Atomic force microscopy of Bacillus spore surface morphology

Zolock, Ruth A.; Li, Guangming; Bleckmann, Charles A.; Burggraf, Larry W.; Fuller, Douglas C.

doi:10.1016/j.micron.2005.11.006

Cited by 32 publications

(30 citation statements)

References 36 publications

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“…The majority of spores from the ⌬Sterne strain ( Fig. 2A) exhibited a smooth raisin-like surface with characteristic ridges similar to those observed by atomic force microscopy (10,48). Many of the bclB-negative spores displayed a rougher, dimpled appearance (Fig.…”

Section: Resultssupporting

confidence: 59%

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

et al. 2007

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Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ⌬Sterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.Bacillus anthracis is a gram-positive, rod-shaped bacterium that causes anthrax, principally in ruminants, and is a major concern as both a zoonotic human pathogen and an agent of bioterrorism (reviewed in reference 25). Anthrax is acquired following contact with B. anthracis spores, which are the infectious form of this organism. The exosporium of B. anthracis and the other members of the Bacillus cereus group of sporeforming bacteria is the most external protein layer enclosing the spore. It consists of an inner basal layer and outer nap region having a hair-like appearance (4,11,20,24,29). The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA (38-42), whereas the basal layer is composed of a number of different proteins in tight and loose associations (35,39). In the past few years, the protein constituents of the exosporium have begun to be elucidated, but there have been few studies of the role of these proteins in the function of the exosporium (2,34,35,38,44).The first spore surface glycoprotein discovered in B. anthracis was referred to as BclA (for Bacillus collagen-like protein anthracis) and is the most prominent protein component of the exosporium (41,42). BclA is the immunodominant antigen located in the filaments of the nap (38). BclA contains an internal tandem repeat region (consisting primarily of GPT repeats containing most of the glycosylation sites) and N -and C-terminal regions (41). These repeats are the primary anchor point for rhamnose oligosaccharides wit...

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Section: Resultssupporting

confidence: 59%

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

et al. 2007

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“…AFM images revealed previously unrecognized germination-induced alterations in spore coat architecture and in the disassembly of outer spore coat rodlet structures. These and other studies (30)(31)(32)(33)(34)(35)(36)(37)(38)(39), including our previous work that showed that the spores of four closely related Bacillus species can be distinguished by AFM surface morphology analysis (37) have proved AFM to be a powerful tool complementary to EM techniques, providing three-dimensional images of native, minimally processed biological samples with nanometer resolution.…”

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confidence: 74%

Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

Xing

Felker

et al. 2014

Appl Environ Microbiol

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Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. Bacterial spores are metabolically dormant cells formed in a process called sporulation, which is generally induced by reduced levels of nutrients in the environment (1-3). Efficient inactivation of spores is of critical importance for a wide range of applications, including biodefense, food safety, environmental protection, and medical device sterilization (4-6). Spores are known to be more resistant to inactivation by heating, radiation exposure, and chemical decontamination than their corresponding vegetative cells. While various methods, including heating, chemical treatment, radiation, and UV treatment, have been used to inactivate spores (4, 6, 7), thermal inactivation is often the method of choice for many applications (8). Thermal inactivation of Bacillus spores in laboratory studies is most often achieved by wet heat in which spores are fully hydrated during heating (9-11) or dry heat in which dry spores are heated on a solid substrate, in an ampoule heated by an oil bath, in a hot air plume, or by infrared heating (5,8,(12)(13)(14)(15)(16)(17)(18)(19)(20). It has long been observed that spores are much less resistant to heat in a well-hydr...

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“…The surface of Bacillus subtilis and Bacillus anthracis spores is populated by ridge structures largely oriented with the long axis of the spore. We previously showed that four closely related species of Bacillus spores can be distinguished by surface morphology analysis (54). Using thermal atomic force microscopy (AFM), we recently characterized thermal effects on the topographic morphology and nanomechanical properties of Bacillus anthracis spores at elevated temperatures (55,56).…”

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Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy

Burggraf

Xing

2016

Appl Environ Microbiol

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The study of structures and properties of bacterial spores is important to understanding spore formation and biological responses to environmental stresses. While significant progress has been made over the years in elucidating the multilayer architecture of spores, the mechanical properties of the spore interior are not known. Here, we present a thermal atomic force microscopy (AFM) study of the nanomechanical properties of internal structures of Bacillus anthracis spores. We developed a nanosurgical sectioning method in which a stiff diamond AFM tip was used to cut an individual spore, exposing its internal structure, and a soft AFM tip was used to image and characterize the spore interior on the nanometer scale. We observed that the elastic modulus and adhesion force, including their thermal responses at elevated temperatures, varied significantly in different regions of the spore section. Our AFM images indicated that the peptidoglycan (PG) cortex of Bacillus anthracis spores consisted of rod-like nanometer-sized structures that are oriented in the direction perpendicular to the spore surface. Our findings may shed light on the spore architecture and properties. IMPORTANCEA nanosurgical AFM method was developed that can be used to probe the structure and properties of the spore interior. The previously unknown ultrastructure of the PG cortex of Bacillus anthracis spores was observed to consist of nanometer-sized rodlike structures that are oriented in the direction perpendicular to the spore surface. The variations in the nanomechanical properties of the spore section were largely correlated with its chemical composition. Different components of the spore materials showed different thermal responses at elevated temperatures. Structures and material properties of bacterial spores play an important role in protecting them against a variety of environmental stresses, such as toxic chemicals (1), radiation (2, 3), and heat (3-5). The current knowledge of spore structures has been obtained largely using transmission electron microscopy (TEM) methods in various forms (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). A mature spore usually shows a concentric multilayer structure, consisting of the core, inner membrane, germ cell wall, cortex, outer membrane, and coat assembly. For Bacillus anthracis spores, there is an exosporium layer that loosely encases the spore. The spore core, which contains mostly DNA, RNA, enzymes, and dipicolinic acid (DPA), is in a dehydrated and metabolically dormant state (21-25). The ability of bacterial spores to survive for long durations, sometimes in harsh environments, has been attributed to the immobilization of essential DNA, RNA, and enzymes by small, acid-soluble spore proteins (SASP) (28-30). It was suggested that the DPA can be intercalated with spore DNA and RNA through covalent bonds, forming a gel-like polymer matrix (26,27). It is worth mentioning that the DPA structure in the spore core is not well understood. The germ cell wall (e.g., inner cortex) and ...

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Atomic force microscopy of Bacillus spore surface morphology

Cited by 32 publications

References 36 publications

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy

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