The distribution of muropeptides formed by muramidase digestion of peptidoglycan from Staphylococcus aureus H was determined by gel-filtration HPLC. The observed crosslinking pattern supports the conclusion that incorporation of peptidoglycan in S. aureus proceeds by a similar mechanism to that proposed earlier for Bacillus megaterium. In this mechanism single glycarepeptide strands are incorporated into the sacculus by crosslinking reactions that take place only between the monomer muropeptide units of the incoming glycopeptide and muropeptides present in the innermost region of wall at the wall-membrane interface : such crosslinking reactions take place only during incorporation and no other crosslinking reactions occur. This assembly process has now been termed restricted monomer addition. The present analysis shows that the distribution of muropeptides in S. aureus peptidoglycan is in excellent agreement with that predicted by this mechanism. We propose that cell wall assembly in S. aureus proceeds via restricted monomer addition without any requirement for the secondary crosslinking reactions that have been suggested to occur in this organism. The high degree of crosslinking in S. aureus, 80% in this study, may result mainly from the freedom for crosslinking provided by the pentaglycine bridge peptide.

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.

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