Katherine E. Wheeler

Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity.

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 (≈30% 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 ≈50 s, and another with a time scale of ≈8 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 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. 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.

Imaging and 3D elemental characterization of intact bacterial spores by high-resolution secondary ion mass spectrometry.

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.

Spatially Resolved Characterization of Water and Ion Incorporation in Bacillus Spores

ABSTRACT 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 (D2O) 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.

Unraveling the architecture and structural dynamics of pathogens by high-resolution in vitro atomic force microscopy

Author(s): Malkin, AJ; Plomp, M; Leighton, TJ; McPherson, A; Wheeler, KE | Abstract: Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, specific cellular processes, environmental dynamics and biotransformation. Copyright 2005, LASPM.