The murG gene of the Escherichia coli chromosome encoding UDP-N-acetylgluco-samine: undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide N-acetylglucosaminyl transferase.

Ikeda, Masato; Wachi, Masaaki; Matsuhashi, Michio

doi:10.2323/jgam.38.53

Cited by 11 publications

(12 citation statements)

References 17 publications

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“…Escherichia coli OV58 (pTA9) over‐expressing MurG [11] was grown at 37°C to log phase in BHI/CAA. Membranes were collected by differential centrifugation (6000× g for 10 min followed by 200 000× g for 1 h) following cell lysis at 20 000 psi using a French press.…”

Section: Methodsmentioning

confidence: 99%

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Goldman¹,

Baizman²,

Longley³

et al. 2000

FEMS Microbiology Letters

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Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

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Section: Methodsmentioning

confidence: 99%

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Goldman¹,

Baizman²,

Longley³

et al. 2000

FEMS Microbiology Letters

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“…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.…”

mentioning

confidence: 92%

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

Bupp

Heijenoort

1993

J Bacteriol

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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...

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“…coli OV58 (pTA9) overexpressing MurG (Ikeda et al, 1992) was grown to an OD '!! 1n0 at 37 mC in a 10 l batch fermenter in BHI\CAA using 1 l min − " aeration with air and 150 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase

et al. 2000

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Moenomycin is a natural product glycolipid that inhibits the growth of a broad spectrum of Gram-positive bacteria. In Escherichia coli, moenomycin inhibits peptidoglycan synthesis at the transglycosylation stage, causes accumulation of cell-wall intermediates, and leads to lysis and cell death. However, unlike Esc. coli, where 5-6 log units of killing are observed, 0-2 log units of killing occurred when Gram-positive bacteria were treated with similar multiples of the MIC. In addition, bulk peptidoglycan synthesis in intact Gram-positive cells was resistant to the effects of moenomycin. In contrast, synthetic disaccharides based on the moenomycin disaccharide core structure were identified that were bactericidal to Gram-positive bacteria, inhibited cell-wall synthesis in intact cells, and were active on both sensitive and vancomycinresistant enterococci. These disaccharide analogues do not inhibit the formation of N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphorylundecaprenol (lipid II), but do inhibit the polymerization of lipid II into peptidoglycan in Esc. coli. In addition, cell growth was required for bactericidal activity. The data indicate that synthetic disaccharide analogues of moenomycin inhibit cell-wall synthesis at the transglycosylation stage, and that their activity on Gram-positive bacteria differs from moenomycin due to differential targeting of the transglycosylation process. Inhibition of the transglycosylation process represents a promising approach to the design of new antibacterial agents active on drug-resistant bacteria.

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The murG gene of the Escherichia coli chromosome encoding UDP-N-acetylgluco-samine: undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide N-acetylglucosaminyl transferase.

Cited by 11 publications

References 17 publications

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

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

Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase

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