INVITRO SYNTHESIS AND O ACETYLATION OF PEPTIDOGLYCAN BY PERMEABILIZED CELLS OF PROTEUS-MIRABILIS

The synthesis and O acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-1-C-14]N-acetyl-D-glucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-C-14]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl-H-3]N-acetyl-D-glucosamine or [glucosamine-1,6-H-3]N-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-6-H-3]N-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-C-14]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of O-linked [C-14]acetate, whereas penicillin G blocked both peptidoglycan synthesis and [C-14]acetate transfer. These results supports the hypothesis that the base-labile O-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N --> O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.