Bacterial endospores possess multiple integument layers, one of which is the cortex peptidoglycan wall. The cortex is essential for the maintenance of spore core dehydration and dormancy and contains structural modifications that differentiate it from vegetative cell peptidoglycan and determine its fate during spore germination. Following the engulfment stage of sporulation, the cortex is synthesized within the intermembrane space surrounding the forespore. Proteins responsible for cortex synthesis are produced in both the forespore and mother cell compartments. While some of these proteins also contribute to vegetative cell wall synthesis, others are sporulation specific. In order for the bacterial endospore to germinate and resume metabolism, the cortex peptidoglycan must first be degraded through the action of germination-specific lytic enzymes. These enzymes are present, yet inactive, in the dormant spore and recognize the muramic-δ-lactam modification present in the cortex. Germination-specific lytic enzymes across Bacillaceae and Clostridiaceae share this specificity determinant, which ensures that the spore cortex is hydrolyzed while the vegetative cell wall remains unharmed. Bacillus species tend to possess two redundant enzymes, SleB and CwlJ, capable of sufficient cortex degradation, while the clostridia have only one, SleC. Additional enzymes are often present that cannot initiate the cortex degradation process, but which can increase the rate of release of small fragments into the medium. Between the two families, the enzymes also differ in the enzymatic activities they possess and the mechanisms acting to restrict their activation until germination has been initiated.
The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ⌬sleB and ⌬ypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. Bacterial spores from the Bacillus and Clostridium genera are metabolically dormant and are known for their extreme resistance to heat, desiccation, UV radiation, chemicals, and other insults (1-3). These resistance properties allow spores to survive in the environment for extended periods and have made eradication from contaminated sites incredibly difficult (4). Spore dormancy and wet heat resistance are largely dependent on spore core dehydration, which is maintained by a thick layer of modified peptidoglycan (PG) known as the cortex (2, 5). While vegetative cell wall PG consists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars, approximately 50% of NAM residues in the cortex are converted to muramic-␦-lactam, while an additional portion of the NAM side chains is generally cleaved to a single L-alanine (6-11).The spore form of Bacillus anthracis is the etiological agent for all types of anthrax infections: inhalational, gastrointestinal, and cutaneous anthrax, as well as the newest form described, injectional anthrax (12, 13). When the spore senses the availability of nutrients, such as when it enters a suitable host, germination is triggered, causing a chain of events that ultimately result in a vegetative cell capable of producing deadly toxins (1, 12). After germinant contact with rec...
In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 ϫ 10 10 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D 10 ) values and irradiation doses required to ensure sterility (D SAL ) to the point at which the probability of detecting a viable spore is 10 Ϫ6 . Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCEThe inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.
Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Endospores produced by members of Gram-positive genera, such as Bacillus and Clostridium, possess extreme resistance properties and can remain in a fully dormant state for years. The dormant state and resistance properties are dependent on the maintenance of the spore core (cytoplasm) in a relatively dehydrated state, and this in turn depends on the intact state of the inner spore membrane and the cortex peptidoglycan (PG) wall surrounding that membrane (1). Upon exposure to nutrient germinants, spores begin to release low-molecular-weight solutes, including a large depot of Ca 2ϩ -dipicolinic acid (Ca 2ϩ -DPA), and take up water (2). Degradation of the cortex PG by germinationspecific lytic enzymes (GSLEs) is required for full expansion of the membrane, full hydration of the core, and resumption of metabolism (3-6). As GSLEs hydrolyze the cortex PG before new protein synthesis can occur, they must be produced during spore formation and held stable and inactive in the dormant spore until germination is triggered (7).Bacillus species possess two major, partially redundant GSLEs: CwlJ and SleB (7). CwlJ is produced in the mother cell of the developing sporangium (8), is associated with the spore coats on the outer surface of the cortex (9-12), and becomes active when exposed to a high concentration of Ca 2ϩ -DPA-normally when that solute is released from the germinating spore (11,13,14). SleB is produced within the developing forespore (15, 16) and is located interior to the cortex in the dormant spore, most likely in close association with the inner spore membrane (10, 17). The mechanisms by which SleB is held inactive during spore dormancy and released to become active during germination are unclear.A potential factor in the regulation of SleB activity is YpeB, which is encoded in an operon with sleB and possesses a transmembrane anchor sequence that should also localize it to the outer surface of the inner spore membrane (15,18,19). SleB and YpeB exhibit codependence for their stable incorporation into the dormant spore (10,18,20). In the absence of their partner protein, both SleB and YpeB are produced and rapidly degraded during spore formation (18). It has also been observed that YpeB is proteolytically processed during spore germination (10), and it has been suggested that this processing could be invol...
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