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.     

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.     

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.     

The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.     

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.     

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.     

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface

The exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen-like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen-like protein, BclB, has been shown to be surface-exposed on B. anthracis spores. We have identified sequences near the N-terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore-associated in a two-step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.     

Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the bacillus anthracis exosporium

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the DeltaexsFA and DeltaexsFA DeltaexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The DeltaexsFA and DeltaexsFA DeltaexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.     

Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.     

Strain discrimination among B. anthracis and related organisms by characterization of bclA polymorphisms using PCR coupled with agarose gel or microchannel fluidics electrophoresis

The bclA gene codes for the protein backbone of the exosporium glycoprotein BclA of B. anthracis. BclA has a central collagen-like region formed by polymorphic GXX repeats and conserved amino- and carboxy-termini. It is noted here that the bclA gene is also present in the genome of Bacillus cereus and Bacillus thuringiensis. There is considerable size heterogeneity among the BclA proteins, both for species and strains, due to different numbers of GPT repeats and [GPT]5GDTGTT repeats (BclA repeats). PCR products that included the entire variable region were analyzed by conventional agarose gel electrophoresis and by micro-channel fluidics (MCF) LabChip to assess differences in molecular weight (MW). Both methods provided discrimination at the strain level for B. cereus group organisms. Results obtained by MCF electrophoresis were superior to conventional agarose gel analysis demonstrating improved reproducibility and much faster analysis time. The expression of a carbohydrate-rich exosporium (corresponding to BclA) in other members of the B. cereus group, in addition to B. anthracis, was also demonstrated ultra-structurally. Analysis of sequence variability within the bclA gene CLR revealed even greater potential for strain and species identification.     

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.     

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.     

Properties and origin of filamentous appendages on spores of Bacillus cereus

Some physical, chemical, and immunological properties of filamentous appendages and the exosporium on the spores of Bacillus cereus were examined for the purpose of elucidating the origin of filamentous appendages. The main components of both filamentous appendages and the exosporium were protein and their amino acid compositions were similar in point of a high content of glycine, alanine, threonine, valine, and acidic amino acids and a low content of basic and sulphur-containing amino acids. Treatment with 1 N NaOH at 50 C solubilized the isolated appendages completely and the isolated exosporia partially. In both preparations the solubilized proteins consisted of highly acidic monomeric subunits with molecular weights between 2,000 and 5,000. Treatment of the spores with 2% 2-mercaptoethanol at 37 C resulted in the isolation of long filamentous appendages without segmentation. When the spores were treated with 10% 2-mercaptoethanol, there was partial destruction of the exosporium as well as detachment of the filamentous appendages. There was a common antigenic component in the exosporium and the tips of the filamentous appendages. Five strains of B. cereus having a common appendage antigen also had a common exosporium antigen, whereas six other strains had neither a common appendage antigen nor a common exosporium antigen. From these facts it was concluded that the filamentous appendages arose from the 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.     

Localization and assembly of the novel exosporium protein BetA of Bacillus anthracis

The exosporium of Bacillus anthracis is comprised of two distinct layers: a basal layer and a hair-like nap that covers the basal layer. The hair-like nap contains the glycoproteins BclA and, most likely, BclB. BclA and BclB are directed to assemble into the exosporium by motifs in their N-terminal domains. Here, we identify a previously uncharacterized putative gene encoding this motif, which we have named betA (Bacillus exosporium-targeted protein of B. anthracis). Like bclA, betA encodes a putative collagenlike repeat region. betA is present in several genomes of exosporium-producing Bacillus species but, so far, not in any others. Using fluorescence microscopic localization of a BetA-enhanced green fluorescent protein (eGFP) fusion protein and immunofluorescence microscopy with anti-BetA antibodies, we showed that BetA resides in the exosporium basal layer, likely underneath BclA. BetA assembles at the spore surface at around hour 5 of sporulation and under the control of BxpB, similar to the control of deposition of BclA. We suggest a model in which BclA and BetA are incorporated into the exosporium by a mechanism that depends on their similar N termini. These data suggest that BetA is a member of a growing family of exosporium proteins that assemble under the control of targeting sequences in their N termini.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus.

Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Each inclusion contained a major polypeptide of about 135,000 daltons with unique antigenic determinants. This subspecies contained only two plasmids, of 98 and 77 megadaltons (MDa). Strains cured of these plasmids produced only the free inclusion. Since the plasmid-cured strains did not contain DNA sequences homologous to plasmid DNA, the gene for the free-inclusion protein must be encoded in the chromosome. In contrast, the enclosed parasporal inclusion was produced only when the plasmid of 98 MDa was present. In addition, transfer of the 98-MDa plasmid to Bacillus cereus resulted in transcipients that produced small inclusions enclosed within the exosporium, and the protein extracted from these inclusions reacted with antibody specific for enclosed inclusion protein of B. thuringiensis subsp. finitimus. Genes in both the chromosome and a plasmid function in the synthesis of distinct parasporal proteins in this subspecies.     

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.