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Balance between Two Transpeptidation Mechanisms Determines the Expression of β-Lactam Resistance in Enterococcus faecium *

Open AccessPublished:June 19, 2002DOI:https://doi.org/10.1074/jbc.M204319200
      Thed,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel β-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation inEnterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala4d-Asx-Lys3) by cross-links resulting froml,d-transpeptidation (l-Lys3d-Asx-Lys3). This was associated with no modification of the level of production of the PBPs or of their affinity for β-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A β-lactam-insensitivel,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a β-lactam-insensitived,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys3d-Asx-Lys3 cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for thel,d-transpeptidase.
      GlcNAc
      N-acetylglucosamine
      MurNAc
      N-acetylmuramic acid
      PBP
      penicillin-binding protein
      MIC
      minimal inhibitory concentration
      RP-HPLC
      reverse-phase high pressure liquid chromatography
      MS
      mass spectrometry
      Boc
      t-butyloxycarbonyl
      Bn
      benzyl
      The synthesis of bacterial cell wall peptidoglycan is a two-stage process. First, the disaccharide peptide monomer unit is assembled in a series of cytoplasmic and membrane reactions (
      • Van Heijenoort J.
      ). InEnterococcus faecium, the resulting unit is composed of N-acetylglucosamine (GlcNAc)1 andN-acetylmuramic acid (MurNAc) substituted by thel-alanyl-γ-d-glutamyl-l- (N ε-d-aspartyl)lysyl-d-alanyl-d-alanine orl-alanyl-γ-d-glutamyl-l-(N ε-d-asparaginyl)lysyl-d-alanyl-d-alanine stem hexapeptide (d-Asx-pentapeptide) (
      • Billot-Klein D.
      • Shlaes D.
      • Bryant D.
      • Bell D.
      • Legrand R.
      • Gutmann L.
      • van Heijenoort J.
      ,
      • De Jonge B.L.M.
      • Gage D.
      • Handwerger S.
      ,
      • Billot-Klein D.
      • Shlaes D.
      • Bryant D.
      • Bell D.
      • van Heijenoort J.
      • Gutmann L.
      ). The final steps of peptidoglycan synthesis involve its transfer through the cytoplasmic membrane, its polymerization to glycan strands by glycosyltransferases, and the cross-linking of stem peptides byd,d-transpeptidases. These latter enzymes catalyze the formation of a peptide bond between the carboxyl ofd-Ala at position 4 of a donor stem peptide and the amino group of the d-asparagine or d-aspartate linked to the ε-amino group of l-Lys at position 3 of an acceptor peptide stem (
      • De Jonge B.L.M.
      • Gage D.
      • Handwerger S.
      ,
      • Billot-Klein D.
      • Shlaes D.
      • Bryant D.
      • Bell D.
      • van Heijenoort J.
      • Gutmann L.
      ,
      • Schleifer K.H.
      • Kandler O.
      ). Thed,d-transpeptidases of E. faeciumthus catalyze the formation of d-Ala4d-Asx-l-Lys3 cross-links after release of the C-terminal d-Ala5 of the donor peptide stem (Fig. 1 A).
      Figure thumbnail gr1
      Figure 1Cross-links generated byd,d-transpeptidases (A) andl,d-transpeptidases (B). d,d-Transpeptidases cleave thed-Ala4-d-Ala5 peptide bond of the donor and forms a d-Ala4d-Asx-l-Lys3 cross-link (boxed). l,d-Transpeptidases cleave the l-Lys3-d-Ala4 bond and forms a l-Lys3d-Asx-l-Lys3 cross-link (boxed). R, glycan strands made of alternating GlcNAc and MurNAc residues.
      β-Lactam antibiotics, which are structural analogues of the C-terminald-Ala4-d-Ala5 end of the peptide stem, act as suicide substrates of thed,d-transpeptidases in an acylation reaction (
      • Ghuysen J.M.
      ). Because transpeptidation is essential to the integrity of the cell wall, these enzymes are the killing target of β-lactams (
      • Ghuysen J.M.
      • Shockman G.D.
      ).d,d-Transpeptidases are multimodular enzymes that combine a C-terminal penicillin-binding domain to an N-terminal glycosyltransferase (class A) or morphogenic (class B) domain (
      • Goffin C.
      • Ghuysen J.M.
      ). Penicillin binding-proteins (PBPs) also include monomodular enzymes with d,d-carboxypeptidase ord,d-endopeptidase activity (
      • Goffin C.
      • Ghuysen J.M.
      ). Among thed,d-transpeptidases of E. faecium, low-affinity PBP5 (class B) is responsible for intrinsic low-level β-lactam resistance. In clinical isolates, acquired high-level resistance to these antibiotics is generally associated with increased production of PBP5 or with amino acid substitutions near the conserved motifs of this protein (
      • Fontana R.
      • Cerini R.
      • Longoni P.
      • Grossato A.
      • Canepari P.
      ,
      • Williamson R., Le
      • Bouguénec C.
      • Gutmann L.
      • Horaud T.
      ,
      • Klare I.
      • Rodloff A.C.
      • Wagner J.
      • Witte W.
      • Hakenbeck R.
      ,
      • Zorzi W.
      • Zhou X.Y.
      • Dardenne O.
      • Lamotte J.
      • Raze D.
      • Pierre J.
      • Gutmann L.
      • Coyette J.
      ,
      • Rybkine T.
      • Mainardi J.L.
      • Sougakoff W.
      • Collatz E.
      • Gutmann L.
      ). Recently, we searched for other resistance mechanisms and obtained after five selection steps a highly ampicillin-resistant mutant, designated D344M512, or briefly M512, from the hypersusceptible E. faecium D344S that does not harbor the pbp5 gene. Analysis of the peptidoglycan structure by reverse-phase HPLC (RP-HPLC) coupled to mass spectrometry revealed substitution ofd-Ala4d-Asx-l-Lys3 cross-links (Fig.1 A) by l-Lys3d-Asx-l-Lys3 cross-links (Fig.1 B) establishing for the first time thatl,d-transpeptidation could by-pass the essential β-lactam-sensitived,d-transpeptidases (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ).
      The presence of the unusual l-Lys3d-Asx-l-Lys3 cross-links in M512 implies that an l,d-transpeptidase cleaves thel-Lys3-d-Ala4 peptide bound of a donor peptide stem and links the α-carboxyl of itsl-Lys3 to the amino group of thed-Asx residue of an acceptor peptide stem (Fig.1 B). Knowledge of this type of enzyme is limited. InEscherichia coli, cross-links generated byl,d-transpeptidation are present in a minority of the muropeptides (<8%) (
      • Pisabarro A.G., De
      • Pedro M.A.
      • Vázquez D.
      ,
      • Driehuis F.
      • Wouters J.T.M.
      ,
      • Glauner B.
      • Höltje J.V.
      • Schwarz U.
      ,
      • Blasco B.
      • Pisabarro A.G.
      • De Pedro M.A.
      ,
      • Höltje J.V.
      • Glauner B.
      ,
      • Höltje J.V.
      ) but the enzyme responsible for their formation has not been studied. Anl,d-transpeptidase activity was detected in crude membrane preparations of Enterococcus hirae (
      • Coyette J.
      • Perkins H.R.
      • Polacheck I.
      • Shockman G.D.
      • Ghuysen J.M.
      ). This enzyme catalyzed in vitro the exchange of the C-terminal d-Ala residue of the dipeptideN α,N ε-acetyl-l-Lys-d-Ala (Ac2-l-Lys-d-Ala) for radioactive d-Ala and, to a lesser extent, ford-Asp, which is the normal in vivo acceptor residue. Although the peptidoglycan was not analyzed (
      • Coyette J.
      • Perkins H.R.
      • Polacheck I.
      • Shockman G.D.
      • Ghuysen J.M.
      ), thel,d-transpeptidase was thought to form l-Lys3d-Asx-l-Lys3 cross-links in vivo.
      We have now characterized specific cellular and biochemical aspects of the peptidoglycan metabolism of the highly resistant mutant E. faecium M512 and of the four intermediary mutants M1, M2, M3, and M4. This included the identification of ampicillin-resistantd,d-carboxypeptidase andl,d-transpeptidase activities, the HPLC and mass spectrometry analyses of the peptidoglycan and of the cytoplasmic precursor pools, the examination of cells by electron microscopy, and the study of their proneness to autolysis. The contribution of Lys3d-Asx-l-Lys3cross-links to peptidoglycan synthesis was found to increase with the level of ampicillin resistance, although no variation of thel,d-transpeptidase activity was detected. Selection for high-level resistance led to production of a β-lactam-insensitive d,d-carboxypeptidase, indicating that the availability of tetrapeptide donor stems was one of the limiting factors forl,d-transpeptidation.

      EXPERIMENTAL PROCEDURES

      Bacterial Strains and Growth Conditions

      Parental strainE. faecium D344S is highly susceptible to ampicillin and derives from E. faecium D344 (
      • Williamson R., Le
      • Bouguénec C.
      • Gutmann L.
      • Horaud T.
      ) by a spontaneous deletion of pbp5 encoding low-affinity PBP5 (
      • Sifaoui F.
      • Arthur M.
      • Rice L.
      • Gutmann L.
      ). E. faecium M1, M2, M3, M4, and M512 are spontaneous mutants of D344S obtained by five successive selection steps on brain heart infusion (Difco) agar containing increasing concentrations of ampicillin as follows. An inoculum of 4 × 109 colony forming units of D344S was plated on agar containing 2-fold increasing concentrations of ampicillin (0.06–4 μg/ml). Mutants appeared after 72 h of incubation on plates containing 0.06, 0.12, 0.25, and 0.5 μg/ml ampicillin (2–10 colonies per plate, frequency of about 10−8). The selection procedure was repeated for one of these mutants, designated M1, that was obtained on the highest ampicillin concentration (0.5 μg/ml). Second step mutants derived from M1 were observed at the same frequency (about 10−8) up to 1 μg/ml ampicillin. One mutant, M2, growing at the latter concentration, was chosen for further selection steps. Using this approach, mutants M3, M4, and M512 were sequentially obtained from plates containing 2, 4, and 512 μg/ml, respectively. The frequencies were about 10−8, 10−8, and 10−6, at the third, fourth, and fifth selection steps, respectively.

      Susceptibility Tests

      Minimal inhibitory concentrations (MICs) were determined on brain heart infusion agar containing 2-fold dilutions of ampicillin (Bristol-Myers Squibb) (
      • Williamson R., Le
      • Bouguénec C.
      • Gutmann L.
      • Horaud T.
      ). Plates were incubated at 37 °C for 24 h. MICs were reproducible (four independent experiments) and did not vary after a longer incubation (48 h instead of 24 h).

      Analysis of Peptidoglycan Structure

      Peptidoglycan was extracted at 100 °C with SDS (4%) from exponentially growing bacteria (A 650 = 0.7), purified after treatment with Pronase and trypsin, and digested with lysozyme and mutanolysin (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ). The resulting muropeptides were reduced with sodium borohydride and separated by RP-HPLC coupled to mass spectrometry (MS and MS/MS) as previously described (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ,
      • Billot-Klein D.
      • Legrand R.
      • Schott B.
      • van Heijenoort J.
      • Gutmann L.
      ). The linear RP-HPLC gradient (0–100% B) was applied between 5 and 45 min and elution in buffer B was continued for an additional 5 min (buffer A, 0.05% trifluoroacetic acid in water; buffer B, 0.035% trifluoroacetic acid and 20% acetonitrile in water) at a flow rate of 0.5 ml/min.

      Preparation of Cytoplasmic and Membrane Extracts

      Bacteria were grown to an A 650 of 0.7, harvested by centrifugation (4,000 × g for 10 min at 4 °C), and washed twice in 10 mm sodium phosphate (pH 7.0). Bacteria were disrupted with glass beads in a cell disintegrator (The Mickle Laboratory Engineering Co., Gromshall, United Kingdom) for 2 h at 4 °C. The extract was centrifuged (5,000 × g for 10 min at 4 °C) to remove cell debris and the supernatant was ultracentrifuged at 40,000 × g for 30 min at 4 °C. The supernatant was saved (cytoplasmic fraction) and the pellet was washed twice in 10 mm sodium phosphate buffer (pH 7.0) (membrane fraction). The protein contents were determined with the Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as standard.

      Synthesis of the Dipeptide Nα,Nε-Acetyl-l-lysyl-d-alanine (Ac2-l-Lys-d-Ala)

      Boc2-l-Lysp-nitrophenylester (
      • Sandrin E.
      • Boissonnas R.A.
      ) was coupled overnight with 1.1 equivalent of d-Ala-OBn p-toluenesulfonate (Novabiochem, Laüfelfingen, Switzerland) in tetrahydrofuran and in the presence of 1.1 equivalent of triethylamine; after the usual work-up, the protected dipeptide derivative Boc2-l-Lys-d-Ala-OBn was obtained as a yellowish solid (yield, 99%). The Boc groups were removed by acidolysis (trifluoroacetic acid/anisole, 10:1 (v/v)) for 30 min; the resulting compound, l-Lys-d-Ala-OBn.2TFA, appeared as a gum (yield, 88%). Acetylation by acetic anhydride in pyridine (3:10 (v/v)) for 24 h and recrystallization from methanol/ethyl acetate provided Ac2-l-Lys-d-Ala-OBn as white crystals (yield, 77%). The Bn group was removed by catalytic hydrogenolysis (5% Pd/C in methanol/acetic acid/water, 50:1:1 (v/v)) for 3 h and the final compound Ac2-l-Lys-d-Ala was obtained as white crystals from acetone (yield, 84%). It was homogeneous by silica gel thin layer chromatography inn-butanol/pyridine/acetic acid/water, 30:10:3:12 (RF0.24) and in n-butanol/acetic acid/water, 3:1:1 (R F 0.35). Amino acid analysis for Ala was 1.00 and Lys was 1.04.

      Assay of l,d-Transpeptidase Activity

      The activity was determined by quantifying the Ac2-l-Lys-d-[14C]Ala formed by the exchange reaction between nonradioactive Ac2-l-Lys-d-Ala andd-[14C]Ala (
      • Coyette J.
      • Perkins H.R.
      • Polacheck I.
      • Shockman G.D.
      • Ghuysen J.M.
      ) becaused-[14C]Asp was not available. The standard assay (50 μl) contained E. faecium membrane or cytoplasmic extracts (75–500 μg of proteins), Ac2-l-Lys-d-Ala (5 mm),d-[14C]Ala (0.15 mm; 2.0 Gbq/mmol, ICN Pharmaceuticals, Orsay, France), 10 mm sodium cacodylate buffer (pH 6.0), and 0.1% Triton X-100 (v/v). The reaction was allowed to proceed at 37 °C and stopped by boiling the samples for 3 min. After centrifugation (10,000 × g, 2 min), 45 μl of the supernatant was analyzed by RP-HPLC at 25 °C on a μ-Bondapak C18 column (3.9 by 300 mm, Waters) with isocratic elution (0.05% trifluoroacetic acid in water/methanol, 9:1 (v/v)) at a flow rate of 0.5 ml/min. Products were detected by the absorbance at 220 nm and by scintillation with a Radioflow Detector (LB508, PerkinElmer Life Sciences) coupled to the HPLC device.

      Inhibition of l,d-Transpeptidase Activity by Ampicillin

      E. faecium membranes (300 μg of proteins) were preincubated for 20 min with ampicillin at 0, 50, 100, 200, 400, 800, 1600, 3200, and 6400 μg/ml in sodium cacodylate (10 mm, pH 6.0) and Triton X-100 (0.1% v/v) (preincubation volume, 25 μl). Kinetics of the exchange reaction catalyzed by thel,d-transpeptidase was performed by adding 25 μl of a solution containing Ac2-l-Lys-d-Ala (final concentration, 5 mm) andd-[14C]Ala (0.15 mm) in sodium cacodylate (final volume, 50 μl; final ampicillin concentration, 0–3200 μg/ml). Aliquots were taken at 0, 25, 45, and 120 min, boiled at 100 °C for 3 min to stop the reaction, and Ac2-l-Lys-d-[14C]Ala was determined by RP-HPLC as described above. The IC50 was defined as the ampicillin concentration that inhibited the reaction by 50% and was deduced from v i /v o =f[I] plots, where v i andv o are the velocity in the presence and absence of ampicillin, respectively, and [I] the final ampicillin concentration. Preliminary experiments showed that the length of the preincubation in the presence of ampicillin (25, 45, or 120 min) did not affect v i /v o.

      Assay of d,d-Carboxypeptidase Activity

      The activity was assayed by quantifying UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala (UDP-MurNAc-tetrapeptide) formed by hydrolysis of the C-terminald-Ala residue of UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala (UDP-MurNAc-pentapeptide) prepared as previously described (
      • Billot-Klein D.
      • Shlaes D.
      • Bryant D.
      • Bell D.
      • Legrand R.
      • Gutmann L.
      • van Heijenoort J.
      ). The assay was performed at 37 °C in a 100-μl mixture containing membrane or cytoplasmic extracts (10–60 μg of proteins), UDP-MurNAc-pentapeptide (1.1 mm), Tris-HCl (50 mm, pH 7.0), and MgCl2 (1 mm). The reaction was stopped by precipitating the proteins with sulfosalicylic acid (0.25 mg). After centrifugation (10,000 × g, 3 min), 90 μl of the supernatant was analyzed by RP-HPLC. UDP-MurNAc-pentapeptide was separated from UDP-MurNAc-tetrapeptide using isocratic elution (50 mm ammonium formate, pH 5.0) at a flow rate of 2 ml/min on a μ-Bondapak C18 column (7.8 × 300 mm, Waters). The products were detected by the absorbance at 262 nm.

      Preparation and Analysis of the Peptidoglycan Precursors Pools

      After growth to an A 650 of 0.7, vancomycin was added to a final concentration of 100 μg/ml to block transglycosylation, and incubation was continued for 30 min. Peptidoglycan precursors were extracted with diluted formic acid (
      • Billot-Klein D.
      • Gutmann L.
      • Collatz E.
      • van Heijenoort J.
      ) and analyzed by RP-HPLC as described above.

      Expression of the vanY Gene in the Strains of E. faecium

      The expression vector pNJ3 (to be described elsewhere) carries a promoter active in enterococci (P 2), two origins of replication active in Gram-negative (oriR pUC) and positive (oriR pAMβ1) bacteria, a gentamicin resistance marker, and the origin of transfer of transposon Tn916. Plasmid pJC1 was constructed by inserting the 1.1-kb fragment of transposon Tn1546 into pNJ3 to place thevanY d,d-carboxypeptidase gene (
      • Arthur M.
      • Depardieu F.
      • Cabanie L.
      • Reynolds P.
      • Courvalin P.
      ) under control of the heterologous promoter P2. Plasmid pJC1(P2 vanY) was introduced by electroporation into JH2–2::Tn916 and subsequently transferred by conjugation to E. faeciumD344S and M3.

      Analysis of PBPs

      PBPs present in E. faecium membrane preparations were labeled with [phenyl-4(n)-3H]benzylpenicillin (2 μg/ml, 777 Gbq/mmol, Amersham Biosciences) as previously described (
      • Williamson R., Le
      • Bouguénec C.
      • Gutmann L.
      • Horaud T.
      ). Competition assays were performed with ampicillin at 0.06, 0.25, 1, and 4 μg/ml (
      • Mainardi J.L.
      • Gutmann L.
      • Acar J.F.
      • Goldstein F.W.
      ).

      Autolysis Assay

      Bacteria from exponential (A 650 = 0.7) and stationary phases were harvested by centrifugation (4,000 × g for 10 min at 4 °C), washed three times with ice-cold distilled water, and incubated at 37 °C in 0.3 m sodium phosphate (pH 7.0) (
      • Cornett J.B.
      • Redman B.E.
      • Shockman G.D.
      ). Turbidity was monitored at 650 nm for 36 h.

      Detection of Autolysins

      Autolytic enzymes were detected according to the method of Beliveau et al. (
      • Beliveau C.
      • Potvin C.
      • Trudel J.
      • Asselin A.
      • Bellmare G.
      ). Bacteria were grown to an A 650 of 0.6 in 10 ml of brain heart infusion broth, washed with distilled water, resuspended in 300 μl of phosphate buffer (50 mm, pH 7.0), treated with 20 μg of mutanolysin and 40 μg of lysozyme, and resuspended in 500 μl of denaturing buffer (2% dithiothreitol, 15% sucrose, 3.8% SDS, w/v). Samples were boiled for 3 min and 60 μl were applied to a SDS-polyacrylamide gel containing 1 mg/ml dry heat-inactivated E. faecium cells. Renaturation of lytic enzymes was obtained by overnight incubation at 37 °C with gentle shaking in 25 mm Tris-HCl (pH 8.0) containing 1% (v/v) Triton X-100.

      Electron Microscopy

      Bacteria were grown to exponential phase in the absence of ampicillin, harvested at a sameA 650 of 0.7, fixed, and stained with 1% uranyl acetate as previously described (
      • Londono-Vallejo J.A.
      • Fréhel C.
      • Stragier P.
      ). Ultrathin sections were contrasted with lead nitrate (
      • Londono-Vallejo J.A.
      • Fréhel C.
      • Stragier P.
      ).

      RESULTS

      Muropeptide Composition of the Peptidoglycans from E. faecium D344S, M1, M2, M3, M4, and M512

      We previously reported identification and quantitative comparison of 34 muropeptides from D344S and M512 by RP-HPLC, MS, and MS-MS (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ). In the present paper, this analysis was extended to the four intermediary mutants (M1, M2, M3, and M4) and to the comparison of the muropeptide composition of peptidoglycan from bacteria grown in the presence or absence of ampicillin. The profiles of monomers were almost identical for the mutants and the parental strain. Variations in the relative proportions of dimer muropeptides generated byd,d-transpeptidation (d-Ala4d-Asx-l-Lys3) versus l,d-transpeptidation (l-Lys3d-Asx-l-Lys3) was the main difference between the muropeptide profiles. For the sake of simplicity, Table I indicates the proportions of muropeptides 13, E, and G that were the most abundant dimers. Muropeptide 13 was the major dimer generated byd,d-transpeptidation and contained a donor tetrapeptide stem and an acceptor tripeptide stem with a d-asparagine branched on the ε-amino group of both lysine residues (Asn-tetra-Asn-tri). Peaks E (Asn-tri-Asn-tri) and G (Asn-tri-Asn-tetra) were the major dimers generated byl,d-transpeptidation.
      Table IProportions (%) of muropeptides 13, E and G in E. faecium strains grown in the presence of various concentrations of ampicillin
      Strains (MIC of ampicillin in μg/ml)Concentration of ampicillin in the culture mediumMuropeptides
      13
      Disaccharide-asparagine-tetrapeptide-asparagine-tripeptide-disaccharide.
      E
      Disaccharide-asparagine-tripeptide-asparagine-tripeptide-disaccharide.
      G
      Disaccharide-asparagine-tripeptide-asparagine-tetrapeptide-disaccharide.
      μg/ml
      D344S (0.06)096.91.21.9
      0.0375.011.513.5
      M1 (0.5)088.68.43.0
      0.1236.034.030.0
      M2 (1)088.19.22.7
      0.2533.034.033.0
      M3 (2)088.39.42.3
      0.527.440.432.2
      M4 (128)071.319.79.0
      0.518.050.032.0
      M512 (>2000)028.542.928.6
      0.56.650.542.9
      32.0
      These conditions reproduce those reported in our previous study (14) that were mistakenly described as growth in the absence of antibiotic since the carry over of the antibiotic from the preculture was not taken into account.
      0.069.430.6
      10000.076.024.0
      Purified peptidoglycan was digested with lysozyme and mutanolysin. After sodium borohydride reduction, muropeptides were identified by RP-HPLC coupled to MS/MS. Values are percentages of the sum of the three prevalent dimers generated by d,d-transpeptidation (muropeptide 13) or l,d-transpeptidation (muropeptides E and G).
      1-a Disaccharide-asparagine-tetrapeptide-asparagine-tripeptide-disaccharide.
      1-b Disaccharide-asparagine-tripeptide-asparagine-tripeptide-disaccharide.
      1-c Disaccharide-asparagine-tripeptide-asparagine-tetrapeptide-disaccharide.
      1-d These conditions reproduce those reported in our previous study (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ) that were mistakenly described as growth in the absence of antibiotic since the carry over of the antibiotic from the preculture was not taken into account.
      In the absence of ampicillin (Table I), muropeptides E and G generated by l,d-transpeptidation were detected in small amounts (3.1%) in D344S, indicating that this mode of transpeptidation was pre-existing in the parental strain. Stepwise increases in the proportion of muropeptides E and G and in the MICs of ampicillin were only detected for the 1st (M1), 4th (M4), and 5th (M512) selection steps. Specifically, the proportion of muropeptides E and G increased from 3.1 (D344S) to 11.4% (M1) at the 1st step, from 11.7 (M3) to 28.7% (M4) at the 4th step, and from 28.7 (M4) to 71.5% (M512) at the 5th step. Selection led to parallel large increases of the ampicillin MICs at each of these steps (8-, 64-, and ≥32-fold, respectively). In contrast, marginal increases of the MICs (2-fold) were observed for the 2nd and 3rd selection steps and the muropeptide composition of mutants M1, M2, and M3 were similar. These observations indicate that three of the five selection steps led to increasedl,d-transpeptidation to the detriment ofd,d-transpeptidation. Activation of thel,d-transpeptidation pathway at these steps was associated with large increases in the ampicillin-resistance level.
      The peptidoglycan structure analysis was repeated for the strains grown in the presence of subinhibitory concentrations of ampicillin to test the effect of PBP inhibition (Table I). For D344S, M1, M2, and M3, the concentration of ampicillin added to culture medium corresponded to the maximum concentration allowing growth. Partial inhibition of thed,d-transpeptidases by ampicillin increased the proportion of dimers generated byl,d-transpeptidation in D344S (from 3.1 to 25%, 8-fold) and in mutants M1, M2, and M3 (6-fold). However, the PBPs remained essential targets in these mutants because higher concentrations of ampicillin inhibited growth. For M4 and M512, thed,d-transpeptidation pathway was almost completely inhibited by ampicillin at 0.5 μg/ml, which corresponds to the subinhibitory concentration tested for M3. Under these conditions, and in contrast to M3, the proportion of muropeptides generated byl,d-transpeptidation reached 82.0 and 93.4% for M4 and M512, respectively. No muropeptides generated byd,d-transpeptidation were detected in the peptidoglycan of M512 grown in the presence of 32 or 1000 μg/ml ampicillin.

      l,d-Transpeptidase Activity

      Dipeptide Ac2-l-Lys-d-Ala was synthesized as described under “Experimental Procedures” to detectl,d-transpeptidase activity based on the exchange reaction (Ac2-l-Lys-d-Ala + d-[14C]Ala ↔ Ac2-l-Lys-d-[14C]Ala + d-Ala) (
      • Coyette J.
      • Perkins H.R.
      • Polacheck I.
      • Shockman G.D.
      • Ghuysen J.M.
      ). l,d-Transpeptidase activity was detected in membrane preparations of D344S (Fig.2), which contained minor amounts of dimers with a l-Lys3d-Asx-l-Lys3 cross-link in its peptidoglycan. This activity was similar for D344S and M512 (23 ± 4 and 32 ± 6 pmol/min/mg of protein, respectively). The concentrations of ampicillin required to inhibit thel,d-transpeptidase activity by 50% (IC50) were also similar for D344S and M512 (about 105 and 110 μg/ml, respectively). Residual activity (about 15–25%) was detected at 1600 and 3200 μg/ml for both strains. Thus, increased synthesis of l-Lys3d-Asx-l-Lys3 cross-links in mutant M512 was not associated with increasedl,d-transpeptidase activity. Neither thel,d-transpeptidase produced by susceptible strain D344S nor that of M512 were inhibited by low concentrations of ampicillin and the IC50 of the antibiotic were similar for the two enzyme preparations.
      Figure thumbnail gr2
      Figure 2l,d-Transpeptidase activity. Membrane preparations were incubated for various time periods with Ac2-l-Lys-d-Ala (5 mm) and d-[14C]Ala (0.15 mm; 2.0 Gbq/mmol). d-[14C]Ala (A) was separated from Ac2-l-Lys-d-[14C]Ala (B) by RP-HPLC. The assay contained the same protein concentration (6 mg/ml) for D344S and M512. Thel,d-transpeptidase activity was mainly present in the membrane fractions, and only low levels were detected in the cytoplasmic fractions (data not shown).

      d,d-Carboxypeptidase Activity

      In the presence of ampicillin (20 μg/ml), d,d-carboxypeptidase activity was detected only in membrane preparations from M4 and M512 (Table II). This enzyme was not inhibited by ampicillin at 2000 μg/ml (data not shown). Membrane preparations from D344S, M1, M2, and M3 contained a 10-fold lowerd,d-carboxypeptidase activity that was totally (>95%) inhibited by ampicillin at 20 μg/ml. These results indicate that the fourth selection step, which generated mutant M4, resulted in high-level production of a β-lactam-insensitived,d-carboxypeptidase in addition to thed,d-carboxypeptidase activity of the putative monofunctional PBPs.
      Table IId,d-Carboxypeptidase activity in membrane preparations
      StrainsActivity (pmol/min/mg protein)
      Without ampicillinWith ampicillin
      20 μg/ml
      D344S42 ± 23<1
      M119 ± 8<1
      M220 ± 5<1
      M325 ± 15<1
      M4250 ± 20210 ± 21
      M512340 ± 67290 ± 72
      Membrane preparations were incubated with UDP-MurNAc-pentapeptide (1.1 mm) in the presence or absence of ampicillin at 20 μg/ml. UDP-MurNAc-tetrapeptide was separated from UDP-MurNAc-pentapeptide by RP-HPLC. The β-lactam sensitive and insensitive activities were mainly present in the membrane fractions, and only low levels were detected in the cytoplasmic fractions (data not shown).
      Low amounts (<10%) of UDP-MurNAc-tetrapeptide were detected in the cytoplasm of D344S and M3 (Table III). In contrast, UDP-MurNAc-tetrapeptide accounted for about half of the cytoplasmic peptidoglycan precursors of M4 (49.5%) and M512 (59.0%). Thus, the β-lactam-insensitived,d-carboxypeptidase, specifically detected in these mutants, generated tetrapeptide stems in vivo.
      Table IIIPools of cytoplasmic peptidoglycan precursors
      StrainsUDP-MurNAc-peptide (%)
      Calculated as percentage of the sum of the peak areas of UDP-MurNAc-pentapeptide and UDP-MurNAc-tetrapeptide.
      TetrapeptidePentapeptide
      D344S8.691.4
      M34.795.3
      M449.550.5
      M51259.041.0
      Cytoplasmic precursors were extracted from enterococcal cells grown to an A 650 of 0.7 and treated with vancomycin (100 μg/ml) for 30 min to accumulate the precursors.
      3-a Calculated as percentage of the sum of the peak areas of UDP-MurNAc-pentapeptide and UDP-MurNAc-tetrapeptide.

      Expression of a Heterologous d,d-Carboxypeptidase Gene in E. faecium D344S and M3

      The vanY gene encodes a metallo-d,d-carboxypeptidase-insensitive to β-lactam inhibition and was originally detected in transposon Tn1546 that confers glycopeptide resistance in enterococci (
      • Arthur M.
      • Depardieu F.
      • Cabanie L.
      • Reynolds P.
      • Courvalin P.
      ). Plasmid pJC1 was constructed by inserting the vanYgene under the control of the P 2 promoter of the shuttle vector pNJ3 to test the influence of elevatedd,d-carboxypeptidase activity on β-lactam resistance. Introduction of pJC1(P 2 vanY) into mutant M3 led to an increase in the MIC of ampicillin (from 2 to 256 μg/ml). A similar increase in the MIC (from 2 to 128 μg/ml) was obtained at the fourth selection step that generated M4 from M3 (TableI). The vector alone had no effect on the level of resistance. Thus, elevated d,d-carboxypeptidase activity was responsible for increased resistance observed at the fourth selection step.
      Plasmid pJC1(P 2 vanY) did not increase the MIC of ampicillin in D344S. Selection of ampicillin-resistant mutants from D344S/pJC1(P 2 vanY) resulted in a highly resistant mutant in three steps (MIC, 64 μg/ml) instead of the four steps necessary to obtain M4 (MIC, 128 μg/ml) from D344S. These observations suggest that production of VanY did not by-pass the requirement for mutations selected at the 1st, 2nd, and 3rd selection steps.

      Binding of β-Lactams to PBPs

      PBP labeling with benzylpenicillin revealed similar SDS-PAGE patterns for D344S and M512, indicating that their level of production was not modified (data not shown). Based on the competition assay, ampicillin at 4 μg/ml saturated all PBPs of D344S and M512. Partial saturation at lower drug concentrations showed that the affinity of the PBPs were not modified in the highly resistant mutant M512.

      Cell Autolysis and Detection of Autolysins

      Autolysis of D344S cells collected from exponential and stationary phases led to a 50% decrease of the A 650 after 9 and 24 h of incubation at 37 °C in phosphate buffer, respectively. In contrast, no significant autolysis of M512 was observed after 36 h. Crude extracts from D344S and M512 contained two major autolysins active on heat-killed E. faecium D344S (data not shown). These enzymes were not active on heat-killed M512 cells. These observations suggested that the mutant M512 was not prone to autolysis because its peptidoglycan containing cross-links generated byl,d-transpeptidation was no longer well recognized by the major E. faecium autolysins.

      Growth Rate and Abnormal Morphology

      The stepwise increase in ampicillin resistance was accompanied by a decrease in growth rate. In the absence of ampicillin in the culture medium, the generation time was 27, 48, 60, 72, 84, and 90 min for D344S, M1, M2, M3, M4, and M512, respectively. For M512, the generation time increased from 90 to 180 min when the ampicillin concentration increased from 750 to 4000 μg/ml. In comparison with parental strain D344S (Fig3 a), electron microscopy of thin sections of M1 and M2 grown in antibiotic-free medium showed no difference in morphology (data not shown). For M3, thickening of the cell wall and abnormalities of the septa were present in about 30% of the cells (Fig. 3 b). For M4 and M512, severe abnormalities were observed in more than 80% of the cells, including cell wall thickening and formation of cell aggregates resulting from disordered septation (Fig. 3, c and d).
      Figure thumbnail gr3
      Figure 3Electron microscopy of thin sections of parental strain D344S and the ampicillin-resistant mutants. D344S cells displayed regular coccoidal shape, symmetric septation, and a thin cell wall (a). Mutants M3 (b), M4 (c), and M512 (d) showed increasing numbers of cells with ultrastructural abnormalities, including formation of cell aggregates, anarchic septation, and cell wall thickening especially at the septum level (arrow). Bar = 0.5 μm.

      DISCUSSION

      In this report, we show that the level of ampicillin resistance in the mutants derived from E. faecium D344S is determined by a balance between the d,d- andl,d-transpeptidation pathways for peptidoglycan cross-linking (Table I). The 1st, 4th, and 5th selection steps that led to mutants M1, M4, and M512, respectively, resulted in large increases in the proportion of muropeptides containing thel-Lys3d-Asx-Lys3cross-links generated by l,d-transpeptidation. Large increases of the ampicillin MICs were observed at these steps.
      Partial inhibition of the d,d-transpeptidases by ampicillin (less or equal to 0.5 μg/ml) increased the proportion of muropeptides with an l-Lys3d-Asx-Lys3 cross-link in all strains. Higher drug concentrations inhibited growth of D344S, M1, M2, and M3, indicating that at least one PBP remained essential in these strains. In contrast, the PBPs were not contributing to peptidoglycan cross-linking in M512 as growth in the presence of a saturating concentration of ampicillin (32 or 1000 μg/ml) led to exclusive synthesis of l-Lys3d-Asx-Lys3 cross-links. In the absence of ampicillin, the two types of cross-links were detected, indicating that the l,d- andd,d-transpeptidation pathways were functional in all strains although their relative contribution varied. Several factors that could affect this balance were examined, revealing that activation of the resistance pathway was not associated with any variation of the l,d-transpeptidase activity or of the PBPs, but to production of ad,d-carboxypeptidase that generated the tetrapeptide donor stems forl,d-transpeptidation.
      The l,d-transpeptidase activity measured by the exchange reaction was similar in D344S and M512 (Fig. 2), indicating that increased production of the enzyme was not required for by-pass of the d,d-transpeptidases. Thel,d-transpeptidase activity was only partially inhibited by high concentrations of ampicillin. The drug concentrations required for 50% inhibition were also similar for the enzyme present in membrane preparations from D344S and M512 (IC50 of about 105 and 110 μg/ml, respectively). Thus, emergence of high-level resistance to ampicillin did not appear to involve any alteration of the l,d-transpeptidase. The following observations may account for the apparent discrepancy between the IC50 (110 μg/ml) and the MIC (>2,000 μg/ml) observed for M512. The generation time of this strain increased from 90 to 180 min when the ampicillin concentration increased from 750 to 4,000 μg/ml, indicating that the drug significantly slowed growth despite the high MIC. A residual l,d-transpeptidase activity was detected in vitro at ampicillin concentrations of 1600 (25%) and 3,200 μg/ml (15%) in membrane preparations of M512. This residual activity could be sufficient to sustain bacterial growth in M512 although at a lower rate. As previously discussed (
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ,
      • Coyette J.
      • Perkins H.R.
      • Polacheck I.
      • Shockman G.D.
      • Ghuysen J.M.
      ,
      • Templin M.F.
      • Ursinus A.
      • Höltje J.V.
      ), the d,d-transpeptidase andl,d-transpeptidase are not expected to be inhibited by β-lactams by the same mechanism. The drugs are suicide substrates of the d,d-transpeptidases because the β-lactam ring is structurally related to thed-Ala-d-Ala moiety of peptidoglycan precursors (
      • Tipper D.J.
      • Strominger J.L.
      ). The l,d-transpeptidases, which cleave thel-Lys-d-Ala rather thand-Ala-d-Ala peptide bond, are therefore not expected to be acylated by the same mechanism.
      Penicillin-binding studies did not reveal any modification of the PBP patterns in M512 (data not shown). This observation implies that in this strain the resistance pathway is dominant as thed,d-transpeptidase appeared to remain functional based on β-lactam acylation. Competition experiments indicated that the acylation of PBPs by ampicillin was not affected by the five-step selection for resistance to this antibiotic because all PBPs were saturated by low concentrations of ampicillin (<4 μg/ml). This observation implies that the β-lactam-sensitived,d-transpeptidase andd,d-carboxypeptidase activities of the PBPs were not playing any essential role in the high level of resistance expressed by M512 (MIC > 2000 μg/ml). In Staphylococcus aureus, methicillin resistance mediated by class B PBP2A requires the glycosyltransferase activity of class A PBP2 (
      • Pinho M.G.
      • de Lencastre H.
      • Tomasz A.
      ). Site-directed mutagenesis of the active-site serine residue of PBP2 showed that the glycosyltransferase module of this protein can function in the absence of its catalytically active d,d-transpeptidase module (
      • Pinho M.G.
      • de Lencastre H.
      • Tomasz A.
      ). In our system, peptidoglycan synthesis may similarly involve cooperation between thel,d-transpeptidase and the glycosyltransferase module of a class A PBP.
      The fourth selection step that generated mutant M4 led to production of a β-lactam-insensitive d,d-carboxypeptidase (Table II) that was active in vivo as shown by the accumulation of UDP-MurNAc-tetrapeptide in the cytoplasm of M4 and M512 (Table III). Selection may have activated a cryptic gene encoding a metallo-d,d-carboxypeptidase because this type of enzyme is not inhibited by β-lactams (
      • Arthur M.
      • Depardieu F.
      • Cabanie L.
      • Reynolds P.
      • Courvalin P.
      ,
      • Wright G.D.
      • Molinas C.
      • Arthur M.
      • Courvalin P.
      • Walsh C.T.
      ,
      • Kimura Y.
      • Takashima Y.
      • Tokumasu Y.
      • Sato M.
      ) and no modification of the patterns of the PBP was observed. In agreement, production of the heterologousd,d-carboxypeptidase VanY increased the level of ampicillin resistance in mutant M3. This effect was not observed in D344S, suggesting that elevatedd,d-carboxypeptidase activity can only increase the level of resistance after modification of other unknown function(s). High-level accumulation of UDP-MurNAc-tetrapeptide (TableIII) suggests that the enzyme hydrolyzes the C-terminald-Ala residue of UDP-MurNAc-pentapeptide in vivo. However, because formation of lipid intermediate I is reversible (
      • Van Heijenoort J.
      ) and translocation of this intermediate may also be reversible (
      • Reynolds P.
      • Ambur O.H.
      • Casadewall B.
      • Courvalin P.
      ), hydrolysis of pentapeptide stems at the outer surface of the cytoplasmic membrane cannot be excluded, as previously discussed for a d,d-carboxypeptidase involved in glycopeptide resistance (
      • Reynolds P.
      • Ambur O.H.
      • Casadewall B.
      • Courvalin P.
      ).
      Taking in account all these observations, production of the β-lactam-insensitive d,d-carboxypeptidase is expected to increase synthesis of l-Lys3d-Asx-Lys3 to the detriment ofd-Ala4d-Asx-Lys3cross-links by two mechanisms (Fig. 4). First, hydrolysis of the C-terminal d-Ala5reduces d,d-transpeptidation because the PBPs require a pentapeptide donor stem. Second, thed,d-carboxypeptidase by generating the tetrapeptide stem donor substrate acts as a new source to increasel,d-transpeptidation. The large increase in the MIC of ampicillin observed at the fourth selection step indicates that the availability of tetrapeptide stems is one of the limiting factors for activation of the resistance pathway. In low-level resistant mutants, the majority of the pentapeptide monomers were used in thed-Ala4d-Asx-Lys3cross-link by the d,d-transpeptidases. Because only small amounts of tetrapeptide stems were generated by thed,d-carboxypeptidase activity of monofunctional PBPs, only a few l-Lys3d-Asx-Lys3 cross-links were formed. Because these latter PBPs were inhibited by ampicillin, thel,d-transpeptidation pathway remains also susceptible to the drug in the absence of another source of tetrapeptide substrate (Table II and Fig. 4).
      Figure thumbnail gr4
      Figure 4Model for activation of thel,d-transpeptidation pathway by production of a β-lactam-insensitived,d-carboxypeptidase. A, in D344S and in the intermediary mutants M1, M2, and M3, d,d-transpeptidation is the major pathway for peptidoglycan cross-linking. Small amounts of tetrapeptide stems were generated by the d,d-carboxypeptidase activity of the monofunctional PBPs and the availability of tetrapeptide donor stems was limiting forl,d-transpeptidation. Ampicillin (Ap) was expected to directly inhibit thed,d-transpeptidases. Ampicillin may also indirectly inhibit the l,d-transpeptidation pathway by blocking the d,d-carboxypeptidase activity of the monofunctional PBPs although thel,d-transpeptidase itself is not a target for this antibiotic. B, mutants M4 and M512 produce a β-lactam-insensitive d,d-carboxypeptidase that activates the l,d-transpeptidation pathway by generating the tetrapeptide donors for thel,d-transpeptidase. The contribution ofd,d-transpeptidation to cross-linking is reduced because the d,d-transpeptidases cannot use tetrapeptide stems as donors. In the presence of ampicillin, this pathway is further reduced.
      By-pass of d,d-transpeptidation is a remarkable example of the flexibility of peptidoglycan synthesis although it was associated with severe defects in peptidoglycan metabolism as shown by increased generation times (up to 4-fold for M512) and cell wall abnormalities (Fig. 3). Impaired activity of the autolysins could be involved in these defects because peptidoglycan generated byl,d-transpeptidation was a poor substrate for hydrolytic enzymes both in vivo and in vitro. Peptidoglycan polymerization is thought to involve multienzyme complexes that include glycosyltransferases,d,d-transpeptidases, and hydrolases (
      • Höltje J.V.
      ). Therefore, it is perhaps not surprising that acquisition of resistance led to impaired growth and required multiple mutations that did not directly affect the catalytic activity of the transpeptidases.

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