Linear, uncross-linked peptidoglycan secreted by penicillin-treated Bacillus subtilis. Isolation and characterization as a substrate for penicillin-sensitive D-alanine carboxypeptidases.

Incubation of growing Bacillus subtilis with penicillin G led to the secretion of a peptidoglycan-related polymer and a nonglycan-bound pentapeptide into the culture medium. The secreted polymer was isolated and characterized as a linear cell wall glycan strand substituted predominantly by uncross-linked pentapeptide side chains. Polymer formation and secretion are most likely the result of continued synthesis and elongation of nascent glycan strands in the absence of subsequent processing by peptidoglycan transpeptidase or D-alanine carboxypeptidase enzymes. The nonglycan-bound pentapeptide, L-Ala-D-iso-Glu-meso-diaminopimelic acid-D-Ala-D-Ala, was probably formed by an N-acetylmuramyl-L-alanine amidase active on the peptide side chains of The uncross-linked polymer. The uncross-linked peptidoglycan polymer was shown to be a good substrate for penicillin-sensitive D-alanine carboxypeptidases purified from membranes of B. subtilis, Bacillus stearothermophilus, and Escherichia coli. D-alanine release was not, however, coupled to the cross-linking of peptide side chains, suggesting that these enzymes do not function as peptidoglycan transpeptidases in vivo. No transpeptidase or D-alanine carboxypeptidase activity was detected in mixtures of high molecular weight penicillin-binding proteins from B. subtilis, B. stearothermophilus, or Staphylococcus aureus. Possible reasons for the inability to demonstrate these activities are discussed. In addition, an N-acetylmuramyl-L-alanine amidase activity which copurifies with penicillin-binding proteins from B. subtilis, S. aureus, and E. coli was partially characterized.