PEPTIDOGLYCAN STRUCTURAL DYNAMICS DURING GERMINATION OF BACILLUS-SUBTILIS-168 ENDOSPORES

Peptidoglycan structural dynamics during endospore germination of Baci llus subtilis 168 have been examined by muropeptide analysis. The firs t germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine, We detected in the spore-associated material new muropeptides which, a lthough they have slightly longer retention times by reversed-phase (R P)-high-pressure liquid chromatography (HPLC) than related ones in dor mant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that t he chemical changes to the muropeptides on germination are minor and a re probably limited to stereochemical inversion. These new muropeptide s account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores o f B. subtilis 168 contains novel muropeptides in addition to those pre sent in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecu lar mass 20 Da lower than those of related reduced muropeptides, These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacter ium, There is also evidence for the activity of a glucosaminidase duri ng the germination process. Quantification of muropeptides in spore-as sociated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, m uramic delta-lactam, is proposed to be a major substrate specificity d eterminant of germination-specific lytic enzymes, allowing cortex hydr olysis without any effect on the primordial cell wall.