Architecture and assembly of the Bacillus subtilis spore coat.

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.

A multistage pathway for human prion protein aggregation in vitro: from multimeric seeds to β-oligomers and nonfibrillar structures.

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared.

Mapping of proteomic composition on the surfaces of bacillus spores by atomic force microscopy-based immunolabeling.

Atomic force microscopy (AFM) provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g., viruses, bacteria, and bacterial spores) at near-molecular resolution in native conditions.

Characterization of spores of Bacillus subtilis that lack most coat layers.

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy.

Protozoal digestion of coat-defective Bacillus subtilis spores produces "rinds" composed of insoluble coat protein.

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.

Spore coat architecture of Clostridium novyi NT spores.

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores.

In vitro high-resolution structural dynamics of single germinating bacterial spores.

Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here, we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures.

Bacillus atrophaeus outer spore coat assembly and ultrastructure.

Our previous atomic force microscopy (AFM) studies successfully visualized native Bacillus atrophaeus spore coat ultrastructure and surface morphology. We have shown that the outer spore coat surface is formed by a crystalline array of approximately 11 nm thick rodlets, having a periodicity of approximately 8 nm. We present here further AFM ultrastructural investigations of air-dried and fully hydrated spore surface architecture.

Architecture and high-resolution structure of Bacillus thuringiensis and Bacillus cereus spore coat surfaces.

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.

Unraveling the architecture of viruses by high-resolution atomic force microscopy.

Atomic force microscopy (AFM) has recently emerged as an effective complement to other structure determination techniques for studying virus structure and function. AFM allows the direct visualization of viruses in a hydrated state and can probe surface topography in unrivaled detail. Moreover, AFM can be used to elucidate dynamic processes associated with the life cycle of viruses in vitro.

The high-resolution architecture and structural dynamics of Bacillus spores.

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization.

Macromolecular crystal growth as revealed by atomic force microscopy.

Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. We can, using AFM, image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development.

Rapid visualization at high resolution of pathogens by atomic force microscopy: structural studies of herpes simplex virus-1.

A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles.