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. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.

Using an automated scanning microscope, we report the surprising result that individual dormant spores of Bacillus thuringiensis grow and shrink in response to increasing and decreasing relative humidity. We simultaneously monitored the size of inorganic calibration particles. We found that the spores consistently swell in response to increased relative humidity, and shrink to near their original size on reexposure to dry air. Although the dispersion of swelling amplitudes within an ensemble of spores is wide (Ϸ30% of the average amplitude), amplitudes for individual spores are highly correlated between different swelling episodes, suggesting that individual spores respond consistently to changes in humidity. We find evidence for two distinct time scales for swelling: one with a time scale of no more than Ϸ50 s, and another with a time scale of Ϸ8 min. We speculate that these two mechanisms may be due to rapid diffusion of water into the spore coat ؉ cortex, followed by slower diffusion of water into the spore core, respectively. Humidity-dependent swelling may account for the greater kill effectiveness of spores by gas-phase chlorine dioxide, formaldehyde, and ethylene oxide at very high relative humidity. In the aftermath of the anthrax attacks in Fall 2001, we looked into measurable physical properties of bacterial endospores that might distinguish Bacillus anthracis from other species. This led to two striking discoveries: The first, which we report in this paper, is that dormant spores are not entirely static but rapidly swell and shrink in response to increases and decreases in relative humidity (RH), with interesting biological implications. The second, which we report in a subsequent paper, is that, when RH and temperature are held constant, accurate measurements of sizes of Bacillus spores can discriminate among species.It has long been known from the high refractive index of spores that the core has a low water content. Neihof et al.(1) measured water sorption and desorption of both freeze-dried and crushed Bacillus subtilis spores as a function of RH. From the sigmoid shape, hysteresis, and similar behavior of normal and crushed spores, they inferred that swelling accompanies sorption and that an anhydrous spore core is not maintained by a water permeability barrier.Studies of the inverse correlation between spore heat resistance and water content have led to the present view that an osmotic pressure within the spore is responsible for maintaining the partly dry core (2, 3). The requisite tension and pressure are thought to be generated in the cortex by peptidoglycan, initially polymerized in an ordered tightly packed conformation, fixed by cross-linking of the glycan chains (3). From experiments with deuterated water, Marshall and Murrell (4) concluded that at least 97% of the water in spores was able to exchange with the environment. In experiments with tritiated water, they found that water exchange in a population of spores as well as of vegetative cells was almost complete in 2-3 min at 0°C. In m...

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. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by NMR. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.atomic force microscopy ͉ cell wall ͉ germination ͉ amyloid ͉ peptidoglycan W hen starved for nutrients, Bacillus and Clostridium cells initiate a series of genetic, biochemical, and structural events that result in the formation of a metabolically dormant endospore (1). Spores can remain dormant for extended time periods and possess a remarkable resistance to environmental insults (i.e., heat, radiation, toxic chemicals, and pH extremes) (1-5) that are lethal to vegetative cells. The resistance and persistence of dormant spores is attributed to a multilayer spore architecture (6). Upon exposure to favorable conditions, spores break dormancy through the process of germination (2,7,8) and eventually reenter the vegetative mode of replication.A comprehensive understanding of the mechanisms controlling spore germination is of fundamental importance both for practical applications related to the prevention of a wide range of diseases by spore-forming bacteria (including food poisoning and pulmonary anthrax), as well as for fundamental studies of cell development. Germination involves an ordered sequence of chemical, degradative, biosynthetic, and genetic events (2, 8).Significant progress has been made in understanding the biochemical and genetic bases for the germination process (2). Germination is triggered by the interaction of germinants with specific receptors (2, 7, 9) in the inner spore core membrane, causing the release of the dipicolinic acid and its replacement by water. Subsequent hydrolysis of the spore cortex, further uptake of water, core expansion, and spore coat hydrolysis allow emergence of the incipient vegetative cell.The role of the spore coat in the germination process is unclear (2, 6) and is the focus of this study. Spore coat structure regulates the permeation of germinant molecules (7-10). It is believed that penetration of germinants proceeds through pores in the coat structure and may involve GerP proteins (9).Atomic force micro...

We present a quantitative, imaging technique based on nanometer-scale secondary ion mass spectrometry for mapping the 3D elemental distribution present in an individual micrometer-sized Bacillus spore. We use depth profile analysis to access the 3D compositional information of an intact spore without the additional sample preparation steps (fixation, embedding, and sectioning) typically used to access substructural information in biological samples. The method is designed to ensure sample integrity for forensic characterization of Bacillus spores. The minimal sample preparation/alteration required in this methodology helps to preserve sample integrity. Furthermore, the technique affords elemental distribution information at the individual spore level with nanometer-scale spatial resolution and high (microg/g) analytical sensitivity. We use the technique to map the 3D elemental distribution present within Bacillus thuringiensis israelensis spores.

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.

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

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.

We present the first direct visualization and quantification of water and ion uptake into the core of individual dormant Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) endospores. Isotopic and elemental gradients in the B. thuringiensis subsp. israelensis spores show the permeation and incorporation of deuterium in deuterated water (D 2 O) and solvated ions throughout individual spores, including the spore core. Under hydrated conditions, incorporation into a spore occurs on a time scale of minutes, with subsequent uptake of the permeating species continuing over a period of days. The distribution of available adsorption sites is shown to vary with the permeating species. Adsorption sites for Li ؉ , Cs ؉ , and Cl ؊ are more abundant within the spore outer structures (exosporium, coat, and cortex) relative to the core, while F ؊ adsorption sites are more abundant in the core. The results presented here demonstrate that elemental abundance and distribution in dormant spores are influenced by the ambient environment. As such, this study highlights the importance of understanding how microbial elemental and isotopic signatures can be altered postproduction, including during sample preparation for analysis, and therefore, this study is immediately relevant to the use of elemental and isotopic markers in environmental microbiology and microbial forensics.