Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3

Abstract

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 Nacetylglucosaminyl -N-acetylmuramyl(pentapeptide) -pyrophosphate-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-MurNAcpentapeptide to the membrane acceptor undecaprenol phosphate, yielding MurNAc(pentapeptide)-pyrophosphate-undecaprenol-(lipid I). Thereafter, a transferase catalyzes the addition of N-acetylglucosamine, yielding lipid II. The translocase also catalyzes the reverse reaction, and the equilibrium is in favor of the formation of UDP-MurNAc-pentapeptide (10,26 cell-free system in which it was allowed to accumulate to a certain extent by incubation of membranes with UDPGlcNAc and radiolabelled UDP-MurNAc-pentapeptide (44).In this study, a set of optimal conditions for a maximal in vitro formation of lipid II with particulate fraction from E. coli was sought, as well as substantial improvements in its purification. A lipid II containing tripeptide Ala-y-D-Glumeso-DAP instead of the pentapeptide was prepared in a similar way. Its possible in vivo presence was investigated. Both types of lipid II were compared as substrates in the in vitro polymerization reactions assayed with PBP lb or PBP 3. The possible physiological significance of such reactions are discussed. MATERUILS AND METHODSBacterial strains, ...

Substrate Specificity and Membrane Topology of Escherichia coli PgpB, an Undecaprenyl Pyrophosphate Phosphatase

“…Therefore, in the presence of only one PAP2 C 55 -PP phosphatase, C 55 -PP and C 55 -P must be efficiently translocated across the inner membrane to reach the sites of dephosphorylation and glycosylation, respectively. This is supported by the fact that a high rate of transbilayer movement of C 55 -P was observed in Micrococcus lysodeikticus (43,44), which has to match the high rate of peptidoglycan synthesis (45). It was hypothesized that the long chain of the carrier lipid enables spontaneous diffusion of the latter across membranes (46).…”

The MurE Synthetase from Thermotoga maritima Is Endowed with an Unusual d-Lysine Adding Activity

“…In the present work, we demonstrated that the D-lysine-containing UDPMurNAc-tripeptide was a good substrate for T. maritima MraY, as good as the conventional UDP-MurNAc-pentapeptide, yielding tripeptide lipid I (Table 3). Such a behavior is not unprecedented; in E. coli, the formation of tripeptide lipid II from UDP-MurNAc-tripeptide was reported in a cell-free system (33), implying that the meso-A 2 pm-containing UDP-MurNAc-tripeptide is a substrate for MraY (and MurG) from this species. Taking into account the usual lack of specificity of MurG and of the glycosyltransferases for the peptide part of the lipid intermediates, it is reasonable to consider that the D-lysine-containing tripeptide is incorporated into the peptidoglycan of T. maritima, as it is the case for the meso-A 2 pm-containing tripeptide into the peptidoglycan of E. coli (33).…”

The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan‐synthesising enzymes

“…The bifunctional nature of the high-molecular-weight PBPs 2 and 3 is still controversial. While the TPase activity of both proteins was proven, the GTase activity could not be demonstrated unambiguously [7]. PBP2 in conjunction with the RodA protein was shown to be involved in cell elongation; PBP3 together with the FtsW protein is believed to play an important role during cell septation [8][9][10][11].…”