Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA, ftsI, or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.The peptidoglycan (murein) layer of the bacterial cell envelope forms a giant, bag-shaped macromolecule, the sacculus, surrounding the cell. The sacculus is a covalently closed structure in which long polysaccharide chains are cross-linked by peptide bridges in a netlike fashion. The sacculus is the principal stress-bearing and shape-maintaining element of the cell wall, and it plays an essential role in bacterial morphogenesis. Metabolism of the sacculus is a complex process not only because of the large number of enzymes and metabolites involved but mostly because of regulatory requirements and topological constraints (1,24,29,37,44).Growth of Escherichia coli occurs by the periodic succession of elongation and division events (1,11). Cells divide at their midpoint once the chromosome is replicated and the initial mass (length) is doubled (15,16). Cell elongation demands the concomitant enlargement of the sacculus, and cell division requires the formation of a transverse septum at the center of the sacculus. As the sacculus supports the turgor pressure of the cell, its enlargement and formation of septa must proceed while avoiding the generation of discontinuities to prevent cell lysis (24,27). In addition, the sacculus is subjected to a dynamic metabolic activity comprising maturation, turnover, and growth phase-dependent structural changes (14, 19-21, 23, 26, 41).Insertion of new precursors by the concerted action of biosynthetic proteins (penicillin-binding proteins [PBPs]) and murein-hydrolytic enzymes promotes the elongation of the sacculus (24, 37, 46). However, a detailed picture of the insertion process is still missing. Incorporation of new precursors is likely to occur in the form of multistrand, cross-linked groups which are first bound to the sacculus in a rela...
