Abstract
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.
Highlights
Spores of bacteria of Bacillus species are formed in sporulation and are metabolically dormant and resistant to a large variety of environmental stress factors
electron microscopy (EM) of ruthenium red stained B. subtilis spores demonstrated the presence of an outermost glycoprotein layer, and it was suggested that this layer is an exosporium that is tightly attached to the coat layer [8]
A combination of EM, fluorescence microscopy, and genetic analysis demonstrated the existence of this outermost glycoprotein layer that was named the spore crust [7]
Summary
Spores of bacteria of Bacillus species are formed in sporulation and are metabolically dormant and resistant to a large variety of environmental stress factors. Work to date has indicated that there are at least four coat layers that can be distinguished by electron microscopy (EM) as well as other means – undercoat, inner coat, outer coat, and an outermost glycoprotein layer called the crust [2,4,7,8]. Several of these individual layers have sublayers, as the inner and outer coats have multiple lamellae. The SpoIIID, GerE and GerR proteins have major effects on the expression of genes encoding coat proteins that are transcribed during sporulation, and this in turn has significant effects on coat properties and morphology [4,5,6]
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