SUMMARY: A wall + membrane preparation from was used to synthesize radioactively labelled 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 β-elimination. Benzylpenicillin (1 μg ml) inhibited cross-linking but not overall synthesis of glycan whereas at concentrations above 10 μg ml overall glycan synthesis was slightly inhibited. In the presence of 100 μg benzylpenicillin ml 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 and 45 disaccharide units were added during a 30 min incubation period. However, if incubation was continued for up to 120 min (in the presence of 100 μg benzylpenicillin ml) a considerable amount of the newly synthesized peptidoglycan was lost from the purified wall because autolytic enzymes were expressed in the wall + membrane preparation after the action of the antibiotic.


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