Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A.

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.     

The crystalline layers in spores of Bacillus cereus and Bacillus thuringiensis studied by freeze-etching and high resolution electron microscopy

The comparative morphology of spores from Bacillus cereus, B. thuringiensis S-9 (wildtype), B. thuringiensis HB 9-1 (acrystaliferous mutant) and B. finitimus, was studied by the freeze-etching technique. Particular attention was given to the three crystalline layers found in these spores, the pitted layer, the parasporal layer, and the basal layer of the exosporium with lattice constants of 9.2 nm, 5.8 nm and 8.0 nm respectively. The parasporal layers, corresponding to the fraction of Fl of Scherrer and Somerville [18] were found to be membraneous sheets laying between spore and exosporium. Analysis of the isolated exosporium by negative staining and digital image processing revealed a lattice structure belonging to the p 6 hexagonal space group with a repeat of 8.0 nm. The unit cell contains 6 subunits, each 3 nm in diameter, which are centered around a hypothetical channel readily penetrated by the stain.     

Ultrastructure of the Exosporium and Underlying Inclusions in Spores of Bacillus megaterium Strains

Spores of selected strains of Bacillus megaterium were prepared by various methods and examined with the electron microscope. An exosporium like that of B. cereus, with a nap and basal layer, was found in spores of a B. megaterium strain that reportedly contains a capsule-like exosporium. The exosporium occasionally appeared to be doubled or have an apical opening. Pili-like filaments were discerned on the surface. Beneath the exosporium were found large deposits of planar inclusions, which in cross section appeared laminated and in surface views consisted of a patchwork of striated packets with a periodicity of approximately 5 nm. The inclusions were usually attached to the exosporium, but in ultrastructure they differed from both the exosporium and coat. In two other strains of B. megaterium, one or two coats occurred but a typical exosporium was not present.     

Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a spore-specific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a DeltaexsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell.     

Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.     

The BclB glycoprotein of Bacillus anthracis is involved in exosporium integrity

Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis DeltaSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.     

Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium

The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.     

Membrane fractions from the outer layers of spores of Bacillus thuringiensis with toxicity to lepidopterous larvae.

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.     

The co-dependence of BxpB/ExsFA and BclA for proper incorporation into the exosporium of Bacillus anthracis

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of two distinct layers, an outer hair-like nap layer and an internal basal layer. The hair-like nap is primarily comprised of the glycosylated collagen-like protein BclA. BclA is found in a trimeric form in close association with many other exosporium proteins in high-molecular weight complexes. We previously had characterized an N-terminal sequence of BclA that is sufficient for incorporation into the exosporium. Here we utilized site-directed mutagenesis to identify BclA residues critical to two steps in this process, positioning of the protein at the site of the developing exosporium basal layer and stable incorporation which includes a proteolytic cleavage of BclA after residue 19. The BxpB (ExsFA) protein is known to be important for proper incorporation of BclA onto the exosporium. BxpB and BclA were found to be expressed at the same time in sporulating cells of B. anthracis and immediately colocalize to high-molecular weight complexes. The BxpB protein was found to be in close proximity to the BclA NTD. BxpB and BclA are co-dependent for exosporium incorporation, with the BclA NTD being sufficient to deliver BxpB to the exosporium.     

Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis

Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that DeltabxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the DeltabxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of DeltabxpB spores and their resistance properties were similar to those of wild-type spores. However, DeltabxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from DeltabxpB spores of a hair-like nap or of enzymes that degrade germinants.     

Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium. Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized. In this study, we identified five major proteins in purified B. anthracis (Sterne strain) exosporia. One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap. Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B. anthracis spore surface. We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response. In addition, the length of the central (GXX)(n) repeat region of BclA appears to be strain specific. Two other unique proteins, BxpA and BxpB, were identified. BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation. In addition, BxpA was found to be cleaved approximately in half. BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island. The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores. Possible functions of the newly identified proteins are discussed.     

Surface hydrophobicity of spores of Bacillus spp

The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.     

The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis

The outermost layer of the Bacillus anthracis spore is the exosporium, which is composed of a paracrystalline basal layer and an external hair-like nap. The filaments of the nap are formed by a collagen-like glycoprotein called BclA, while the basal layer contains several different proteins. One of the putative basal layer proteins is ExsY. In this study, we constructed a DeltaexsY mutant of B. anthracis, which is devoid of ExsY, and examined the assembly of the exosporium on spores produced by this strain. Our results show that exosporium assembly on DeltaexsY spores is aberrant, with assembly arrested after the formation of a cap-like fragment that covers one end of the forespore-always the end near the middle of the mother cell. The cap contains a normal hair-like nap but an irregular basal layer. The cap is retained on spores prepared on solid medium, even after spore purification, but it is lost from spores prepared in liquid medium. Microscopic inspection of DeltaexsY spores prepared on solid medium revealed a fragile sac-like sublayer of the exosporium basal layer, to which caps were attached. Examination of purified DeltaexsY spores devoid of exosporium showed that they lacked detectable levels of BclA and the basal layer proteins BxpB, BxpC, CotY, and inosine-uridine-preferring nucleoside hydrolase; however, these spores retained half the amount of alanine racemase presumed to be associated with the exosporium of wild-type spores. The DeltaexsY mutation did not affect spore production and germination efficiencies or spore resistance but did influence the course of spore outgrowth.     

Collagen-Like Glycoprotein BclS Is Involved in the Formation of Filamentous Structures of the Lysinibacillus sphaericus Exosporium

Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.     

Efficacy of a Fluorescent-Antibody Procedure for Identifying Bacillus cereus in Foods

One hundred and seventeen strains of Bacillus were examined by the fluorescent-antibody technique by using the globulin fraction of serum prepared against spores of B. cereus T. All but one strain of the 59 B. cereus tested fluoresced at the exosporium surface. Fluorescent staining of B. anthracis, B. thuringiensis, and B. mycoides was also observed. Absorption of the globulin fraction with B. anthracis and B. mycoides resulted in the elimination of staining of these organisms. Absorption with B. thuringiensis ATCC 10792 removed antibodies reacting with 6 of the strains of B. thuringiensis tested. Absorption with B. thuringiensis var. galleriae removed antibodies against B. cereus to such a degree that the globulin fraction was unusable.     

Assembly of the outermost spore layer: pieces of the puzzle are coming together

Certain endospore‐forming soil dwelling bacteria are important human, animal or insect pathogens. These organisms produce spores containing an outer layer, the exosporium. The exosporium is the site of interactions between the spore and the soil environment and between the spore and the infected host during the initial stages of infection. The composition and assembly process of the exosporium are poorly understood. This is partly due to the extreme stability of the exosporium that has proven to be refractive to existing methods to deconstruct the intact structure into its component parts. Although more than 20 proteins have been identified as exosporium‐associated, their abundance, relationship to other proteins and the processes by which they are assembled to create the exosporium are largely unknown. In this issue of Molecular Microbiology, Terry, Jiang, and colleagues in Per Bullough's laboratory show that the ExsY protein is a major structural protein of the exosporium basal layer of B. cereus family spores and that it can self‐assemble into complex structures that possess many of the structural features characteristic of the exosporium basal layer. The authors refined a model for exosporium assembly. Their findings may have implications for exosporium formation in other spore forming bacteria, including Clostridium species.     

ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus

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.     

Current physical and SDS extraction methods do not efficiently remove exosporium proteins from Bacillus anthracis spores

Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these methods. Sonication and bead beating methods for exosporium layer removal were also examined. A combination of genetic and physical methods is the most effective for isolating proteins found in the spore exosporium.     

YwdL in Bacillus cereus: its role in germination and exosporium structure

In members of the Bacillus cereus group the outermost layer of the spore is the exosporium, which interacts with hosts and the environment. Efforts have been made to identify proteins of the exosporium but only a few have so far been characterised and their role in determining spore architecture and spore function is still poorly understood. We have characterised the exosporium protein, YwdL. ΔywdL spores have a more fragile exosporium, subject to damage on repeated freeze-thawing, although there is no evidence of altered resistance properties, and coats appear intact. Immunogold labelling and Western blotting with anti-YwdL antibodies identified YwdL to be located exclusively on the inner surface of the exosporium of B. cereus and B. thuringiensis. We conclude that YwdL is important for formation of a robust exosporium but is not required to maintain the crystalline assembly within the basal layer or for attachment of the hairy nap structure. ΔywdL spores are unable to germinate in response to CaDPA, and have altered germination properties, a phenotype that confirms the expected defect in localization of the cortex lytic enzyme CwlJ in the coat.