Polar Septum Research Articles

Serine affects engulfment during the sporulation process in Clostridium perfringens strain SM101

ObjectivesAlthough Clostridium perfringens sporulation is a key event in the pathogenesis of food-borne illness, the molecules and underlying mechanisms responsible for regulating sporulation are incompletely understood. The present study sought to identify amino acids that affect sporulation in C. perfringens strain SM101. MethodsA C. perfringens strain was cultured in the chemically defined medium deficient in an amino acid. The bacterial growth was determined by spectrophotometrically measuring culture turbidity and by calculating colony-forming unit. Morphological characteristics were assessed by phase-contrast microscopy with fluorescent staining and by electron microscopy. ResultsThe amino acids Arg, Cys, Gly, His, Ile, Leu, Met, Phe, Thr, Trp, Tyr, and Val were important for sporulation, and furthermore, Ser reduced sporulation. The mechanism underlying Ser-induced prevention of sporulation was assessed morphologically. The numbers of bacterial cells in sporulation stage II were significantly higher in the presence than in the absence of Ser. In the presence of Ser, almost all cells were in stage II−III, characterized by polar septation−early engulfment, and did not proceed to late engulfment. ConclusionsThese results suggest that Ser accelerated the early stage of sporulation of C. perfringens strain SM101, but disturbed the engulfment process, resulting in reduction of sporulation. To the best of our knowledge, this is the first study reporting that an amino acid affects engulfment during the C. perfringens sporulation process.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.

The Transcription Factor CpcR Determines Cell Fate by Modulating the Initiation of Sporulation in Bacillus thuringiensis.

Bacillus thuringiensis is a bacterium capable of differentiating into a spore, a dormant and highly resistant cellular form. During the sporulation process, this bacterium produces insecticidal toxins in the form of a crystal inclusion, usually in the sporulating cell. We previously reported that the B. thuringiensis LM1212 strain can differentiate into two distinct subpopulations of sporeformers and crystal producers and that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a typical B. thuringiensis strain. The transcription factor CpcR was characterized as the regulator responsible for this phenotype. Here, we examined how CpcR interacts with the sporulation network to control the cell differentiation. We found that the sporulation process was inhibited prior to polar septum formation and that Spo0A activity was impaired in the presence of cpcR in strain LM1212. Using bioinformatics and genetic tools, we identified a gene positively controlled by CpcR encoding a putative phosphatase of the Spo0E family known to specifically dephosphorylate phosphorylated Spo0A (Spo0A-P). We showed that this protein (called Spo0E1) is a negative regulator of sporulation and that variations in spo0E1 expression can modulate the production of spores. Using fluorescent reporters to follow gene expression at the single-cell level, we correlated expression of cpcR and sporulation genes to the formation of the two differentiated subpopulations. IMPORTANCE Formation of spores is a paradigm for study of cell differentiation in prokaryotes. Sporulation initiation is governed by a gradual increase in the level and activity of the master regulator Spo0A. Spo0A is usually indirectly phosphorylated by a multicomponent phosphorelay, and modulation of this phosphorelay system is a critical aspect of Bacillus physiology. Though we know that this phosphorelay system is usually affected by two negative regulatory mechanisms, i.e., rap genes and spo0E family genes, the regulatory mechanisms controlling the transcription of these genes are poorly understood. Here, we report that the transcription factor CpcR positively regulates a spo0E family gene and that variations in spo0E expression can modulate the production of spores in B. thuringiensis. This work emphasizes the diversity in modes of sporulation and illustrates the diversity in the strategies employed by bacteria to control this differentiation pathway and ensure their survival.

Shaping an Endospore: Architectural Transformations During Bacillus subtilis Sporulation.

Endospore formation in Bacillus subtilis provides an ideal model system for studying development in bacteria. Sporulation studies have contributed a wealth of information about the mechanisms of cell-specific gene expression, chromosome dynamics, protein localization, and membrane remodeling, while helping to dispel the early view that bacteria lack internal organization and interesting cell biological phenomena. In this review, we focus on the architectural transformations that lead to a profound reorganization of the cellular landscape during sporulation, from two cells that lie side by side to the endospore, the unique cell within a cell structure that is a hallmark of sporulation in B. subtilis and other spore-forming Firmicutes. We discuss new insights into the mechanisms that drive morphogenesis, with special emphasis on polar septation, chromosome translocation, and the phagocytosis-like process of engulfment, and also the key experimental advances that have proven valuable in revealing the inner workings of bacterial cells.

Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase.

Formation of a division septum near a randomly chosen pole during sporulation in Bacillus subtilis creates unequal sized daughter cells with dissimilar programs of gene expression. An unanswered question is how polar septation activates a transcription factor (σ(F)) selectively in the small cell. We present evidence that the upstream regulator of σ(F), the phosphatase SpoIIE, is compartmentalized in the small cell by transfer from the polar septum to the adjacent cell pole where SpoIIE is protected from proteolysis and activated. Polar recognition, protection from proteolysis, and stimulation of phosphatase activity are linked to oligomerization of SpoIIE. This mechanism for initiating cell-specific gene expression is independent of additional sporulation proteins; vegetative cells engineered to divide near a pole sequester SpoIIE and activate σ(F) in small cells. Thus, a simple model explains how SpoIIE responds to a stochastically-generated cue to activate σ(F) at the right time and in the right place.

Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA.

Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.

How SpoVM Interacts with Lipid Bilayers and Bacterial Cell Membranes

SpoVM, a small protein with 26 residues, is essential for spore formation in Bacillus subtili. SpoVM is produced in the mother cell chamber of the sporangium during the process of sporulation and is recruited to the polar septum after the sporangium undergoes asymmetric division. There are reports suggesting that SpoVM localizes to the surface of the forespore by sensing membrane curvature. Besides its transformation from random coils to α-helices when binding to a lipid bilayer, the molecular mechanism of how SpoVM interacts with membranes has not been clarified. For example, we found that the curvature dependence of the binding affinity is difficult to detect. By using single giant unilamellar vesicle method, we found that SpoVM binds to the membrane and expands the surface area of membrane at low concentration with no molecular leakage from the vesicle. However, above the critical concentration, SpoVM causes leakage of the content dye from the GUV. The critical concentrations for different lipid compositions including phosphoethanolamine, phosphatidylglycerol, phosphatidylcholine and cardiolipin were further investigated. Surprisingly, the critical concentration for GUVs with 30% PE is more than six times higher than for other lipid compositions without PE. To understand how SpoVM interact with bacterial membranes, fluorescent microscopy was used to directly observe the effect of SpoVM on Escherichia coli in real time. The result showed that SpoVM makes both the outer membrane and the cytoplasmic membrane of Escherichia coli permeable to Sytox Green above a critical concentration. Oriented circular dichroism results show that SpoVM changes the orientation of its helical axis from parallel to perpendicular with respect to the plane of bilayers. Our results suggest that the SpoVM-membrane interaction follows the same pattern as many other helical antimicrobial peptides, such as magainin, melittin and islet amyloid polypeptides.

An oscillating Min system in Bacillus subtilis influences asymmetrical septation during sporulation

The Min system plays an important role in ensuring that cell division occurs at mid-cell in rod-shaped bacteria. In Escherichia coli, pole-to-pole oscillation of the Min proteins specifically inhibits polar septation. This system also prevents polar division in Bacillus subtilis during vegetative growth; however, the Min proteins do not oscillate in this organism. The Min system of B. subtilis plays a distinct role during sporulation, a process of differentiation which begins with an asymmetrical cell division. Here, we show that oscillation of the E. coli Min proteins can be reproduced following their introduction into B. subtilis cells. Further, we present evidence that the oscillatory behaviour of the Min system inhibits sporulation. We propose that an alternative Min system mechanism avoiding oscillation is evolutionarily important because oscillation of the Min system is incompatible with efficient asymmetrical septum formation and sporulation.

Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σF from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 Å spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded β-sheets are packed against one another and flanked by α-helices in an αββα arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling.

Modeling Three-Dimensional Spatial Regulation of Bacterial Cell Division (Dissertation)

AbstractMany important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the previously reported properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally.

A new multicompartmental reaction-diffusion modeling method links transient membrane attachment of E. coli MinE to E-ring formation

Many important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the previously reported properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-009-9047-2) contains supplementary material, which is available to authorized users.

Expression ofEscherichia coli Min system inBacillus subtilisand its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli, dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.

A new multicompartmental reaction-diffusion modeling method links transient membrane attachment of E. coli MinE to E-ring formation

AbstractMany important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the established properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally.

Characterization and subcellular localization of a bacterial flotillin homologue

The process of endospore formation in Bacillus subtilis is complex, requiring the generation of two distinct cell types, a forespore and larger mother cell. The development of these cell types is controlled and regulated by cell type-specific gene expression, activated by a sigma-factor cascade. Activation of these cell type-specific sigma factors is coupled with the completion of polar septation. Here, we describe a novel protein, YuaG, a eukaryotic reggie/flotillin homologue that is involved in the early stages of sporulation of the Gram-positive model organism B. subtilis. YuaG localizes in discrete foci in the membrane and is highly dynamic. Purification of detergent-resistant membranes revealed that YuaG is associated with negatively charged phospholipids, e.g. phosphatidylglycerol (PG) or cardiolipin (CL). However, localization of YuaG is not always dependent on PG/CL in vivo. A yuaG disruption strain shows a delay in the onset of sporulation along with reduced sporulation efficiency, where the spores develop to a certain stage and then appear to be trapped at this stage. Our results indicate that YuaG is involved in the early stage of spore development, probably playing a role in the signalling cascade at the onset of sporulation.

SpoIIQ Anchors Membrane Proteins on Both Sides of the Sporulation Septum in Bacillus subtilis

During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation.

DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis.

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.

Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (Pi), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of Pi-supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that Pi was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that Pi acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.

Bacteriophages: How Bacterial Spores Capture and Protect Phage DNA

A recent study explains how bacterial spores capture and protect phage DNA, which remains free in the host cytoplasm but is unable to initiate the virulence pathway that leads to lysis of actively growing bacterial cells.

Genetic Dissection of the Sporulation Protein SpoIIE and Its Role in Asymmetric Division inBacillus subtilis

SpoIIE is a dual-function protein in Bacillus subtilis that contributes to the switch from medial to polar cell division during sporulation and is responsible for activating the cell-specific transcription factor sigma(F). SpoIIE consists of an N-terminal domain with 10 membrane-spanning segments (region I), a C-terminal phosphatase domain (region III), and a central domain (region II) of uncertain function. To investigate the role of SpoIIE in polar division, we took advantage of a system for efficiently producing polar septa during growth in a SpoIIE-dependent manner using cells engineered to produce the sporulation protein in response to an inducer. The results show that regions II and III play a critical role in polar septum formation and that specific amino acid substitutions in those regions affect the abilities of SpoIIE both to promote polar division and to localize to the division machinery. Additionally, we show that neither the phosphatase function of SpoIIE nor the N-terminal, membrane-spanning region is needed for the switch to asymmetric division.