Summary Bacillus anthracis , the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino‐terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen‐like surface protein was named BclA (for Bacillus c ollagen‐ l ike protein of anthracis ). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor σ K , which is required for a variety of events late in sporulation, was found upstream from the bclA gene. A bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.

We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (

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 ⌬exsFA and ⌬exsFA ⌬exsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The ⌬exsFA and ⌬exsFA ⌬exsFB 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.

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. Endospores of the Bacillus cereus family, which includesBacillus anthracis and Bacillus thuringiensis, are enveloped by a large, balloon-like layer known as the exosporium (11,19). B. subtilis, the paradigm sporeformer, does not have this distinct layer, although the B. subtilis spore coat may have a tight-fitting outermost layer that can be visualized after extraction of coat material with urea or mercaptoethanol (23). Scanning electron microscopy has revealed that the exosporium is composed of two layers-a paracrystalline basal layer with hexagonal periodicity and a "hairy nap" outer layer (11). The exosporium is chemically complex and is composed of 53% protein, 20% amino and neutral polysaccharides, 18% lipids, and ϳ4% ash (17).Multiple proteins from the exosporium of both B. cereus and B. anthracis have been identified (22,24,31); the majority do not have homologues in B. subtilis. For example, the surface layers of B. cereus, B. anthracis, and B. thuringiensis spores all contain glycoproteins with characteristic collagen-like repeat regions (5,10,27). Of these, the B. anthracis glycoprotein BclA is an essential component of the hairy nap layer of the exosporium (27, 29).Analysis of genome sequence information from B. cereus ATCC 14579 and B. anthracis Ames identified a cluster of genes near bclA implicated in exosporium production, including the region whose genes were designated yjbX-exsYyjcA-yjcB-exsFA-cotY (31). The ExsY and CotY proteins are homologues (ca. 35% amino acid identity) of the B. subtilis coat proteins CotY and CotZ (37). ExsY and CotY proteins have both been detected in purified exosporium from B. anthracis endospores by N-terminal sequencing of separated peptides (22). ExsY and CotY are very sim...