Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein

The gram-positive bacterium Bacillus subtilis forms remarkably resistant endospores when it is faced with starvation (36). This process, known as sporulation, produces metabolically dormant spores that are resistant to heat, radiation, and desiccation, as well as to predation by the protozoan Tetrahymena thermophila (17, 26, 36). These spores remain dormant until they sense that nutrients are available, at which time they can undergo germination and outgrowth to change back into growing cells (35). Several layers are assembled around the developing forespore during sporulation. The outermost layer of spores of most species is the coat, a multilayer structure made of more than 50 proteins in B. subtilis spores (7-9, 13). It is thought that the coat proteins together provide resistance to potentially toxic chemicals, as well as predation, but the coat has limited involvement in resistance to heat, radiation, or mechanical disruption (14, 17, 36). It has been difficult to comprehensively study the proteins of the spore coat, as at least 30% of the total coat protein is insoluble (7, 8). Because proteins can become insoluble when they are covalently cross-linked, it is thought that a number of coat proteins participate in this type of interaction (1, 13). SodA, a superoxide dismutase that may catalyze the formation of dityrosine bridges, and Tgl, a bacterial transglutaminase that forms ɛ-(γ-glutamyl)-lysine isopeptide bonds, have been suggested as proteins that may catalyze cross-link formation in the spore coat (12, 18, 19, 24, 40). Two main layers of the B. subtilis spore coat have been visualized by electron microscopy. The outer spore coat is thick and layered, while the inner spore coat is composed of several fine lamellae (9). Proper assembly of these layers is dependent on a number of morphogenetic proteins, including SpoIVA, SpoVID, SafA, CotE, CotH, and CotO, as well as the transcription factor GerE, and loss of any one of these proteins alters spore coat assembly, as well as the final coat structure (13). According to the models of spore coat assembly that have been described, during sporulation SpoIVA is produced in the mother cell immediately after asymmetric division and assembles around the forespore surface (34). Once SpoIVA has assembled, a CotE ring, whose formation is SpoIVA dependent, assembles ∼75 nm from SpoIVA (10, 28, 33). The space between SpoIVA and CotE is called the matrix, and as sporulation continues, the matrix becomes the inner spore coat (10), while the outer spore coat forms around the CotE ring, leaving this protein sandwiched between the two layers once coat assembly is complete (21). While coat proteins can be synthesized in the absence of CotE, the outer coat cannot be assembled (21), and without a properly assembled outer coat the spore is vulnerable to chemicals and lytic enzymes (7, 17, 39). Although a coat-defective spore is sensitive to protozoal predation, at least part of the spore is resistant, as a residue that resembles the coat remains after protozoal digestion (17). Such residues, called “rinds,” appear to be hollow, spherical, or hemispherical structures when they are examined by electron and phase-contrast microscopy (17). In this work, we used atomic force microscopy (AFM) and chemical analyses to probe the structure of wild-type and cotE rinds from B. subtilis spores.