Architecture and High-Resolution Structure of Bacillus thuringiensis and Bacillus cereus Spore Coat Surfaces

Abstract

We have utilized atomic force microscopy (AFM) to visualize the native surface topography and ultrastructure of Bacillus thuringiensis and Bacillus cereus spores in water and in air. AFM was able to resolve the nanostructure of the exosporium and three distinctive classes of appendages. Removal of the exosporium exposed either a hexagonal honeycomb layer (B. thuringiensis) or a rodlet outer spore coat layer (B. cereus). Removal of the rodlet structure from B. cereus spores revealed an underlying honeycomb layer similar to that observed with B. thuringiensis spores. The periodicity of the rodlet structure on the outer spore coat of B. cereus was approximately 8 nm, and the length of the rodlets was limited to the cross-patched domain structure of this layer to approximately 200 nm. The lattice constant of the honeycomb structures was approximately 9 nm for both B. cereus and B. thuringiensis spores. Both honeycomb structures were composed of multiple, disoriented domains with distinct boundaries. Our results demonstrate that variations in storage and preparation procedures result in architectural changes in individual spore surfaces, which establish AFM as a useful tool for evaluation of preparation and processing "fingerprints" of bacterial spores. These results establish that high-resolution AFM has the capacity to reveal species-specific assembly and nanometer scale structure of spore surfaces. These species-specific spore surface structural variations are correlated with sequence divergences in a spore core structural protein SspE.

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).…”

“…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.…”

“…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).…”

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

“…AFM studies had previously been done on spore surfaces (28)(29)(30), but the exosporium appeared to be featureless (28) or to have a granular texture (30), as the hairy nap present on the exosporium would not have revealed the underlying ordered structure of the basal layer. Because we had fragmented the exosporium as well as looked at B. cereus family strains with and without the hairy nap (but with identical basal layer architecture), we were able to image both the outer and inner side of the crystalline basal layer and this has revealed the well-ordered crystalline lattice.…”

Multi-Probe Investigation of Proteomic Structure of Pathogens

“…We have directly visualized, for the first time, species-specific high-resolution native structures of bacterial endospores including the exosporium and crystalline layers of the spore coat of four Bacillus. (4)(5)(6)(7)(8) and Clostridium novyi-NT (10) spore species in their natural environment, namely air and fluid. We found that strikingly different species-dependent spore coat structures are a consequence of nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat (4-6).…”