Bacillus atrophaeus Outer Spore Coat Assembly and Ultrastructure

Abstract

Our previous atomic force microscopy (AFM) studies successfully visualized nativeBacillus atrophaeus spore coat ultrastructure and surface morphology. We have shown that the outer spore coat surface is formed by a crystalline array of ~11 nm thick rodlets, having a periodicity of ~8 nm. We present here further AFM ultrastructural investigations of air-dried and fully hydrated spore surface architecture. In the rodlet layer, planar and point defects, as well as domain boundaries, similar to those described for inorganic and macromolecular crystals, were identified. For several Bacillus species, rodlet structure assembly and architectural variation appear to be a consequence of species-specific nucleation and crystallization mechanisms that regulate the formation of the outer spore coat. We propose a unifying mechanism for nucleation and self-assembly of this crystalline layer on the outer spore coat surface.3

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

“…The former finding suggested that the coating layers of the endospores had a pronounced propensity for accumulating the staining dye. Our observation for surface accumulation of ThT was consistent with the abundance of ␤-sheet proteins found on the endospore surfaces (23,(60)(61)(62)(63).…”

“…These facts provide the rationale of this study for staining endospores with ThT, which targets their endogenous protein structures. The images of the ThT-stained endospores showed bright contours outlining their elliptical shapes, which is consistent with the dye collecting in the surface regions of the spores (where the high ␤-protein content has been demonstrated [60][61][62][63]), and with an increase in the fluorescence quantum yield of the dye bound to these endospore surface regions. Our evidence does not rule out the possibility for weakly fluorescent ThT present in the interior of the stained endospores.…”

“…Despite the established pronounced affinity of ThT for ␤-amyloid types of protein structures prevalent in endospores and in the cell walls of vegetative bacteria (17,20,42,(60)(61)(62)(63), binding of the positively charged chromophore (Fig. 1) to a range of other macromolecular structures presents a limitation for employing ThT as a stain for biomedical and environmental samples.…”

“…This study does not provide a direct answer. Indeed, ThT targets ␤-amyloid-type proteins with high affinity (43), and bacterial endospores are coated with ␤-strand proteins as evident, for example, from high-resolution atomic force microscopy (AFM) images (60)(61)(62)(63). These facts provide the rationale of this study for staining endospores with ThT, which targets their endogenous protein structures.…”

“…High-resolution scanning probe images, for example, revealed significant abundance of ␤-sheet amyloid-like proteins on the surfaces of bacterial endospores (60)(61)(62)(63). Amyloid folds are, indeed, intricate components of bacterial cell walls and endospore coatings (17,20,23,42,(60)(61)(62)(63).…”