JOURNAL OF BACrERIOLOGY, Apr. 1994, p. 2398-24050021-9193/94/$04.00+0Copyright X) 1994, American Society for Microbiology

Modification of Peptidoglycan Precursors Is a Common Feature of theLow-Level Vancomycin-Resistant VANB-Type Enterococcus D366 andof the Naturally Glycopeptide-Resistant Species Lactobacillus casei,

Pediococcus pentosaceus, Leuconostoc mesenteroides,and Enterococcus gallinarum

D. BILLOT-KLEIN,' L. GUTMANN,'* S. SABLE,2 E. GUITTET,2 AND J. VAN HEIJENOORT3

Laboratoire de Microbiologie Medicale, 15, rue de l'Ecole de Medecine, 75270 Paris Cedex 06,1 Laboratoire de RMN,Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette,

and Unite de Recherche Associee 1131 du Centre National de la Recherch Scientifique, BiochimieMoleculaire et Cellulaire, Universite Paris-Sud, 91405 Orsay Cedex,3 France

Received 22 November 1993/Accepted 7 February 1994

The biochemical basis for the acquired or natural resistance of various gram-positive organisms toglycopeptides was studied by high-pressure liquid chromatographic analysis of their peptidoglycan UDP-MurNAc-peptide precursors. In all cases, resistance was correlated with partial or complete replacement of theC-terminal D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide by a new precursor with a modified Cterminus. Nuclear magnetic resonance analysis by sequential assignment showed that the new precursorencountered in Enterococcus faecium D366, a strain belonging to the VANB class, which expresses low-levelresistance to vancomycin, was UDP-MurNAc-L-Ala--y-D-Glu-L-Lys-D-Ala-D-lactate, identical to that previ-

ously found in the VANA class, which expresses high-level resistance to vancomycin. High-pressure liquidchromatographic analyses, composition determinations, and digestion by R39 D,D-carboxypeptidase demon-strated the exclusive presence of the new precursor in LactobaciUlus casei and Pediococcus pentosaceus, which are

naturally highly resistant to glycopeptides. The low-level natural resistance of Enterococcus gaUlinarum tovancomycin was found to be associated with the synthesis of a new precursor identified as a UDP-MurNAc-pentapeptide containing a C-terminal D-serine. The distinction between low and high levels of resistance toglycopeptides appeared also to depend on the presence or absence of a substantial residual pool of a

D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide.

Glycopeptides are very important antibiotics widely usedagainst gram-positive organisms in the clinical setting. Theirmechanism of action is based essentially on their capacity tobind specifically to the D-alanyl-D-alanine C terminus of thedisaccharide pentapeptide (24), which is the structural mono-

mer unit of cell wall peptidoglycan (27). The formation of sucha complex inhibits peptidoglycan synthesis by interfering withthe transglycosylation and transpeptidation reactions leadingto nascent peptidoglycan (27). It was previously assumed thatexcept under very abnormal growth conditions (15), the D-

alanyl-D-alanine terminal dipeptide was a feature present inthe precursors of the peptidoglycans of all bacteria (28).Recently, clinical isolates of enterococci which have acquiredresistance to glycopeptides (19, 29, 34) have been separatedinto two major classes, VANA and VANB. In the VANA class,the genes responsible for the resistance are plasmid borne andlead to inducible cross-resistance between vancomycin andteicoplanin (5, 29). In the VANB class, the resistance genes are

chromosome borne and associated with inducible resistance tovancomycin only (34). There is a third class, designatedVANC, which corresponds to natural resistance to vancomycinand to which Enterococcus gallinarum belongs (30, 33).

It has been shown that the resistance in the VANA andVANB classes is linked to the synthesis of a new modifiedpentapeptide precursor associated with a decrease in thequantity of the normal pentapeptide precursor (7). The new

* Corresponding author. Phone: 33-1-43292863. Fax: 33-1-43256812.

precursor has been biochemically identified only in the VANAclass (2, 16, 22) and ends in D-lactate instead of D-alanine. Thisis explained by the presence of two new enzymes: an ox-ketoacid dehydrogenase (VanH) (6, 10) which catalyzes the syn-

thesis of D-lactate from pyruvate and a ligase (VanA) (9, 10)which catalyzes the formation of depsipeptide D-Ala-D-lactate(D-Ala-D-Lac), which is subsequently linked to the UDP-MurNAc-tripeptide. This modification in the structure of theUDP-MurNAc-pentapeptide precursor is accepted in the sub-sequent steps of peptidoglycan synthesis, including the poly-merization reactions. The resistance to glycopeptides couldthen be explained by the fact that vancomycin does not bind tothe modified disaccharide pentapeptide unit, as suggested byits very low affinity for the N-acetyldepsipeptide (10).

Taking into account all of these results, we were promptedto investigate (i) whether the biochemical basis of the glyco-peptide resistance in the VANB class resembles that ofVANAand (ii) whether the presence of precursors not ending inD-Ala-D-Ala would explain the resistance in different lacticacid bacterial species which are naturally resistant to glycopep-tides. We therefore studied E. faecium D366, the prototypeVANB strain (3, 7, 34); E. gallinarum, which is naturallyresistant to a moderate level of vancomycin and belongs to theVANC class (30); and one strain each of Leuconostoc mesen-

teroides, Pediococcus pentosaceus, and Lactobacillus casei,which are known to be naturally highly resistant to glycopep-tides (23, 32). The peptidoglycan in all of these organismscontains lysine in its peptide moiety (18, 28).

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MATERIALS AND METHODS

Bacterial strains, growth conditions, and antibiotics. E.faecium D366 (VANB) and E. gallinarum SC1 (VANC) werepreviously described (3, 33). L. casei ATCC 393, L. mesen-teroides ATCC 8293, and P. pentosaceus ATCC 33316 wereobtained from the Institut Pasteur Collection. Enterococciwere grown in BH1 broth (Difco) and the other strains weregrown in MRS (Diagnostic Pasteur). MICs were determined inliquid medium at 37°C by a dilution method with inocula of 105CFU/ml. MICs were read after 18 h of incubation. Antimicro-bial agents were kindly provided as follows: vancomycin, EliLilly & Co., Saint-Cloud, France; teicoplanin, Marrion-MerrellDow, Cincinnati, Ohio. When necessary, precursors wereaccumulated for 30 min after addition of vancomycin at 100j.g/ml to an exponential-phase culture grown beforehand to anoptical density at 650 nm of 0.4.

Chemicals. Different standard compounds were used. UDP-MurNAc-L-Ala--y-D-Glu-L-Lys, prepared as described by Itoet al. (17), was kindly provided by M. Guinand (Laboratoire deChimie Biologie, Universite de Lyon I, Villeurbanne, France),and UDP-MurNAc-L-Ala-,y-D-Glu-L-Lys-D-Ala-D-Ala wasaccumulated in Staphylococcus aureus by incubation in thepresence of vancomycin and extracted as described by Parkand Chatterjee (25). UDP-MurNAc-tetrapeptide was obtainedfrom UDP-MurNAc-pentapeptide in the presence of the D,D-carboxypeptidase from Actinomadura sp. strain R39, whichspecifically catalyzes the hydrolysis of D,D peptides, esters, andthioesters (1, 20). The substrate (3 to 20 nmol) and the enzyme(0.25 p.g) were incubated for 15 min at 30°C in a final volumeof 30 ,ul containing 50 mM Tris-HCl (pH 7.5) and 4 mMMgCl2.

Quantification of muramic acid, amino acids, and D-lactate.The muramic acid and amino acid contents in different high-pressure liquid chromatography (HPLC) fractions were deter-mined with a Biotronik LC2000 automatic amino acid analyzerafter hydrolysis in 6 N HCl at 95°C for 16 h. D-Lactate wasassayed with a D-lactic acid-L-lactic acid kit (BoehringerMannheim) by UV determination of NADH formation in thepresence of D-lactate dehydrogenase and NAD.

Extraction and quantification of peptidoglycan precursors.Extraction of cytoplasmic peptidoglycan precursors was carriedout in accordance with the method of Bochner and Ames (8).In brief, 200 ml of an exponential-phase culture at an opticaldensity at 650 nm of 0.4 was centrifuged at 11,000 x g and30°C and washed once when grown in the presence of vanco-mycin. A 47-ml volume of ice-cold 1.1 M formic acid wasrapidly added to the pellet (final pH 2), and nucleotides wereextracted by incubation for 30 min at 4°C before centrifugation(15,000 x g for 20 min, 4°C). The supernatant was lyophilized.Subsequent identification and quantification of the precursorswere carried out as previously described (7, 13, 21), with slightmodifications. Different buffers were used to separate theprecursors by HPLC with a p.Bondapak C18 column (3.9 or 7.8by 300 mm). The UDP-MurNAc-tripeptide was separated byusing 50 mM ammonium formate, pH 5 (buffer 1). UDP-MurNAc-tetrapeptide-D-lactate was separated by using 10mM ammonium acetate, pH 6 (buffer 2), or 10 mM ammoniumacetate, pH 5, with a 0 to 2% acetonitrile gradient (buffer 3).The UDP-MurNAc-pentapeptide could be separated by usingany one of the three buffers. The UDP-MurNAc-tripeptide,UDP-MurNAc-tetrapeptide, and UDP-MurNAc-pentapeptidewere located with UDP-MurNAc-peptide standards (13). Af-ter isolation of the corresponding peaks, analysis of the aminoacid and muramic acid contents showed that the UV-absorbingmaterial was composed mainly of the respective precursors.

TABLE 1. MICs of glycopeptides for the strains used in this studyMIC (>Lg/ml)

Strain ClassVancomycin Teicoplanin

E. faecium D366 VANB 32 0.5L. casei ATCC 393 >512 512L. mesenteroides ATCC 8293 >512 512P. pentosaceus ATCC 33316 >512 512E. gallinarum SC1 VANC 8-16 0.5

Identification of UDP-tetrapeptide-D-lactate and UDP-tet-rapeptide-D-serine. UDP-tetrapeptide-D-lactate in E. faeciumD366 was identified as follows. Precursors were extracted froma 5-liter culture grown in the presence of 4 ptg of vancomycinper ml to express the resistance and separated by HPLC withbuffer 3. Peak I, corresponding to the new precursor previouslydescribed (7), was isolated by using buffer 1 and repurifiedtwice by using buffer 2. The purified precursor (ca. 40 nmol)was subjected to nuclear magnetic resonance (NMR) analysis.The identified precursor UDP-MurNAc-tetrapeptide-D-

lactate of E. faecium D366 was subsequently used as thestandard in HPLC analyses of the precursors from the otherstrains studied. Two types of experiments were performed withthe isolated precursors: determination of amino acid andmuramic acid contents and incubation with R39 D,D-car-boxypeptidase. The latter allowed (i) visualization after sepa-ration by HPLC of the disappearance of the precursor peakand the appearance of a UDP-MurNAc-tetrapeptide peak and(ii) identification of the released C-terminal residue (15 to 20nmol of the substrate was necessary for detection of D-lactate,and 2 to 3 nmol was necessary for detection of serine).Two-dimensional NMR. Rotating frame nuclear Overhauser

effect spectroscopy (ROESY) and nuclear Overhauser effectspectroscopy (NOESY) experiments (mostly in H20) wereperformed on a Bruiker AMX600 apparatus. The water signalwas suppressed by using the watergate pulse sequence (26, 31)after the read pulse of the two-dimensional experiments. Alldata sets, with a spectral width of 12.07 ppm, were collected as512 and 2,048 points in the tl and t2 dimensions, respectively.For ROESY, NOESY, and homonuclear Hartmann-Hahnspectroscopy (HOHAHA) experiments, mixing times were setat 300, 300, and 50 ms, respectively. Seventy-two scans werecollected for the ROESY experiment with the normal UDP-MurNAc-pentapeptide precursor. Sixty-four and 296 scanswere collected for the NOESY experiments with the normaland the new precursors, respectively, and 16 and 96 scans werecollected for the corresponding HOHAHA experiments. Co-sine and squared-cosine apodization functions were used alongthe tl and t2 dimensions, respectively, in all experiments, alongwith one level of zero filling in both dimensions. AdditionalHOHAHA spectra were obtained in D20.

RESULTS

Antibiotic susceptibilities. The MICs of the glycopeptidesfor the different strains are presented in Table 1. L. casei, L.mesenteroides, and P. pentosaceus, which are known to benaturally resistant, showed a high level of resistance to bothvancomycin and teicoplanin. E. faecium D366, belonging to theinducible VANB class, expressed a moderate level of resis-tance to vancomycin and was sensitive to teicoplanin (34). E.gallinarum SC1, belonging to the VANC class, resembled E.faecium D366, as far as MICs were concerned, but wasconsidered naturally resistant only to vancomycin.

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Ec

C4

c

u

D

time (min)

I

D

2

X.... it- *

3

3

I..4 .. .. ;...1time (min)

FIG. 1. Separation of UDP-MurNAc-peptide precursors of E. fae-cium D366 by HPLC. A sample of 1/20 of the total extract from 1 literof culture induced in the presence of 4 ,ug of vancomycin per ml was

applied to a p.Bondapak C18 column using buffer 3 (A). Elution of thenew precursor in buffer 2 before (B) and after (C) incubation with theR39 D,D-carboxypeptidase and of the UDP-MurNAc-pentapeptidebefore (D) and after (E) incubation with the R39 D,D-carboxypepti-dase. Peaks: 1, new precursor; 2, UDP-MurNAc-pentapeptide; 3,UDP-MurNAc-tetrapeptide.

Identification of UDP-MurNAc-tetrapeptide-D-lactate invancomycin-induced E. faecium D366. We have previouslyshown (7) that a new precursor appeared upon vancomycininduction in E. faecium D366 belonging to the VANB class.This new precursor coeluted from the HPLC column with theinduced precursor present in the VANA class which was sincewell characterized as UDP-MurNAc-tetrapeptide-D-lactate(2, 16, 22). To identify the new precursor from E. faecium D366unambiguously, large amounts of peak I (Fig. 1), correspond-ing to the new precursor, were purified. On HPLC, it was

eluted later than the standard UDP-MurNAc-pentapeptideprecursor, suggesting that it was a less polar compound (Fig.1). The specific cleavage of the C terminus of the purifiedprecursor by R39 D,D-carboxypeptidase led to formation of theUDP-MurNAc-tetrapeptide (Fig. 1). This suggested that thenew precursor was composed at least of the UDP-MurNAc-tetrapeptide, linked by a D,D linkage to an unidentified residueat its C terminus. This was substantiated by the presence in thenew precursor of muramic acid, alanine, glutamic acid, andlysine in a ratio of 1:1.7:1.2:0.8.NMR characterization of the new precursor. The structure

of the new precursor was assessed by various NMR experi-ments. The method used for analysis of the spectra was basedon the general approach described by K. Wutrich (35) andknown as sequential assignment. It consists of identification ofthe spin systems by the HOHAHA experiment, followed bycorrelation of these systems by sequential NOE connectivities.At first, this strategy was tested on the normal pentapeptideprecursor, UDP-MurNAc-L-Ala- - D - Glu-L-LyS-D -Ala-D-Ala. Some of the most important cross peaks in the NOESYexperiments are shown in Fig. 2. These revealed the successionof the various residues identified by the HOHAHA experi-ments and the locations of the linkages (side chain or mainchain) between them. An additional correlation between the H

proton of the penultimate alanine and the NH proton of thelast alanine was revealed by a ROESY experiment with a300-ms mixing time (data not shown), corresponding to con-nection 5 in Fig. 2. This correlation was, however, absent in theNOESY experiment, probably because of increased flexibilityof the latter residue.

In the new precursor, the various spin systems were easilyidentified by a HOHAHA experiment with D20. A newCH-CH3 spin system replaced one of the alanines of thenormal pentapeptide precursor, and the chemical shift of thecorresponding H proton shifted from 4.01 to 4.75 ppm andsuggested that this residue was a lactate with a free carboxy-late. Only five NHs were detected in H20, compared with sixin the normal pentapeptide precursor. The characteristic NOEcross peaks were very similar to those of the normal pentapep-tide precursor for the first five residues of the sequence, asshown in Fig. 2, and involved all of the observed NHs, exceptthe NH of the glucosamine part (indicated as Glc in Fig. 2) ofthe N-acetylmuramic acid residue. The missing NH corre-sponded to the last residue. These observations suggest re-placement of the last alanine residue by a lactate in themodified precursor, with an ester linkage to the rest of thepeptide. As seen in Table 2, the main chemical shift differencesbetween the two products were found in the H of the lactateresidue at the last position of the peptide (as expected) and inthe NH region for the alanine adjacent to that residue.Noticeably, no NOEs characteristic of a peculiar foldingsolution were identified.From these overall analyses, it was concluded that the new

precursor present in inducible class VANB E. faecium D366was UDP-MurNAc-L-Ala--y-D-GlU-L- Lys-D-Ala-D-lactate,which is identical to that found in the VANA class (2, 16, 22).

Identification of the peptidoglycan precursors from L. caseiATCC 393, P. pentosaceus ATCC 33316, and L. mesenteroidesATCC 8293. L. casei ATCC 393, P. pentosaceus ATCC 33316,and L. mesenteroides ATCC 8293 are naturally highly resistantto glycopeptides, and precursor pools from strains grown in theabsence of vancomycin were analyzed. To obtain better sepa-ration of the peaks, elutions were performed with two buffersystems: buffer 1 for separation of the UDP-MurNAc-tripep-tide and the UDP-MurNAc-tetrapeptide and buffer 2 forseparation of UDP-MurNAc-tetrapeptide-D-lactate (Fig. 3).Peaks were located by using standards including the UDP-MurNAc-tetrapeptide-D-lactate purified from vancomycin-in-duced E. faecium D366. Small quantities of the UDP-Mur-NAc-tripeptide were present in the different extracts (Fig. 3and Table 3). Interestingly, no UDP-MurNAc-pentapeptide orUDP-MurNAc-tetrapeptide was found. In contrast, a peakcorresponding to UDP-MurNAc-tetrapeptide-D-lactate waspresent in large amounts in L. casei extracts and in smalleramounts in P. pentosaceus extracts, while none was detectablein L. mesenteroides extracts. To identify these precursorsfurther as UDP-MurNAc-tetrapeptide-D-lactate, they weresubjected to the action of the R39 D,D-carboxypeptidase. In thetwo former cases, there was complete disappearance of thepresumed UDP-MurNAc-tetrapeptide-D-lactate peak andappearance of a peak eluting with a retention time expectedfor the UDP-MurNAc-tetrapeptide (data not shown). Theseresults, i.e., coelution with the UDP-MurNAc-tetrapeptide-D-lactate standard, presence of muramic acid in the peak, andtransformation into the UDP-MurNAc-tetrapeptide after di-gestion with the R39 D,D-carboxypeptidase, strongly suggestedthat the material was in fact UDP-MurNAc-tetrapeptide-D-lactate. This was confirmed by determination of the muramicacid, amino acid, and D-lactate contents. Muramic acid, ala-nine, glutamic acid, and lysine were found in ratios of 1.2:1.8:

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PEPTIDOGLYCAN PRECURSORS IN Vanr BACTERIA 2401

a)

go<|o_w* 0~~~~,

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8.50 8.00 ppm

b)

8.50

2O H H O

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8.00 ppmFIG. 2. Extracts from a set of NMR experiments (at 600 MHz) showing the NH to aliphatic proton connectivities on the normal

UDP-MurNAc-pentapeptide precursor (upper two spectra) and the new precursor (lower two spectra). Panels: a and c, HOHAHA experiments;b and d, NOESY experiments. The most significant cross peaks used for sequential assignment and structure determination are numbered, andthe corresponding assignments are indicated on the two structural diagrams at the right. The NH of the C-terminal alanine is indicated by thesymbol A* in panel a. An additional correlation (marked 5) appeared in the ROESY experiment at the frequency of this NH on the normalpentapeptide precursor but was absent in the NOESY experiment. In the spectra, Glc stands for the glucosamine residue, and in the two formulas,G stands for the N-acetylglucosamine residue.

1:0.9 and 0.9:1.7:1:0.8 for the precursors from L. casei and P.pentosaceus, respectively. The presence of D-lactate was as-sayed after release of the C-terminal residue from the precur-sor by the R39 D,D-carboxypeptidase. A sufficient amount ofpurified material for the assay was secured from the extracts ofboth organisms: 16 nmol of D-lactate was released from 18nmol of purified nucleotide precursors.

Identification of the peptidoglycan precursors from E. galli-narum SCI. E. gallinarum is considered naturally resistant tovancomycin. In contrast to L. mesenteroides, P. pentosaceus,

and L. casei, its vancomycin MIC is low and it is susceptible toteicoplanin (Table 1). In the extract of E. gallinarum SC1grown in the absence of antibiotic, a significant amount of theUDP-MurNAc-pentapeptide was found (Fig. 4 and Table 4).Since the presence of a normal pool of this precursor could notexplain the low-level resistance to vancomycin, additionalexperiments with vancomycin were performed. The precursorpool was analyzed after a brief (30-min) treatment with a highconcentration (100 pg/ml) of vancomycin (Fig. 4 and Table 4).The UDP-MurNAc-pentapeptide pool had increased about

TABLE 2. Representative chemical shifts of the two precursors in H20 at 20°C

Normal precursor New precursorResidue'

NH Hot Hp H-y Others NH Ha HB H-y Others

Lac 4.13 1.31 4.10 1.31Ala 7.96 4.15 1.35 7.96 4.13 1.36Glu 7.86 4.06 1.79, 2.06 2.20, 2.20 7.87 4.09 1.79, 2.07 2.19, 2.19Lys 8.22 4.10 1.71, 1.71 1.38, 1.38 1.59, 1.59, 8.19 4.11 1.71, 1.71 1.39, 1.39 1.59, 1.59,

2.91, 2.91 2.91, 2.91Ala 8.27 4.23 1.27 8.46 4.33 1.34Ala/Lac 7.90 4.01 1.24 4.75 1.34

" Residues are tabulated in accordance with the primary sequence, towards the C terminus.

2.00 -

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E

time (Sin)

FIG. 3. Separation of UDP-MurNAc-peptide precursors of L. caseiATCC 393 (A and B), P. pentosaceus ATCC 333316 (C and D), and L.mesenteroides ATCC 8293 (E and F). One-twentieth of the total extractfrom 1 liter of culture was applied to a ,uBondapak C18 column usingbuffer 1 (A, C, and E) or buffer 3 (B, D, and F). Peaks: 4, UDP-MurNAc-tripeptide; 3, UDP-MurNAc-tetrapeptide; 2, UDP-Mur-NAc-pentapeptide; 1, UDP-MurNAc-tetrapeptide-D-lactate.

fourfold compared with that of the culture grown in theabsence of vancomycin. No UDP-MurNAc-tetrapeptide-D-lactate precursor was detected, while a new peak which elutedbefore the UDP-MurNAc-pentapeptide was present inamounts almost equal to that of the UDP-MurNAc-pentapep-tide. Analysis of this purified peak showed that it comigratedwith the UDP-MurNAc-tetrapeptide and contained muramicacid, alanine, glutamic acid, and lysine in a ratio of 0.7:1.9:

1.1:1, corresponding to the composition expected for theUDP-MurNAc-tetrapeptide.Another approach was to analyze the precursor pools of E.

gallinarum grown in the presence of vancomycin at a concen-tration below the MIC (4 ,ug/ml) (Fig. 4 and Table 4). Thethreefold decrease of the UDP-MurNAc-pentapeptide poolwas associated with the appearance of a large amount of theUDP-MurNAc-tetrapeptide, and no UDP-MurNAc-tetrapep-tide-D-lactate was detectable. More surprising was the appear-ance of a new muramic acid-containing peak eluting betweenthe UDP-MurNAc-tetrapeptide and the UDP-MurNAc-pen-tapeptide. Analysis of this new precursor showed that muramicacid, alanine, glutamic acid lysine, and serine were present in aratio of 1.1:1.8:1:1:0.8, suggesting that it was composed of aserine-containing pentapeptide. When this purified precursorwas incubated with the R39 D,D-carboxypeptidase, it disap-peared completely while equal amounts of the UDP-MurNAc-tetrapeptide and free serine were detected as separate compo-nents by HPLC or amino acid analysis.

Therefore, the peak was identified as a UDP-MurNAc-pentapeptide with a D-serine at the C terminus. Similar resultswere obtained, although with increased quantities of eachprecursor, when the culture grown in the presence of 4 ,ug ofvancomycin per ml was exposed to 100 ,ug of vancomycin perml to accumulate the precursors (Table 4).

DISCUSSION

High-level resistance of E. faecium and E. faecalis of theVANA class to glycopeptides is linked to the acquisition ofgenes which allow synthesis of a new nucleotide precursor thatends in the depsipeptide D-Ala-D-Lac and has a very lowaffinity for the glycopeptides (4). For the VANB class, vanco-mycin MICs are generally lower than those observed for theVANA class, although they may reach those determined forthe VANA class (4, 14). Since the sequence of the VANBligase gene showed 77% homology with that of the VANAligase gene (12, 14), it was of interest to determine whether thenew precursor in the VANB class resembled that present in theVANA class. In a previous study, we had shown that the newprecursors from strains belonging to the two classes eluted withthe same retention time after HPLC (7). In this study, wedemonstrated that the structure of the new precursor presentin class VANB E. faecium D366 was UDP-MurNAc-tetrapep-tide-D-lactate, which is identical to that produced in strains ofthe VANA class, which expresses high level of resistance toglycopeptides. The moderate level of resistance to vancomycinin E. faecium D366 may be explained by the residual pool ofthe normal UDP-MurNAc-pentapeptide which is still presentand which leads to peptidoglycan intermediates with a Cterminus able to bind vancomycin (7). It must be stressed thatnow the structure of peptidoglycan precursors can be readilyand unambiguously determined by NMR, even under high-dilution conditions (ca. 0.1 mM). The watergate pulse se-quence with the 1-9-19-19-9-1 composite 1800 pulse (26, 31)was used at the end of all two-dimensional experiments withH20 as the water suppression technique and proved extremelyrobust and efficient under these extreme circumstances.

In this context, it was highly relevant to determine themolecular basis of the high-level natural resistance to glyco-peptides in L. casei, L. pentosaceus, and L. mesenteroides,which are all lactic acid bacteria. For L. casei and P. pentosa-ceus, the total absence of the UDP-MurNAc-pentapeptide andthe presence of significant amounts of UDP-MurNAc-tet-rapeptide-D-lactate, which leads to intermediates with a D-Ala-D-Lac terminus having low glycopeptide affinity, explained

-le...* ....*....V, . .. ...;6-14 1....*....a....* 16....t....A.... ..d,. ..L.- ...............I..................

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PEPTIDOGLYCAN PRECURSORS IN Vanr BACTERIA 2403

TABLE 3. Pool levels of peptidoglycan precursors in the different strains

Pool level (nmola/liter of culture)Strain Vancomycin MIC

UDP-MurNAc- UDP-MurNAc- UDP-MurNAc- (p.g/ml)tripeptide pentapeptide tetrapeptide-D-lactate

E. faecium D366 (VANB)b 1,125 86 95 32L. casei ATCC 393 51 <1 465 >512P. pentosaceus ATCC 33316 21 <1 62 >512L. mesenteroides ATCC 8293 10 <1 <1 >512

a Nanomoles of muramic acid.b Induced in the presence of vancomycin (7).

the high resistance to these antibiotics. From these results, itcan be hypothesized that these bacteria encode a ligase whichcatalyzes preferentially, or exclusively, the synthesis of D-Ala-D-lactate. Presumably, no D-alanyl-D-alanine is formed in theseorganisms. The UDP-MurNAc-tripeptide pool was small, andthis is in contrast to the very high level of accumulation of this

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2I

time (min)

FIG. 4. Separation of UDP-MurNAc-peptide precursors of E. galli-narum. One-twentieth of the total extract from 1 liter of culture wasapplied to a p.Bondapak C18 column using buffer 2. Panels: A, extractsfrom E. gallinarum SC1 grown in the absence of antibiotics; B, E.gallinarum SC1 after accumulation of precursors; C, E. gallinarum SC1grown in the presence of 4 p.g of vancomycin per ml; D, same as inpanel C after accumulation (1/40 of the total extract). Peaks: 3,UDP-MurNAc-tetrapeptide; 2', UDP-MurNAc-pentapeptide with aD-serine at the C terminus; 2, UDP-MurNAc-pentapeptide; 1, UDP-MurNAc-tetrapeptide-D-lactate.

pool observed in enterococci of the VANA and VANB classes(7). This suggested that contrary to the latter cases, thedepsipeptide D-Ala-D-lactate in the naturally resistant speciesis readily added to the UDP-MurNAc-tripeptide.

In L. mesenteroides, neither the normal pentapeptide nor theD-lactate precursors were found in the absence of accumula-tion. It is hardly possible that peptidoglycan is polymerizedonly from the UDP-MurNAc-tripeptide. Therefore, one couldassume that in this organism small D-lactate precursor amountsare synthesized and immediately integrated into peptidogly-can, explaining the absence of detection of the pool. Analternative explanation could be that some other, undetectedprecursor exists. However, when the R39 D,D-carboxypeptidasewas used on total extracts of L. mesenteroides, no obviouschanges in the elution profile were found (data not shown),suggesting that at least no precursor ending in a D,D linkagewas present in detectable amounts.

For E. gallinarum, which expresses only low-level resistanceto vancomycin, the situation differed, since no D-lactate pre-cursor was present and only the new UDP-MurNAc-pentapep-tide ending with D-serine was detected when the bacteria weregrown in the presence of vancomycin concentrations below theMIC. The mechanism of resistance could relate to the pres-ence of this new pentapeptide precursor ending in D-serine. Ifthis is the case, the constitutive character of the naturalresistance (33) of this organism is doubtful since synthesis ofthe D-serine precursor was observed only after growth atvancomycin concentrations below the MIC and not in theaccumulation experiment done with the organism grown in theabsence of vancomycin.The low-level resistance to vancomycin could be explained

by a lesser affinity of vancomycin for the D-Ala-D-Ser terminithan for the normal D-Ala-D-Ala termini and by the presenceof a still appreciable pool of the normal precursor. Furtherstudies are necessary to define the affinity of vancomycin, aswell as that of teicoplanin, to which the strain remains suscep-tible, for the D-alanyl-D-serine moiety. Inactivation of theVanC ligase from E. gallinarum, which has ca. 37% homologywith the VanA and VanB ligases, was associated with loss ofresistance (11). It would be interesting to know whether thenew precursor is affected in this mutagenized strain. The largetetrapeptide precursor pool observed upon vancomycin treat-ment could be explained by the presence of an inducibleD,D-carboxypeptidase activity (33) acting on pentapeptide pre-cursors at high pool levels. The possibility that the tetrapeptideprecursor at a high pool level may also be used for peptidogly-can synthesis cannot be excluded.

In conclusion, the presence of a D-lactate-containing pepti-doglycan precursor appears as a common mechanism for bothinducible acquired and constitutive natural resistances to gly-copeptides. However, as clearly shown in E. gallinarum, other

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2404 BILLOT-KLEIN ET AL.

TABLE 4. Pool levels of peptidoglycan precursors in E. gallinarum SC1

Pool level (nmolA/liter of culture)Growth withvancomycin UDP-MurNAc- UDP-MurNAc- UDP-MurNAc- UDP-MurNAc- UDP-MurNAc-

(I.g/ml) tripeptide tetrapeptide pentapeptide tetrapeptide- pentapeptideu-lactate (serine)'

None 62 < 1 140 < 1 <5None + 100' NDd 511 559 <1 <54 ND 145 50 < 1 954 + 100c ND 1,547 111 <1 608

a Nanomoles of muramic acid.b UDP-MurNAc-pentapeptide with a D-serine at the C terminus.Vancomycin (100 ,ug/ml) was added for 30 min at the end of the culture.

d ND, not determined.

modifications of the peptidoglycan precursors are associatedwith glycopeptide resistance.

ACKNOWLEDGMENTSWe thank J. M. Frere for the gift of R39 D,D-carboxypeptidase.This work was supported by grants from the Institut National de la

Sante et de la Recherche Medicale (CRE 930603 and CRE 900314)and from the Centre National de la Recherche Scientifique (Imabio-1992 and URA 1131).

ADDENDUM IN PROOF

After this paper was submitted, the presence of a precursorending in D-lactate was described for L. mesenteroides and L.casei by Handwerger et al. (16a).

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