The activity of penicillin-binding protein 3 of Escherchia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits. Kraus et al. (13) shows that the structure of peptidoglycan synthesized by either ethertreated E. coli cells or isolated envelopes was different from that of peptidoglycan synthesized by the living cell.It is well known that PBP 3 from E. coli participates in cell division. Mutations affecting PBP 3 or selective inhibition of this PBP by 13-lactams impairs cell septation and produces filamentation of E. coli cells (29). Botta and Park, by selective inhibition of PBP 3 during defilamentation (a period of active septation), demonstrated that PBP 3 was involved in septal peptidoglycan synthesis (5). It is now believed that PBP 3 synthesizes, at least in part, the septal peptidoglycan. So far, little or nothing is known about either the activity of this PBP or the structure of septal peptidoglycan. A recent study by Olijhoek et al. (22) has shown that the degree of peptidoglycan cross-linkage was higher during septation. From these data, it was inferred that septal peptidoglycan is of a more cross-linked nature, with a higher ratio of both dimeric and trimeric subunits (12, 13 In this communication, we present biochemical data on the activity of PBP 3 both in vivo and in vitro, with regard to the substrates it uses and the products yielded, as well as an outline of the structure of the septal peptidoglycan of E. coli. A partial account of this study has appeared elsewhere (26). MATERIALS AND METHODSBacterial strains and culture conditions. E. coli PA3092 (Fthr-J leu-6 thi-J argHI thy his-i trp-J str-9 lacYJ malAl xyl-7 mtl-2 melffhuA2 supE44) and PAT84 (like PA3092, but dapA lysA sulB [27a]). Cells were grown aerobically in L broth (16) supplemented with D-glucose (4 mg/ml) and, for PAT84 strain, 2,6-meso-diaminopimelic acid (5 mg/liter). Other conditions varied from one experiment to another and are defined below.Detection of PBPs in cell extracts and cell envelopes. Usually, PBPs were detected in whole-cell extracts. Cells were collected from the cultures by rapid cooling and centrifugation at low speed. Pelleted cells were suspended in a small volume of ice-cold 20 mM sodium phosphate buffer, pH 7.0. Suspended cells (usually about 0.5 ml) were subjected to a 10-s burst of sonication in a 150-V sonic oscillator (MSE Instruments, Crawley, U.K.). Sonic treatment was carried out with a 1/8-in. (3 mm) probe at an amplitude of 6 ,um (peak to peak at...
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