Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic ␦-lactam. Production of muramic ␦-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic ␦-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic ␦-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic ␦-lactam. CwlD and PdaA are necessary and sufficient for muramic ␦-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.Bacterial endospores can maintain a dormant, highly resistant state for long periods and then, under favorable conditions, rapidly germinate to produce vegetative cells. Spore dormancy and heat resistance are dependent on the relative dehydration of the spore core (6, 24, 28). Spore core dehydration requires the integrity of a thick spore peptidoglycan wall, and peptidoglycan hydrolysis is required for rehydration and resumption of spore core metabolism during spore germination (27,31).The spore peptidoglycan is composed of two contiguous layers that are synthesized between the two membranes surrounding the developing forespore. The inner layer, the germ cell wall, makes up only 10 to 15% of the total spore peptidoglycan (23) and is apparently synthesized by proteins expressed on the surface of the inner forespore membrane (22, 35). The germ cell wall has a structure resembling the peptidoglycan of the vegetative cell wall (23), is maintained during spore germination to serve as the initial wall of the outgrowing spore (5), and appears to function as a template for proper synthesis of the outer spore peptidoglycan layers, the cortex (22). The cortex makes up Ͼ80% of the spore peptidoglycan (23), is synthesized by proteins present on the surface of the outer forespore membrane (10, 35), is rapidly degraded...