Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between Nacetylglucosamine and muramic--lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lytic enzymes and their regulators are attractive targets for development of germination inhibitors to kill spores and for development of activators to cause loss of resistance properties for decontamination of spore release sites. Bacterial endospores are metabolically dormant and are highly resistant to many treatments that rapidly kill vegetative cells. The resistance properties are dependent on the relative dehydration of the spore core or cytoplasm and the high concentrations of calcium dipicolinic acid (DPA) and other core solutes (26). When exposed to a nutrient-rich environment, spores initiate germination and outgrowth into vegetative cells. Nutrient germinant molecules bind to Ger protein family receptors in the inner forespore membrane surrounding the spore core and trigger biophysical changes (20). The spore becomes partially rehydrated as water enters the spore core, and the calcium DPA that was in the core is released into the environment. To complete the germination process and resume metabolism (11, 24), the spore must go through a second stage, which is more biochemical (20). This involves activation of spore cortex peptidoglycan (PG) lytic enzymes, which are present in an inactive state within the dormant spore. As the cortex is degraded, the cell expands to its full size and regains full metabolic activity. Due to the key role that cortex lytic enzymes play in germination, they are potential targets for spore decontamination methods that could involve either irreversible inactivation or gratuitous activation of the enzymes. The latter would lead to premature spore germination into vegetative forms that are much more easily killed. PG consists of glycan strands with alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are cross-linked by peptide side chains located on the NAM residues (Fig. 1). While the PG structure in vegetative cells can vary between species, primarily in the amino acid composition of the peptide side chains, spore PG structure has been found to be highly conserved across species (3, 30). The peptide side chains contain L-alanine, D-glutamate, meso-diaminopimelic acid (Dpm), and D-alanine. The cross-linking occurs between the Dpm on one strand and the D-alanine of a tetrapeptide on an adjacent strand. The spore PG is composed of the inner, germ cell wall layer (10 to 20% of the PG), which resembles the PG of vegetative cells (6, 19), and the cortex, which has important and unique chemical modifications (2, 3, 5, 23, 28, 30). In the cortex, approximately 50% of the muramic acid residues (the residues on every alternate disaccharide) have their peptide side chains completely removed, and these NAM residues are converted to muramic--lactam. In the Bacillus species previously examined, 25% of the cortex muramic acid residues have tetrapeptide side chains, while the majority of the remaining residues have their peptides cleaved to single L-alanine residues. Both the shortening and the removal of peptide side chains cause a decrease in cross-linking