GerM is required to assemble the basal platform of the SpoIIIA–SpoIIQ transenvelope complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. An early step in the assembly of this transenvelope complex (called the A–Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell–cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A–Q complex and a hub for the localization of mother cell and forespore proteins.     

The SpoIIQ‐SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis

Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc‐binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQ^H120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ^H120S, binds Zn²⁺, and metal absence alters the SpoIIQ‐SpoIIIAH complex in vitro. Possibly, SpoIIQ^H120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post‐engulfment, forespore and mother cell‐specific gene expression, suggesting a channel‐like function. Both engulfment and a channel‐like function may be ancestral functions of SpoIIQ‐SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms.     

Stably bridging a great divide: localization of the SpoIIQ landmark protein in Bacillus subtilis

Many bacterial proteins involved in fundamental processes such as cell shape maintenance, cell cycle regulation, differentiation, division and motility localize dynamically to specific subcellular regions. However, the mechanisms underlying dynamic protein localization are incompletely understood. Using the SpoIIQ protein in Bacillus subtilis as a case study, two reports present important novel insights into how a protein finds its right place at the right time and remains stably bound. During sporulation, SpoIIQ localizes in clusters in the forespore membrane at the interface that separates the forespore and mother cell and functions as a landmark protein for SpoIIIAH in the mother cell membrane. The extracellular domains of SpoIIQ and SpoIIIAH interact directly effectively bridging the gap between the two membranes. Here, SpoIIQ localization is shown to depend on two pathways, one involves SpoIIIAH, the second involves two peptidoglycan‐degrading enzymes SpoIIP and SpoIID; and, SpoIIQ is only delocalized in the absence of all three proteins. Importantly, in the absence of SpoIIIAH, SpoIIQ apparently localizes normally. However, FRAP experiments demonstrated that SpoIIQ is not stably maintained in the clusters in this mutant. Thus, a second targeting pathway can mask significant changes in the localization of a protein.     

Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.     

Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a transenvelope secretion complex that connects the mother cell and developing spore. The forespore protein SpoIIQ and the mother‐cell protein SpoIIIAH interact across the double membrane septum and are thought to assemble into a channel that serves as the basement layer of this specialized secretion system. SpoIIQ is absolutely required to recruit SpoIIIAH to the sporulation septum on the mother‐cell side, however the mechanism by which SpoIIQ is localized has been unclear. Here, we show that SpoIIQ localization requires its partner protein SpoIIIAH and degradation of the septal peptidoglycan (PG) by the two cell wall hydrolases SpoIID and SpoIIP. Our data suggest that PG degradation enables a second mother‐cell‐produced protein to interact with SpoIIQ. Cells in which both mother‐cell anchoring mechanisms have been disabled have a synergistic sporulation defect suggesting that both localization factors function in the secretion complex. Finally, we show that septal PG degradation is critical for the assembly of an active complex. Altogether, these results suggest that the specialized secretion system that links the mother cell and forespore has a complexity approaching those found in Gram‐negative bacteria and reveal that the sporulating cell must overcome similar challenges in assembling a transenvelope complex.     

A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels

During spore formation in Bacillus subtilis, sigma(E)-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of sigma(G) in the forespore by a pathway of intercellular signalling that is composed of multiple proteins of unknown function. Here, we confirm that the vegetative protein SpoIIIJ, the forespore protein SpoIIQ and eight membrane proteins (SpoIIIAA through SpoIIIAH) produced in the mother cell under the control of sigma(E) are ordinarily required for intercellular signalling. In contrast, an anti-sigma(G) factor previously implicated in the pathway is shown to be dispensable. We also present evidence suggesting that SpoIIIJ is a membrane protein translocase that facilitates the insertion of SpoIIIAE into the membrane. In addition, we report the isolation of a mutation that partially bypasses the requirement for SpoIIIJ and for SpoIIIAA through SpoIIIAG, but not for SpoIIIAH or SpoIIQ, in the activation of sigma(G). We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of sigma(G). Finally, based on the similarity of SpoIIIAH to a component of type III secretion systems, we speculate that signalling is mediated by a channel that links the mother cell to the forespore.     

The SpoIIQ landmark protein has different requirements for septal localization and immobilization

Bacillus subtilis sporulation depends on the forespore membrane protein SpoIIQ, which interacts with the mother cell protein SpoIIIAH at the septum to localize other sporulation proteins. It has remained unclear how SpoIIQ localizes. We demonstrate that localization of SpoIIQ is achieved by two pathways: SpoIIIAH and the SpoIID, SpoIIM, SpoIIP engulfment proteins. SpoIIQ shows diffuse localization only in a mutant lacking both pathways. Super‐resolution imaging shows that in the absence of SpoIIIAH, SpoIIQ forms fewer, slightly larger foci than in wild type. Surprisingly, photobleaching experiments demonstrate that, although SpoIIQ localizes without SpoIIIAH, it is no longer immobilized, and is therefore able to exchange subunits within a localized pool. SpoIIQ mobility is further increased by the additional absence of the engulfment proteins. However an enzymatically inactive SpoIID protein immobilizes SpoIIQ even in the absence of SpoIIIAH, indicating that complete septal thinning is not required for SpoIIQ localization. This suggests that SpoIIQ interacts with both SpoIIIAH and the engulfment proteins or their peptidoglycan cleavage products. They further demonstrate that apparently normal localization of a protein without a binding partner can mask dramatic alterations in protein mobility. We speculate that SpoIIQ assembles foci along the path defined by engulfment proteins degrading peptidoglycan.     

Forespore engulfment mediated by a ratchet-like mechanism

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.     

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.     

Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis

In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood. SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression. We find that SpoIIQ dynamically localizes to the sporulation septum, tracks the engulfing mother cell membrane, assembles into helical arcs around the forespore and is finally degraded. Retention of SpoIIQ in the septum requires one or more mother cell-expressed proteins. We also observed that any forespore-expressed membrane protein initially localizes to the septum and later spreads throughout the forespore membrane, suggesting that membrane protein insertion occurs at the forespore septal region. This possibility provides an attractive mechanism for how activation of mother cell-specific gene expression is restricted to adjacent sister cells, since direct insertion of the signaling protein SpoIIR into the septum would spatially restrict its activity. In keeping with this hypothesis, we find that SpoIIR localizes to the septum and is transiently expressed.     

spoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis

A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ , which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.     

Structural insights into ring-building motif domains involved in bacterial sporulation

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for “Ring-Building Motifs”. During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the β-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.     

Bistable Forespore Engulfment in Bacillus subtilis by a Zipper Mechanism in Absence of the Cell Wall

To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in 60 of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.