Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

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.