Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism.

The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a "rind" that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon.