The ExsA Protein of Bacillus cereus Is Required for Assembly of Coat and Exosporium onto the Spore Surface

Abstract

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 cortexbinding 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 coatassociated 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. Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensisare very closely related (16,22), and the possession of an exosporium is a major characteristic of this group. This outermost layer of the spore is the least understood of all the spore integuments. The paradigm of sporeformers, B. subtilis, lacks a distinct, separate exosporium, although there has been a report that a very outermost tightly fitting layer of the spore coat can be visualized after extracting some of the coat material from spores with urea-mercaptoethanol and might be considered an exosporium (26). The exosporium of Bacillus cereus is first observed as a small lamellar structure in the mother cell cytoplasm in proximity to, but not in contact with, the outer forespore membrane; it is synthesized concurrently with the spore coat, although the two structures are clearly separate within the mature spore (16). The exosporium contains a hexagonal crystal-like basal layer and a hairy-nap outer layer (9). It has been estimated as containing 53% protein, 20% amino and neutral polysaccharide, 18% lipids, and approximately 4% ash. The whole structure makes up approximately 2% of the dry weight of the spore (14). A number of the proteins from the exosporia of B. cereus (7, 37) and B. anthracis (25,28,30) have been identified, most notably the B. anthracis surface-exposed glycoprotein antigen BclA (30, 31).The hydrophobic properties of Bacillus me...

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…Although it is named as a putative exosporium protein and involved in proper exosporium assembly, ExsA is not likely a true exosporium component. It possesses a conserved Nterminal cortex binding domain which suggests that it resides in the inner coat layer of the spore (41). ExsA displays sequence similarity to the B. subtilis SafA spore protein that functions in spore coat assembly.…”

“…These have been identified through the isolation of mutants that fail to stably attach the exosporium and from which, as a consequence, the exosporium is released as sheets separate from the mature spores upon lysis of the mother cells. Proteins implicated in exosporium attachment include the spore coat protein CotE and the exosporium-or interspace-localized proteins CotY, ExsA, ExsB, ExsY, and ExsM (1,(41)(42)(43)(44)(45). Whether these proteins participate directly in exosporium attachment or indirectly, through proper positioning of proteins actually involved in the attachment, has not been established.…”

“…Spores of an exsA mutant of B. cereus exhibited decreased hydrophobicity and increased permeability to lysozyme and were blocked at a late stage of germination (41). Truncation mutations resulted in defects in anchoring of the exosporium as well as outer spore coat layers.…”

The Clostridium difficile Exosporium Cysteine (CdeC)-Rich Protein Is Required for Exosporium Morphogenesis and Coat Assembly

“…In some species such as Bacillus anthracis and Bacillus cereus, the outermost layers are formed by the exosporium, which is composed of at least 21 protein species forming a paracrystalline basal layer and a hair-like nap composed mainly of collagen-like proteins, such as BclA and BclB (6,9,10). The components of the spore coat and exosporium layers are synthesized in the mother cell and subsequently assembled on the developing forespore (6).…”

“…However, bioinformatic analysis has shown that these proteins are absent in other species that have an exosporium layer (i.e., C. difficile) (6). Below the exosporium is the spore coat, where several morphogenetic factors essential for the proper assembly of the coat of Bacillus subtilis, B. anthracis, and B. cereus spores include ExsA (SafA orthologue), CotE, ExsB (CotG analogue), ExsY (CotZ orthologue), CotB, CotY, and ExsK (a cysteine-rich protein related to CotX) (10,11,14). However, the ultrastructure of C. difficile 630's spore exosporium layer is significantly different from that of B. anthracis spores (7,8,17) and bioinformatic and proteomic analyses (6,18) indicate that the exosporium proteins of B. anthracis and B. cereus are absent in C. difficile, suggesting that the exosporium of C. difficile spores is made of proteins that are highly divergent.…”