A Quality-Control Mechanism Removes Unfit Cells from a Population of Sporulating Bacteria.
ABSTRACT: Recent discoveries of regulated cell death in bacteria have led to speculation about possible benefits that apoptosis-like pathways may confer to single-celled organisms. However, establishing how these pathways provide increased ecological fitness has remained difficult to determine. Here, we report a pathway in Bacillus subtilis in which regulated cell death maintains the fidelity of sporulation through selective removal of cells that misassemble the spore envelope. The spore envelope, which protects the dormant spore's genome from environmental insults, uses the protein SpoIVA as a scaffold for assembly. We found that disrupting envelope assembly activates a cell death pathway wherein the small protein CmpA acts as an adaptor to the AAA+ ClpXP protease to degrade SpoIVA, thereby halting sporulation and resulting in lysis of defective sporulating cells. We propose that removal of unfit cells from a population of terminally differentiating cells protects against evolutionary deterioration and ultimately loss of the sporulation program.
Project description:Spores of Bacillus subtilis are dormant cell types that are formed when the bacterium encounters starvation conditions. Spores are encased in a shell, termed the coat, which is composed of approximately seventy different proteins and protects the spore's genetic material from environmental insults. The structural component of the basement layer of the coat is an exceptional cytoskeletal protein, termed SpoIVA, which binds and hydrolyzes ATP. ATP hydrolysis is utilized to drive a conformational change in SpoIVA that leads to its irreversible self-assembly into a static polymer in vitro. Here, we characterize the middle domain of SpoIVA, the predicted secondary structure of which resembles the chemotaxis protein CheX but, unlike CheX, does not harbor residues required for phosphatase activity. Disruptions in this domain did not abolish ATP hydrolysis, but resulted in mislocalization of the protein and reduction in sporulation efficiency in vivo. In vitro, disruptions in this domain prevented the ATP hydrolysis-driven conformational change in SpoIVA required for polymerization and led to the aggregation of SpoIVA into particles that did not form filaments. We propose a model in which SpoIVA initially assumes a conformation in which it inhibits its own aggregation into particles, and that ATP hydrolysis remodels the protein so that it assumes a polymerization-competent conformation.
Project description:We report the cloning and characterization of the Bacillus subtilis sporulation locus spoIVA, mutations at which cause an unusual defect in spore formation in which the coat misassembles as swirls within the mother cell. We show that spoIVA is a single gene of 492 codons that is capable of encoding a polypeptide of 55 kDa. Transcription of spoIVA is induced at about the second hour of sporulation by the regulatory protein sigma E from two closely spaced promoters designated P1 and P2. Experiments in which the upstream promoter P1 was removed show that transcription of spoIVA from P2 is sufficient for efficient spore formation. Based on these and other findings, we infer that the spoIVA gene product is a morphogenetic protein; we discuss its role in the deposition of coat polypeptides around the developing forespore.
Project description:Mature spores of the bacterium Bacillus subtilis are encased by two concentric shells: an inner shell (the 'cortex'), made of peptidoglycan; and an outer proteinaceous shell (the 'coat'), whose basement layer is anchored to the surface of the developing spore via a 26-amino-acid-long protein called SpoVM. During sporulation, initiation of cortex assembly depends on the successful initiation of coat assembly, but the mechanisms that co-ordinate the morphogenesis of both structures are largely unknown. Here, we describe a sporulation pathway involving SpoVM and a 37-amino-acid-long protein named 'CmpA' that is encoded by a previously un-annotated gene and is expressed under control of two sporulation-specific transcription factors (?(E) and SpoIIID). CmpA localized to the surface of the developing spore and deletion of cmpA resulted in cells progressing through the sporulation programme more quickly. Overproduction of CmpA did not affect normal growth or cell division, but delayed entry into sporulation and abrogated cortex assembly. In those cells that had successfully initiated coat assembly, CmpA was removed by a post-translational mechanism, presumably in order to overcome the sporulation inhibition it imposed. We propose a model in which CmpA participates in a developmental checkpoint that ensures the proper orchestration of coat and cortex morphogenesis by repressing cortex assembly until coat assembly successfully initiates.
Project description:Mutations in the spoIVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a sigma E-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spoIVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spoIVA mutations, would be in accord with the hypothesis that sigma E is only active after septation and in the mother cell compartment.
Project description:Some bacteria, such as Bacillus subtilis, withstand starvation by forming dormant spores that revive when nutrients become available. Although sporulation and spore revival jointly determine survival in fluctuating environments, the relationship between them has been unclear. Here we show that these two processes are linked by a phenotypic "memory" that arises from a carry-over of molecules from the vegetative cell into the spore. By imaging life histories of individual B. subtilis cells using fluorescent reporters, we demonstrate that sporulation timing controls nutrient-induced spore revival. Alanine dehydrogenase contributes to spore memory and controls alanine-induced outgrowth, thereby coupling a spore's revival capacity to the gene expression and growth history of its progenitors. A theoretical analysis, and experiments with signaling mutants exhibiting altered sporulation timing, support the hypothesis that such an intrinsically generated memory leads to a tradeoff between spore quantity and spore quality, which could drive the emergence of complex microbial traits.
Project description:Bacterial spores are dormant cells that are encased in a thick protein shell, the "coat," which participates in protecting the organism's DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis, 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM's localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.
Project description:The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.
Project description:The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation.IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.
Project description:We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium Bacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like spoIVA, are under the control of the mother cell transcription factor sigmaE. One such gene was spoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.
Project description:Endospore formation by Bacillus subtilis is a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.