-Deacetylases required for muramic-δ-lactam production are involved in sporulation, germination, and heat resistance.

Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses. Bacterial spores are multilayered structures, one of which is a peptidoglycan layer called the cortex, containing muramic-δ-lactams that are synthesized by at least two bacterial enzymes, the muramoyl-l-alanine amidase CwlD and the -deacetylase PdaA. This study focused on the spore cortex of , a Gram-positive, toxin-producing anaerobic bacterial pathogen that can colonize the human intestinal tract and is a leading cause of antibiotic-associated diarrhea. Using ultra-HPLC coupled with high-resolution MS, here we found that the spore cortex of the 630Δ strain differs from that of Among these differences, the muramic-δ-lactams represented only 24% in , compared with 50% in CD630_14300 and CD630_27190 were identified as genes encoding the -deacetylases PdaA1 and PdaA2, required for muramic-δ-lactam synthesis. In a mutant, only 0.4% of all muropeptides carried a muramic-δ-lactam modification, and muramic-δ-lactams were absent in the cortex of a double mutant. Of note, the mutant exhibited decreased sporulation, altered germination, decreased heat resistance, and delayed virulence in a hamster infection model. These results suggest a much greater role for muramic-δ-lactams in than in other bacteria, including In summary, the spore cortex of contains lower levels of muramic-δ-lactams than that of , and PdaA1 is the major -deacetylase for muramic-δ-lactam biosynthesis in , contributing to sporulation, heat resistance, and virulence.