The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

The Bacillus anthracis endospore loses resistance properties during germination when its cortex peptidoglycan is degraded by germination-specific lytic enzymes (GSLEs). Although this event normally employs several GSLEs for complete cortex removal, the SleB protein alone can facilitate enough cortex hydrolysis to produce vulnerable spores. As a means to better understand its enzymatic function, SleB was overexpressed, purified, and tested in vitro for depolymerization of cortex by measurement of optical density loss and the solubilization of substrate. Its ability to bind peptidoglycan was also investigated. SleB functions independently as a lytic transglycosylase on both intact and fragmented cortex. Most of the muropeptide products that SleB generates are large and are potential substrates for other GSLEs present in the spore. Study of a truncated protein revealed that SleB has two domains. The N-terminal domain is required for stable peptidoglycan binding, while the C-terminal domain is the region of peptidoglycan hydrolytic activity. The C-terminal domain also exhibits dependence on cortex containing muramic-␦-lactam in order to carry out hydrolysis. As the conditions and limitations for SleB activity are further elucidated, they will enable the development of treatments that stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca 2؉ -dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore's integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca 2؉ -DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-δ-lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft.

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