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Cell division in Escherichia coli is mediated by a large protein complex called the divisome. Most of the divisome proteins have been identified, but how they assemble onto the Z ring scaffold to form the divisome and work together to synthesize the septum is not well understood. In this review, we summarize the latest findings on divisome assembly and activation as well as provide our perspective on how these two processes might be regulated. [ABSTRACT FROM AUTHOR]

Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the resilient peptidoglycan ( PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low- GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin-binding proteins ( PBPs) between septal and side-wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent-vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2 D and 3 D- SIM (structured illumination) immunofluorescence microscopy ( IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3 D- SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines. [ABSTRACT FROM AUTHOR]

Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis. [ABSTRACT FROM AUTHOR]

Dynamic oscillation of the Min system in Escherichia coli determines the placement of the division plane at the midcell. In addition to stimulating MinD ATPase activity, we report here that MinE can directly interact with the membrane and this interaction contributes to the proper MinDE localization and dynamics. The N-terminal domain of MinE is involved in direct contact between MinE and the membranes that may subsequently be stabilized by the C-terminal domain of MinE. In an in vitro system, MinE caused liposome deformation into membrane tubules, a property similar to that previously reported for MinD. We isolated a mutant MinE containing residue substitutions in R10, K11 and K12 that was fully capable of stimulating MinD ATPase activity, but was deficient in membrane binding. Importantly, this mutant was unable to support normal MinDE localization and oscillation, suggesting that direct MinE interaction with the membrane is critical for the dynamic behavior of the Min system. [ABSTRACT FROM AUTHOR]

NDR kinases are important for growth and differentiation and require interaction with MOB proteins for activity and function. We characterized the NDR kinases and MOB activators in Neurospora crassa and identified two NDR kinases (COT1 and DBF2) and four MOB proteins (MOB1, MOB2A, MOB2B and MOB3/phocein) that form two functional NDR–MOB protein complexes. The MOB1–DBF2 complex is not only essential for septum formation in vegetative cells and during conidiation, but also functions during sexual fruiting body development and ascosporogenesis. The two MOB2-type proteins interact with both COT1 isoforms and control polar tip extension and branching by regulating COT1 activity. The conserved region directly preceding the kinase domain of COT1 is sufficient for the formation of COT1–MOB2 heterodimers, but also for kinase homodimerization. An additional N-terminal extension that is poorly conserved, but present in most fungal NDR kinases, is required for further stabilization of both types of interactions and for stimulating COT1 activity. COT1 lacking this region is degraded in a mob-2 background. We propose a specific role of MOB3/phocein during vegetative cell fusion, fruiting body development and ascosporogenesis that is unrelated to the three other MOB proteins and NDR kinase signalling. [ABSTRACT FROM AUTHOR]

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-β-1,3-glucanase and the Agn1 endo-α-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for β-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Δ mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein. [ABSTRACT FROM AUTHOR]

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)β-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1+ shut-off and bgs1Δ cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)β-d-glucan and primary septum formation. bgs1+ shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)β-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Δ cells, where there is no Bgs1p. The septum analysis of bgs1+ -repressed cells indicates that linear (1,3)β-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)β-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die. [ABSTRACT FROM AUTHOR]

The conversion of a growing cell into an endospore in Bacillus subtilis involves a phagocytic-like process in which the developing spore (the forespore) is wholly engulfed by the adjacent mother cell. A prerequisite for engulfment is the removal of peptidoglycan from the septum that separates the forespore from the mother cell, a process that depends on the autolysin SpoIID and two proteins of unknown function, SpoIIM and SpoIIP. Here we present evidence that SpoIIP is also an autolysin, that it acts in tandem with SpoIID, and that all three proteins are in a complex with each other. We further show that the members of the complex exhibit a hierarchical relationship in which SpoIIM is responsible for localization to the septal membrane, SpoIIP localizes to the septal membrane by interacting with SpoIIM, and SpoIID, in turn, localizes by interacting with SpoIIP. Finally, we show that localization of SpoIIM depends on a fourth protein SpoIIB, raising the possibility that the complex contains an additional component and creating an overall hierarchy of the form: SpoIIB→SpoIIM→SpoIIP→SpoIID. [ABSTRACT FROM AUTHOR]

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region. [ABSTRACT FROM AUTHOR]

In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-tike arrays in developing aerial hyphae, with an average spacing of about 1.3 µm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 µm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae. [ABSTRACT FROM AUTHOR]

Nuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time-lapse video microscopy during hyphal extension. Hyphae elongated at 0.1-1.2µm min-1 and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http://www.blacksci.co.uk/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented. [ABSTRACT FROM AUTHOR]

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at alt time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for σWhiG-dependent promoters and EσWhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network. [ABSTRACT FROM AUTHOR]

During sporulation in Bacillus subtilis a small pre-spore ceil is formed by an asymmetric ceil division. Pre-spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spoOJ gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere-like apparatus that anchors the oriC region of the prespore chromosome in the pole of the cell. [ABSTRACT FROM AUTHOR]

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-σE to σE in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why σE is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, σF, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell. [ABSTRACT FROM AUTHOR]