Studies of the Formation of Peptide Cross-Links in the Cell Wall Peptidoglycan of Streptococcus fczecaZis*

A method was developed to label specifically the glycan chains of the cell wall peptidoglycan of Streptococcus fuecalis ATCC 9790 with [‘“Cl acetate. The formation of peptide cross-links (a) during exponential growth, (b) after valine starvation and wall thickening, and (c) during regrowth after 2 hours of valine starvation, was studied using continuous, pulse and pulse-chase labeling of the peptidoglycan with both [%]acetate and [3H]lysine. After labeling, walls were isolated, digested with the muramidase of Chalaropsis B, and the “free” peptidoglycan fragments (75 to 90% of the total peptidoglycan) were then fractionated on columns of Sephadex G-50, G-50, and G-25 in series into disaccharide-peptide monomer and peptide cross-linked bisdisaccharide-peptide dimer, trisdisaccharide-peptide trimer, and higher oligomer fractions. Peptidoglycan made during valine starvation and wall thickening was found to be slightly more cross-linked than peptidoglycan made during exponential growth. Pulse and pulse-chase experiments indicated that peptide cross-linking continued for an unexpectedly long time after incorporation of precursors into insoluble peptidoglycan.