Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination

This study examined the structure of peptidoglycan from dormant Bacillus megaterium KM spores and how it changes during germination. Researchers used reverse-phase HPLC, mass spectrometry, and amino acid analysis to identify 21 different muropeptides in the spore cortex. They found that 44.2% of muramic acid residues carry δ-lactam groups, 28.8% carry single L-alanine, and 25.1% carry tetrapeptides, with very low cross-linking at only 2.2% of muramic acid residues. During germination triggered by L-alanine, novel muropeptides appeared, indicating the activity of at least two hydrolytic enzymes: an N-acetylglucosaminidase and a lytic transglycosylase. A salt extract from germinated spores caused germination-like changes in permeabilized spores, with the N-acetylglucosaminidase being the predominant enzyme. The B. megaterium peptidoglycan structure was largely similar to B. subtilis, though with some notable differences including greater de-N-acetylation and unsubstituted muramic acid residues.

Key findings

• B. megaterium spore peptidoglycan contains 44.2% δ-lactam-substituted muramic acid, 28.8% single L-alanine, and 25.1% tetrapeptide residues, with very low cross-linking at 2.2% per muramic acid.
• During germination, two main hydrolytic enzymes are activated: an N-acetylglucosaminidase and a lytic transglycosylase, which generate characteristic novel muropeptides.
• B. megaterium spore peptidoglycan shares 76% of muropeptides with B. subtilis but contains unique structural features including de-N-acetylated amino sugars and unsubstituted muramic acid residues.
• Muropeptides with unsubstituted N-acetylmuramic acid likely represent intermediates in δ-lactam formation, suggesting a two-step biosynthetic process.