Murein Biosynthesis and O-Acetylation of N-Acetylmuramic Acid during the Cell-Division Cycle of Proteus mirabilis

The murein of Proteus mirabilis is unique with respect to the O-acetylation of part of its N-acetylmuramic acid residues. Working with synchronized cells and providing radioactive N-acetylglucosamine with the medium, the murein was pulse-labelled in vivo for 10-min periods at different times of the cell cycle. After isolation of murein and enzymatic cleavage with endo-N, O-diacetylmuramidase from Chalaropsis, the distribution of radioactivity between uncrosslinked murein subunits (monomers) and the peptide-crossliitked dimers was analyzed. The following results were obtained. 1Murein synthesized at different times during the cell cycle did not show significant variation regarding the degree of crosslinkage and the degree of O-acetylation. However, the degree of O-acetylation of pulse-labelled murein was found to be about sevenfold lower than that of murein from continuously labelled asynchronous cultures. 2A pulse-chase experiment revealed that only non-O-acetylated murein subunits were incorporated into the growing murein sacculus, part of which became O-acetylated subsequently. Since a certain amount of newly incorporated subunits was lost during the chase, which reappeared in the sacculus about one generation later, limited murein turnover during the cell cycle became evident. 3The degree of crosslinkage of both the O-acetylated as well as the non-O-acetylated newly synthesized murein increased during subsequent growth. 4The labelled peptide-crosslinked dimers were cleaved by an endopeptidase isolated from P. mirabilis. The distribution of radioactivity between the resulting non-O-acetylated and O-acetylated monomers indicated that most of the mono-O-acetylated dimer fraction carried radioactivity exclusively in the non-O-acetylated subunit which was not O-acetylated during subsequent murein biosynthesis. 5Since only a small portion of the labelled mono-O-acetylated dimer fraction carried radioactivity in both subunits, and only at specific times of murein biosynthesis, it was concluded that thin portion of the labelled mono-O-acetylated dimer fraction represented an intermediate in the O-acetylation process.From these results a defined sequence of steps of murein biosynthesis is proposed.