Synthesis and assembly of a novel glycan layer in Myxococcus xanthus spores.
ABSTRACT: Myxococcus xanthus is a Gram-negative deltaproteobacterium that has evolved the ability to differentiate into metabolically quiescent spores that are resistant to heat and desiccation. An essential feature of the differentiation processes is the assembly of a rigid, cell wall-like spore coat on the surface of the outer membrane. In this study, we characterize the spore coat composition and describe the machinery necessary for secretion of spore coat material and its subsequent assembly into a stress-bearing matrix. Chemical analyses of isolated spore coat material indicate that the spore coat consists primarily of short 1-4- and 1-3-linked GalNAc polymers that lack significant glycosidic branching and may be connected by glycine peptides. We show that 1-4-linked glucose (Glc) is likely a minor component of the spore coat with the majority of the Glc arising from contamination with extracellular polysaccharides, O-antigen, or storage compounds. Neither of these structures is required for the formation of resistant spores. Our analyses indicate the GalNAc/Glc polymer and glycine are exported by the ExoA-I system, a Wzy-like polysaccharide synthesis and export machinery. Arrangement of the capsular-like polysaccharides into a rigid spore coat requires the NfsA-H proteins, members of which reside in either the cytoplasmic membrane (NfsD, -E, and -G) or outer membrane (NfsA, -B, and -C). The Nfs proteins function together to modulate the chain length of the surface polysaccharides, which is apparently necessary for their assembly into a stress-bearing matrix.
Project description:The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3? spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3? spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.
Project description:To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.
Project description:The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ?cotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ?cotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ?cotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.
Project description:Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.
Project description:Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.
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 exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation.
Project description:Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE-/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat.
Project description:Bacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment.
Project description:The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.