Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins rPBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes charicterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.Cell wall synthetic enzymes are integral membrane proteins. The fact that these proteins form covalent complexes with radioactive penicillin allows the quick detection and quantitation of these important membrane proteins without need of purification or other complex strategies that are used for thti detection and titration of other protein membrane coniponnts. The availability of such a sensitive and specific assay opens up the possibility of studying the biosynthesis, membrane incorporation, and regulation of this set of protein molecules of great biological importance.The catalytic functions of cell wall synthetic enzymes (penicillin-binding protein [PBPs]) involve a strong topographic element: these enzymes, while anchored in the plasma membrane, catalyze the building and rearrangement of covalent bonds in the macromolecular sheets of cell wall exterior to the plasma membrane. In the bacteria studied in this report, this incorporation reaction is restricted to a limited number of anatomical areas of the cell suirface, most commonly at the cell equator (1, 6). PBPs are minor protein constituents of the plasma membrane which have been estimated to represent about 3,000 molecules per cell in Escherichia coli (17) and about 20,000 molecules in pneumococci (21). However, only 1,000 molecules per cell in E. coli and 2/3 of the total number of PBPs in pneumococci appear to perform essential functions. Additional studies haveshown that apparently normal wall synthesis (and cell multiplication) can continue with less than a full complement of these essential PBPs (3, 22), indicating either that these proteins are present in excess or that only some fraction of all the proteins estimated by the PBP assay are functional. The purpose of the studies described in this paper was to test this point. Our experimental strategy exploited conditions of penicillin tolerance in which cell wall synthetic enzymes could be fully acylated with penicillin without killing or lysis of the cells. We carefully exam...