Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci

Abstract

Cytoplasmic precursors of the peptidoglycan biosynthetic pathway were purified from vancomycin‐treated, glycopeptide‐sensitive and ‐resistant strains of Enterococcus faecium. Resistance was due to production of a modified precursor, UDP‐MurNAc‐l‐Ala‐d‐Glu‐l‐Lys‐d‐Ala‐d‐lactate, where lactate was identified on the basis of mass of the precursor and on its ability to act as a substrate for d‐lactate dehydrogenase after release from the precursor. The presence of the d‐lactate residue instead of d‐alanine in the terminal position would hinder formation of a vancomycin‐precursor complex, without preventing incorporation of the precursor into mature peptidoglycan.

The vanG glycopeptide resistance operon from Enterococcus faecalis revisited

“…Extraction and analysis of peptidoglycan precursors was performed as described (Messer and Reynolds, 1992; Reynolds et al ., 1994a). Enterococci were grown in BHI medium in the absence or presence of vancomycin (4 µg ml −1 ) to the midexponential phase (A 600 = 1).…”

“…The activities were determined from suitable dilutions of cytoplasmic and membrane fractions in a final volume of 45 µl. The assay mixture contained Bis Trispropane (50 mM, pH 7.5), 10 mM l ‐serine or l ‐alanine and 15 µl of the diluted fraction as enzyme preparation and was incubated at 37°C for 30 min d ‐amino acids produced by racemase activity were assayed using a d ‐amino acid oxidase assay (Messer and Reynolds, 1992) with d ‐serine or d ‐alanine as standards. Protein concentration was determined according to the method of Bradford (1976) with bovine serum albumin as standard.…”

Requirement of the VanY and VanX D,D‐peptidases for glycopeptide resistance in enterococci

“…Extraction and analysis of peptidoglycan precursors were performed essentially as described previously (Messer and Reynolds, 1992; Reynolds et al ., 1994). Enterococci were grown in broth supplemented with 0.5% yeast extract and appropriate antibiotics to mid‐exponential phase (OD 600 = 1), ramoplanin (9 μg ml −1 ) was added and incubation was continued for 0.5 mean generation time.…”

Gene vanXYC encodes d,d ‐dipeptidase (VanX) and d,d‐carboxypeptidase (VanY) activities in vancomycin‐resistant Enterococcus gallinarum BM4174

“…In a final volume of 30 μl, the assay mixture contained, 100 mM PO 4 buffer (pH 7.2) ( E. coli preparations) or 100 mM Tris‐HCl (pH 7.2) (enterococcal preparations), 10 mM d ‐Ala‐ d ‐N (where N is another amino acid, VanX activity) or substrates ending in acyl‐ d ‐Ala‐ d ‐N (VanY activity), and 10 μl of a suitable dilution of the cytoplasmic or membrane fraction so that less than 10% of the substrate was converted to product during the incubation (30 min at 37°C for VanX activity; 40 min at 37°C for VanY activity). The concentration of d ‐Ala or d ‐Ser released was measured using the d ‐amino acid oxidase assay with appropriate standards (Messer and Reynolds, 1992). A more sensitive assay, using 2,2′‐azinodi(3‐ethylbenzthiazoline sulphonate) instead of o ‐dianisidine as the chromogenic reagent, was adopted when the activity was low (Granier et al ., 1994).…”