of E. coli FtsZ in R. meliloti resulted in the same branched morphology, as did overproduction of R. meliloti FtsZ in Agrobacterium tumefaciens. These results suggest that in these normally rod-shaped species and perhaps others, branching and swelling are default pathways for increasing mass when cell division is blocked.     

Cell division in morphological mutants ofAgmenellum quadruplicatum,strain BG-1

Summary Two types of filamentous mutants were derived from the unicellular blue-green alga,Agmenellum, by brief exposure to nitrosoguanidine. The parent exhibits constrictive division analogous to that of the enteric bacteria. The septate mutant exhibits septal division which is almost identical to that observed in all filamentous blue-green algae thus far described. In this mutant, the two outer wall layers fail to invaginate, leaving the daughter cells connected. The coenocytic filamentous mutant divides sporadically by both of these methods. The nuclear region of this mutant appears continuous throughout the length of the filament. It is suggested that the non-septate mutant is impaired in the coordination of cytological events leading to cell division.     

The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli

Fission of bacterial cells involves the co-ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram-negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG-binding OM (lipo)proteins. The Tol-Pal system is energized by proton motive force and is well conserved in Gram-negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein-PG and protein-protein interactions. Although the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells, its precise role has remained unclear. We show that all five components accumulate at constriction sites in Escherichia coli and that mutants lacking an intact system suffer delayed OM invagination and contain large OM blebs at constriction sites and cell poles. We propose that Tol-Pal constitutes a dynamic subcomplex of the division apparatus in Gram-negative bacteria that consumes energy to establish transient trans-envelope connections at/near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction.     

Pattern of unequal cell division and development in Caulobacter crescentus

The pattern of asymmetric division has been examined in Caulobacter crescentus (gram-negative aquatic bacteria) by determining the position of the “division site” on cells of different ages. Measurements of cell width and length at this site, which corresponds to the point of eventual cell separation, were made on electron micrographs of cells stained with phosphotungstic acid. The results show that (i) the division site is formed early in the cell cycle and it constitutes the first visible feature on the growing stalked cell to differentiate the incipient swarmer cell, (ii) the division site is located asymmetrically (closer to the swarmer pole than the stalked pole) on the dividing cell, (iii) its position relative to the stalked and swarmer poles does not change during the cell cycle, and (iv) division is consequently unequal, with the swarmer cell always smaller than the stalked cell. The implications of these findings for general models of unequal cell division and stem cell development are discussed.     

Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge.

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with [meso-3H]diaminopimelic acid ([3H]Dap). The second method was autoradiography of cells pulse-labeled with [3H]Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surface components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of [3H]Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific beta-lactam antibiotic furazlocillin did not affect [3H]Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.     

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

DipM,a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus

In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C‐terminal lysostaphin‐like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.     

Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.     

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.     

Cell division in baeocyte producing cyanobacteria

Summary An ultrastructural examination of cell division in two baeocyte producing cyanobacteria,Pleurocapsa minor andDermocarpa violaceae, reveals two distinct patterns of binary (transverse) fission. Septate binary fission, inPleurocapsa minor, involves centripetal synthesis and deposition of the mucopolymer cell wall layer (L 2). The ingrowth of the cytoplasmic membrane and L 1 cell wall layer, along with the synthesis of the L 2 cell wall layer, results in the formation of a prominent septum. Partitioning of the cell occurs by the constriction of the outer cell wall layers (L 3 and L 4) through the septum. InDermocarpa violaceae, constrictive binary fission occurs by the simultaneous ingrowth or constriction of the cytoplasmic membrane and all cell wall layers (L1, L2, L3, L4). Septate and constrictive binary fission may proceed symmetrically (medially) or asymmetrically (nonmedially). Multiple fission occurs regularly inDermocarpa violaceae and provides for a rapid means of reproduction when compared to binary fission. Successive radial and tangential divisions of the protoplast result in formation of many small daughter cells (baeocytes). The process of multiple fission is similar to septate binary fission with reduced septa being formed. However, constriction of the outer cell wall layers, through the septa, proceeds concurrently with septum formation.