Membrane Intermediates in the Peptidoglycan Metabolism of Escherichia coli: Possible Roles of PBP 1b and PBP 3

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively.Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification.When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified.Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP lb or PBP 3.With PBP lb, tripeptide lipid II was used as efficiently as pentapeptide lipid II.It should be stressed that the in vitro PBP lb activity accounts for at best to 2 to 3% of the in vivo synthesis.With PBP 3, no polymerization was observed with either substrate.Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed.In particular, it is speculated that the transglycosylase activity of PBP lb could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.It is now established that in Escherichia coli there are at least two distinct modes for the insertion of newly polymerized peptidoglycan material, one for elongation and the other for septation (30).Several proteins have been described as directly involved in the polymerization reactions (34, 47).In particular, certain penicillin-binding proteins (PBPs) were found to catalyze in vitro polymerization reactions with N- acetylglucosaminyl -N-acetylmuramyl(pentapeptide) -pyro- phosphate-undecaprenol as a substrate (17,18,22, 29,39,41).In vivo, this membrane intermediate (lipid II) is formed from UDP-N-acetylmuramoyl-pentapeptide by a two step process (34).First, a translocase catalyzes the transfer of the phospho-MurNAc-pentapeptide moiety of UDP-MurNAc- pentapeptide to the membrane acceptor undecaprenol phos- phate, yielding MurNAc(pentapeptide)-pyrophosphate-undecaprenol-(lipid I).Thereafter, a transferase catalyzes the addition of N-acetylglucosamine, yielding lipid II.The trans- locase also catalyzes the reverse reaction, and the equilibrium is in favor of the formation of UDP-MurNAc-pentapeptide (10, 26).The irreversibility of the transferase reaction allows for the in vivo formation of lipid II.Recently, the translocase and the transferase have been identified as the products of gene mraY and gene murG, respectively (15, 26).From the data of Ramey and Ishiguro (33), the ratio of lipid I to lipid II in growing cells can be estimated at 1:1.5.More recently, the UDP-MurNAc-pentapeptide/lipid I/lipid II ratio was estimated at ca. 300:1:3 (20) or 140:1:2.7 (26).Considering that there are ca.105 molecules of UDP-Mur- NAc-pentapeptide per cell (23), there would thus be only 1,000 to 2,000 molecules of lipids II and less than 700 of lipid I.Such extremely low pool levels explain why the isolation and purification of lipids I and II directly from cells is not an easy matter.However, lipid II has been isolated from a