The degree of peptidoglycan 0-acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild-base-released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation-exchange amino acid analysis. The /3-~-N,6-O-diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20-52.8%, relative to the total muramic acid concentration. Each of the 0-acetylated peptidoglycans was found to be resistant to solubilization by both human and hen eggwhite lysozymes and for hen egg-white lysozyme, the extent of this resistance was dependent upon the degree of 0-acetylation. The steady-state parameters, K, and V, for the hen-egg-white-lysozyme-catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25°C. Values of K , for the different peptidoglycan samples were found to increase with increasing 0-acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [d(dG*)], a consequence of increasing 0-acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.Peptidoglycan is a heteropolymer of distinct composition and structure, associated uniquely with eubacterial cell walls. Proteus mirabilis peptidoglycan is comprised of equimolar amounts of GlcNAc, N-acetylmuramic acid (MurNAc), Lalanine, D-glutamic acid, L-meso-diaminopimelic acid and Dalanine. The cross-linking of neighbouring peptides is direct (chemotype Aly) and involves the meso-diaminopimelic acid and D-alanyl residues. The extent of cross-linking (33%) is typical of most Gram-negative bacteria [l]. Thus, the peptidoglycan of P. mirabilis is characteristic of the Enterobacteriaceae but for one notable and significant difference, the presence of extensive 0-acetylation [2]. Peptidoglycan 0-acetylation occurs at the 6-hydroxyl group of MurNAc residues producing the corresponding N,O-diacetylmuramyl derivatives, and this modification confers resistance to the hydrolytic action of many muramidases, including hen egg-white lysozyme (HEWL) and human lysozymes [3 -61.The phenomenon of peptidoglycan 0-acetylation was first observed independently by two different groups of researchers over 30 years ago [7, 81, and its role in conferring lysozyme resistance was demonstrated soon after [6]. Since then, 0-acetylation of peptidoglycan has been observed in a diverse group of bacteria which includes some important pathogens, both Gram positive (e.g. Staphylococcus aureus [9, 101) and Gram negative (e. g. Neisseria gonorrhoeae [3] and P. mirabilis strains of N . gonorrhoeae and characterized by 34-52% 0-acetylation, was shown to be resistant to hydrolysis by HEWL to the extent of 34-59% [12]. On the other hand, O-acetyldeficient peptidoglycan, isolated from N . gonorrhoea RD5 was completely solubilized and degraded to low-molecular-mass fragments. In the study, resistance was defined as the percen...