The final step of peptidoglycan subunit assembly in Escherichia coli occurs in the cytoplasm

Abstract

The murG gene ofEscherichia coli encodes the N-acetylglucosaminyltransferase responsible for the final step in the formation of the lipid-linked disaccharide-pentapeptide subunit of peptidoglycan. Using trypsin to probe maxicell spheroplasts, we show that this enzyme is peripherally associated with the inner face of the cytoplasmic membrane. Therefore, the peptidoglycan subunit is completely assembled before it traverses the cytoplasmic membrane.The final steps of Escherichia coli peptidoglycan subunit assembly occur on a lipid carrier molecule located in the cytoplasmic membrane (8, 18). First, phospho-MurNAc-might also be used in this reaction (18). Next, GlcNAc (Nacetylglucosamine) is transferred from UDP to the lipid-linked MurNAc-pentapeptide (or MurNAc-tripeptide). The MurNAc-(pentapeptide/tripeptide)-GlcNAc subunit thus formed is then polymerized into peptidoglycan in the periplasm (2). These lipid-linked subunit assembly reactions were previously shown to be associated both with the inner membrane and with a membrane fraction which probably corresponds to zones of adhesion between the inner and outer membranes (7).Although the biochemical details of these reactions have been known for some time, the precise point at which cytoplasmic synthesis becomes periplasmic synthesis was not known. Recently, the GlcNAc transferase was identified as the product of the murG gene of E. coli (6,12). In that work (12), Mengin-Lecreulx et al. also showed that the MurG protein was membrane associated. Here we show that the GlcNAc transferase is associated with the cytoplasmic face of the inner membrane and that therefore the entire peptidoglycan subunit is assembled before being transported across the cytoplasmic membrane.To assess the topology of the GlcNAc transferase with respect to the cytoplasmic membrane of E. coli, we used trypsin to probe maxicell spheroplasts. Sodium dodecyl sulfate (SDS) sample buffer was added to each sample, the samples were boiled and run on SDS-10% polyacrylamide gels. The gels were treated with Amplify (Amersham), and exposure was carried out for 1 to 2 days at -70'C. Proteins were identified by comparison with strains not containing the indicated plasmids and by comparison with 14C-methylated molecular weight markers (Amersham) (data not shown).Using this approach, we found that the GlcNAc transferase was insensitive to trypsin unless trypsin gained access to the cytoplasm. Access could be gained either by permeabilizing the membrane with Triton X-100 or by the use of very high concentrations (.1 mg/ml) of trypsin in the absence of the detergent. Preliminary experiments showed that permeabilization of the cytoplasmic membrane by the addition of 1% Triton X-100 to maxicell spheroplasts resulted in increased sensitivity of the MurG protein to tryptic digestion (data not shown).To demonstrate that the increased sensitivity of the GlcNAc transferase to trypsin in the presence of Triton X-100 was a consequence of the disruption of the membrane rather than denaturation of the transferase, we per...

Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: Minimal structural requirements for intermolecular complexation and fibril formation

“…However, ramoplanin differs from transglycosylase inhibitors such as moenomycin and mersacidin in that it not only inhibits TGase activity, but also generates a metabolic block at the level of the MurG reaction, forcing a buildup of cellular pools of Lipid I (12,13). Inhibition of MurG activity might be explained simply if the ramoplanin-Lipid II complex was an inhibitor of MurG, because the enzyme is peripherally associated with the inner face of the cytoplasmic membrane (23). Alternatively, should either ramoplanin or Lipid I encounter the other because of membrane translocation, an inhibitory complex could form.…”

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

“…Cell wall synthesis begins in the cytoplasm, where a set of highly conserved enzymes catalyze the formation of the lipid-linked precursor lipid II, which is composed of undecaprenyl-pyrophosphate (UndPP) linked to N-acetylglucosamine-N-acetylmuramic acid pentapeptide. Lipid II is synthesized on the inner face of the cytoplasmic membrane (1). The molecule is then translocated to the outer face of the membrane, where the disaccharide-peptide monomer is incorporated into the existing PG by cell wall synthetic machineries composed of penicillin-binding proteins and additional factors (2).…”

A Bacillus subtilis gene encoding a protein similar to nucleotide sugar transferases influences cell shape and viability

“…The chains are formed by polymerization of disaccharide units that are assembled on a lipid carrier molecule at the inner surface of the cytoplasmic membrane (11,12,18). Synthesis of the disaccharide is catalyzed by MurG, a UDP-N-acetylglucosamine:Nacetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase, which transfers GlcNAc from the nucleotide sugar UDP-GlcNAc to undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide to form undecaprenylpyrophosphoryl-MurNAc-(pentapeptide)GlcNAc (2,14). Here we report that B. subtilis contains a gene, ypfP, that influences cell shape and viability, and whose inferred product resembles MGDG synthase of C. sativus and shows similarity to some conserved regions of MurG proteins.…”