Proteins involved in formation of the outermost layer of Bacillus subtilis spores

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.     

Architecture and Assembly of the Bacillus subtilis Spore Coat

Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism.     

Interactions between Bacillus subtilis early spore coat morphogenetic proteins

When challenged by stresses such as starvation, the soil bacterium Bacillus subtilis produces an endospore surrounded by a proteinaceous coat composed of >70 proteins that are organized into three main layers: an amorphous undercoat, lightly staining lamellar inner coat and electron-dense outer coat. This coat protects the spore against a variety of chemicals or lysozyme. Mutual interactions of the coat's building blocks are responsible for the formation of this structurally complex and extraordinarily resistant shell. However, the assembly process of spore coat proteins is still poorly understood. In the present work, the main focus is on the three spore coat morphogenetic proteins: SpoIVA, SpoVID and SafA. Direct interaction between SpoIVA and SpoVID proteins was observed using a yeast two-hybrid assay and verified by coexpression experiment followed by Western blot analysis. Coexpression experiments also confirmed previous findings that SpoVID and SafA directly interact, and revealed a novel interaction between SpoIVA and SafA. Moreover, gel filtration analysis revealed that both SpoIVA and SpoVID proteins form large oligomers.     

Dynamics of the assembly of a complex macromolecular structure--the coat of spores of the bacterium Bacillus subtilis

The coat is the outermost layer of spores of many Bacillus species, and plays a key role in these spores' resistance. The Bacillus subtilis spore coat contains > 70 proteins in four distinct layers: the basement layer, inner coat, outer coat and crust. In this issue of Molecular Microbiology, McKenney and Eichenberger study the dynamics of spore coat assembly using GFP-fusions to 41 B. subtilis coat proteins. A key finding in the work is that formation of the spore coat is initiated by the apparently simultaneous assembly of foci of proteins from all four coat layers on the developing spore just as forespore engulfment by the mother cell begins. The expansion of these foci before completion of forespore engulfment then sets up the scaffold to which coat proteins added later in sporulation are added. This study provides new understanding of the mechanism of the assembly of a multi-protein, multi-lamellar structure.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE

The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat's outer layer. We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.     

Formation of the Dictyostelium spore coat

The spore coat forms as a rigid extracellular wall around each spore cell during culmination. Coats purified from germinated spores contain multiple protein species and an approximately equal mass of polysaccharide, consisting mostly of cellulose and a galactose/N-acetylgalactosamine polysaccharide (GPS). All but the cellulose are prepackaged during prespore cell differentiation in a regulated secretory compartment, the prespore vesicle. The morphology of this compartment resembles an anastomosing, tubular network rather than a spherical vesicle. The molecules of the prespore vesicles are not uniformly mixed but are segregated into partially overlapping domains. Although lysosomal enzymes have been found in the prespore vesicle, this compartment does not function as a lysosome because it is not acidic, and a common antigen associated with acid hydrolases is found in another, acidic vesicle population. All the prespore vesicle profiles disappear at the time of appearance of their contents outside of the cell; this constitutes an early stage in spore coat formation, which can be detected both by microscopy and flow cytometry. As an electron-dense layer, the future outer layer of the coat, condenses, cellulose can be found and is located immediately beneath this outer layer. Certain proteins and the GPS become associated with either the outer or inner layers surrounding this middle cellulose layer. Assembly of the inner and outer layers occurs in part from a pool of glycoproteins that is shared between spores, and unincorporated molecules loosely reside in the interspore matrix, a location from which they can be easily washed away. When the glycosylation of several major protein species is disrupted by mutation, the coat is assembled, but differences are found in its porosity and the extractibility of certain proteins. In addition, the retention or loss of proteolytic fragments in the mutants indicates regions of spore coat proteins that are required for association with the coat. Comparative examination of the macrocyst demonstrates that patterns of molecular distributions are not conserved between the macrocyst and spore coats. Thus spore coat assembly is characterized by highly specific intermolecular interactions, leading to saturable associations of individual glycoproteins with specific layers and the exclusion of excess copies to the interspore space.     

Alteration by heat activation of enzymes localized in spore coats of Bacillus cereus

Heat activation (70 degrees C for 20 min) resulted in alteration in structural proteins and enzymes found in Bacillus cereus spore coats. The three notable changes were increased glycosylation of coat proteins, alteration in polypeptide pattern on sodium dodecyl sulfate - polyacrylamide gels, and an increase in free SH groups of proteins. About three polypeptides leaked out in small quantities from the spore coats during heat activation. The extraction of five spore coat associated enzyme activities was followed during the coat stripping procedures, which left the cortex and core intact. Two of these activities, L-alanine dehydrogenase and purine nucleoside hydrolase, were solubilized when the undercoat was extracted by 1,4-dithioerythritol (DTE) at pH 9.8. Three other activities, a protease, a corticolytic enzyme, and purine nucleoside phosphorylase, were solubilized by both DTE alone and DTE plus urea at pH 9.8. The DTE plus urea extraction removed the two more insoluble coat layers, the outer cross-patch, and the inner pitted layers. Mutants deficient in the cross-patch layer contained normal amounts of the protease, corticolytic, and purine nucleoside phosphorylase activities suggesting their association with the pitted layer. In intact spores all five enzymes were found to be stable to the heat activation treatment. However, extracted and partially purified preparations of protease, purine nucleoside phosphorylase, and L-alanine dehydrogenase were heat sensitive. Similar preparations of corticolytic enzyme and purine nucleoside hydrolase were stable to the heat activation conditions.     

Bacillus subtilis spore coat assembly requires cotH gene expression.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.     

Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat

During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA. We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.     

FORMATION AND STRUCTURE OF THE SPORE OF BACILLUS COAGULANS

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.     

Role of SP65 in assembly of the Dictyostelium discoideum spore coat

Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.     

Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis

We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.     

Ultrastructural studies of sporulation in Bacillus sphaericus.

Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.     

Morphogenesis of the Bacillus anthracis spore

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.