Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation

The composition and fine structure of the vegetative cell wall peptidoglyca n from Bacillus subtilis were determined by analysis of its constituent mur opeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subt ilis vegetative cell peptidoglycan have the free carboxylic group of diamin opimelic acid amidated, Anhydromuropeptides and products missing a glucosam ine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to en d glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. T his amount is, however, dependent on the composition of the growth media, P otential attachment sites for anionic polymers to peptidoglycan occur on do minant muropeptides and account for 2.1% of the total. B. subtilis peptidog lycan is incompletely digested by lysozyme due to de-N-acetylation of gluco samine, which occurs on 17.3% of muropeptides. The cross-linking index of t he polymer changes with the growth phase, It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, res pectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers, The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weigh t PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan ma turation.