Biosynthesis of Peptidoglycan in Wall plus Membrane Preparations from Micrococcus luteus: Direction of Chain Extension, Length of Chains and Effect of Penicillin on Cross-linking

Abstract

A wall + membrane preparation from M. luteus was used to synthesize radioactively labeled peptidoglycan. The newly synthesized peptidoglycan either was cross-linked by transpeptidation to existing wall or remained associated with the membrane fraction but was not cross-linked. The average biosynthetic chain lengths, calculated from the ratio of free reducing groups of muramic acid to total muramic acid, were 66 disaccharide units for cross-linked and 26 disaccharide units for the uncross-linked material. The latter value was confirmed by the release of lactyl peptide side chains by .beta.-elimination. Benzylpenicillin (1 .mu.g ml-1) inhibited cross-linking but not overall synthesis of glycan but at concentrations above 10 .mu.g ml-1 overall glycan synthesis was slightly inhibited. In the presence of 100 .mu.g benzylpenicillin ml-1 the incorporation of disaccharide units to existing walls decreased to 25% of the control. This residual incorporation represented extension by transglycosylation of peptidoglycan already cross-linked to existing walls. Chains with an average length of between 30-45 disaccharide units were added during a 30 min incubation period. If incubation was continued for up to 120 min (in the presence of 100 .mu.g benzylpenicillin ml-1) a considerable amount of the newly synthesized peptidoglycan was lost from the purified wall becaue autolytic enzymes were expressed in the wall + membrane preparation after the action of the antibiotic.