Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

Abstract

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.The Bacillus subtilis spore is encased within a complex multilayered protein structure known as the coat, whose role is to protect the spore against bactericidal enzymes and chemicals, such as lysozyme and chloroform, and to influence the spore's ability to germinate in response to appropriate germinants. However, the recent finding that a component of the B. subtilis coat has laccase activity (20) suggests that the coat may have other, so far unexplored, roles. The coat is composed of a heterogeneous group of over 25 polypeptides arranged into three main structural layers: a diffuse undercoat, a laminated lightly staining inner layer, and a thick electron-dense outer coat. Several of these polypeptides have been studied, and their structural genes (cot genes) have been identified. Expression of all cot genes is governed by a cascade of four transcription factors, acting specifically in the mother cell compartment of the sporangium in the sequence sigma E-SpoIIID-sigma K-GerE, with sigma E and sigma K being RNA polymerase sigma factors and SpoIIID and GerE being DNA-binding proteins acting in conjunction with sigma E-and sigma K-driven RNA polymerase (5, 11).

Effects of Phosphorelay Perturbations on Architecture, Sporulation, and Spore Resistance in Biofilms ofBacillus subtilis

“…To visualize actual spore formation within the colonies, we constructed a full translational fusion of the gene encoding the abundant spore coat protein CotC (21) to the gfp gene under control of its own promoter. A C-terminal GFP fusion was constructed instead of an N-terminal fusion to avoid possible loss of the GFP marker by removal of the N-terminal part of the protein due to posttranslational processing (25). Correct timing and localization of the P cotC -CotC-GFP fusion to the forespore were confirmed by single-cell fluorescence microscopy (Fig.…”

Assembly and Function of a Spore Coat-Associated Transglutaminase of Bacillus subtilis

“…Nevertheless, about 30% of the total spore coat protein is refractory to extraction, forming an insoluble highly cross-linked fraction (5,10,30). Several coat proteins appear to be cross-linked, and the available evidence suggests that both reversible and irreversible cross-links occur within the coat (5,10,11,12,14,20,21,42). In a classical study, Pandey and Aronson (30) have suggested the existence of o,o-dityrosine cross-links in purified coat material, an observation confirmed by more recent work (5).…”

The Bacillus subtilis spore coat provides “eat resistance” during phagocytic predation by the protozoan Tetrahymena thermophila

“…The B. subtilis spore coat is composed of several distinct layers and contains Ն30 proteins, almost all of which are spore-specific gene products (5)(6)(7)(8)(9)(10)(11)(12). There is extensive cross-linking of a number of proteins in the coats, at least in part by a transglutaminase and perhaps by the formation of dityrosine cross-links as well (13)(14)(15)(16)(17)(18).…”