Architecture and High-Resolution Structure of <i>Bacillus thuringiensis</i> and <i>Bacillus </i><i>c</i><i>ereus </i>Spore Coat Surfaces

We have utilized atomic force microscopy (AFM) to visualize the native surface topography and ultrastructure of <i>Bacillus thuringiensis</i> and<i> Bacillus cereus</i> 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 (<i>B. thuringiensis</i>) or a rodlet outer spore coat layer (<i>B. cereus</i>). Removal of the rodlet structure from <i>B. cereus</i> spores revealed an underlying honeycomb layer similar to that observed with <i>B. thuringiensis </i>spores. The periodicity of the rodlet structure on the outer spore coat of <i>B. cereus</i> was ∼8 nm, and the length of the rodlets was limited to the cross-patched domain structure of this layer to ∼200 nm. The lattice constant of the honeycomb structures was ∼9 nm for both <i>B. cereus</i> and <i>B. thuringiensis </i>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.