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• W2090927759 abstract "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-Ala4 →d-Asx-Lys3) by cross-links resulting froml,d-transpeptidation (l-Lys3 →d-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-Lys3→ d-Asx-Lys3 cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for thel,d-transpeptidase. 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-Ala4 →d-Asx-Lys3) by cross-links resulting froml,d-transpeptidation (l-Lys3 →d-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-Lys3→ d-Asx-Lys3 cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for thel,d-transpeptidase. N-acetylglucosamine N-acetylmuramic acid penicillin-binding protein minimal inhibitory concentration reverse-phase high pressure liquid chromatography mass spectrometry t-butyloxycarbonyl 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 (1Van Heijenoort J. Nat. Prod. Rep. 2001; 18: 503-519Crossref PubMed Scopus (358) Google Scholar). 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) (2Billot-Klein D. Shlaes D. Bryant D. Bell D. Legrand R. Gutmann L. van Heijenoort J. J. Bacteriol. 1997; 179: 4684-4688Crossref PubMed Google Scholar, 3De Jonge B.L.M. Gage D. Handwerger S. Microb. Drug Resist. 1996; 2: 225-229Crossref PubMed Scopus (15) Google Scholar, 4Billot-Klein D. Shlaes D. Bryant D. Bell D. van Heijenoort J. Gutmann L. Biochem. J. 1996; 313: 711-715Crossref PubMed Scopus (31) Google Scholar). 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 (3De Jonge B.L.M. Gage D. Handwerger S. Microb. Drug Resist. 1996; 2: 225-229Crossref PubMed Scopus (15) Google Scholar, 4Billot-Klein D. Shlaes D. Bryant D. Bell D. van Heijenoort J. Gutmann L. Biochem. J. 1996; 313: 711-715Crossref PubMed Scopus (31) Google Scholar, 5Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar). Thed,d-transpeptidases of E. faeciumthus catalyze the formation of d-Ala4 →d-Asx-l-Lys3 cross-links after release of the C-terminal d-Ala5 of the donor peptide stem (Fig. 1 A). 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 (6Ghuysen J.M. Trends Microbiol. 1994; 2: 372-380Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Because transpeptidation is essential to the integrity of the cell wall, these enzymes are the killing target of β-lactams (7Ghuysen J.M. Shockman G.D. Lieve L. Bacterial Membranes and Wall. Marcel Dekker, New York1973: 37-130Google Scholar).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 (8Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar). Penicillin binding-proteins (PBPs) also include monomodular enzymes with d,d-carboxypeptidase ord,d-endopeptidase activity (8Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar). 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 (9Fontana R. Cerini R. Longoni P. Grossato A. Canepari P. J. Bacteriol. 1983; 155: 1343-1350Crossref PubMed Google Scholar, 10Williamson R., Le Bouguénec C. Gutmann L. Horaud T. J. Gen. Microbiol. 1985; 131: 1933-1940PubMed Google Scholar, 11Klare I. Rodloff A.C. Wagner J. Witte W. Hakenbeck R. Antimicrob. Agents Chemother. 1992; 36: 783-787Crossref PubMed Scopus (43) Google Scholar, 12Zorzi W. Zhou X.Y. Dardenne O. Lamotte J. Raze D. Pierre J. Gutmann L. Coyette J. J. Bacteriol. 1996; 178: 4948-4957Crossref PubMed Google Scholar, 13Rybkine T. Mainardi J.L. Sougakoff W. Collatz E. Gutmann L. J. Infect. Dis. 1998; 178: 159-163Crossref PubMed Scopus (97) Google Scholar). 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-Ala4 →d-Asx-l-Lys3 cross-links (Fig.1 A) by l-Lys3 →d-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 (14Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The presence of the unusual l-Lys3 →d-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%) (15Pisabarro A.G., De Pedro M.A. Vázquez D. J. Bacteriol. 1985; 161: 238-242Crossref PubMed Google Scholar, 16Driehuis F. Wouters J.T.M. J. Bacteriol. 1987; 169: 97-101Crossref PubMed Google Scholar, 17Glauner B. Höltje J.V. Schwarz U. J. Biol. Chem. 1988; 263: 10088-10095Abstract Full Text PDF PubMed Google Scholar, 18Blasco B. Pisabarro A.G. De Pedro M.A. J. Bacteriol. 1988; 170: 5224-5228Crossref PubMed Google Scholar, 19Höltje J.V. Glauner B. Res. Microbiol. 1990; 141: 75-89Crossref PubMed Scopus (35) Google Scholar, 20Höltje J.V. Microbiol. Mol. Biol. Rev. 1998; 62: 181-203Crossref PubMed Google Scholar) but the enzyme responsible for their formation has not been studied. Anl,d-transpeptidase activity was detected in crude membrane preparations of Enterococcus hirae (21Coyette J. Perkins H.R. Polacheck I. Shockman G.D. Ghuysen J.M. Eur. J. Biochem. 1974; 44: 459-468Crossref PubMed Scopus (29) Google Scholar). 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 (21Coyette J. Perkins H.R. Polacheck I. Shockman G.D. Ghuysen J.M. Eur. J. Biochem. 1974; 44: 459-468Crossref PubMed Scopus (29) Google Scholar), thel,d-transpeptidase was thought to form l-Lys3 →d-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 Lys3 → d-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. Parental strainE. faecium D344S is highly susceptible to ampicillin and derives from E. faecium D344 (10Williamson R., Le Bouguénec C. Gutmann L. Horaud T. J. Gen. Microbiol. 1985; 131: 1933-1940PubMed Google Scholar) by a spontaneous deletion of pbp5 encoding low-affinity PBP5 (22Sifaoui F. Arthur M. Rice L. Gutmann L. Antimicrob. Agents Chemother. 2001; 45: 2594-2597Crossref PubMed Scopus (70) Google Scholar). 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. Minimal inhibitory concentrations (MICs) were determined on brain heart infusion agar containing 2-fold dilutions of ampicillin (Bristol-Myers Squibb) (10Williamson R., Le Bouguénec C. Gutmann L. Horaud T. J. Gen. Microbiol. 1985; 131: 1933-1940PubMed Google Scholar). 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). 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 (14Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The resulting muropeptides were reduced with sodium borohydride and separated by RP-HPLC coupled to mass spectrometry (MS and MS/MS) as previously described (14Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 23Billot-Klein D. Legrand R. Schott B. van Heijenoort J. Gutmann L. J. Bacteriol. 1997; 179: 6208-6212Crossref PubMed Google Scholar). 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. 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. Boc2-l-Lysp-nitrophenylester (24Sandrin E. Boissonnas R.A. Helv. Chim. Acta. 1963; 46: 1637-1669Crossref Scopus (71) Google Scholar) 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. 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 (21Coyette J. Perkins H.R. Polacheck I. Shockman G.D. Ghuysen J.M. Eur. J. Biochem. 1974; 44: 459-468Crossref PubMed Scopus (29) Google Scholar) 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. 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. 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 (2Billot-Klein D. Shlaes D. Bryant D. Bell D. Legrand R. Gutmann L. van Heijenoort J. J. Bacteriol. 1997; 179: 4684-4688Crossref PubMed Google Scholar). 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. 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 (25Billot-Klein D. Gutmann L. Collatz E. van Heijenoort J. Antimicrob. Agents Chemother. 1992; 36: 1487-1490Crossref PubMed Scopus (34) Google Scholar) and analyzed by RP-HPLC as described above. 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 (26Arthur M. Depardieu F. Cabanie L. Reynolds P. Courvalin P. Mol. Microbiol. 1998; 30: 819-830Crossref PubMed Scopus (90) Google Scholar) 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. 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 (10Williamson R., Le Bouguénec C. Gutmann L. Horaud T. J. Gen. Microbiol. 1985; 131: 1933-1940PubMed Google Scholar). Competition assays were performed with ampicillin at 0.06, 0.25, 1, and 4 μg/ml (27Mainardi J.L. Gutmann L. Acar J.F. Goldstein F.W. Antimicrob. Agents Chemother. 1995; 39: 1984-1987Crossref PubMed Scopus (110) Google Scholar). 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) (28Cornett J.B. Redman B.E. Shockman G.D. J. Bacteriol. 1978; 133: 631-640Crossref PubMed Google Scholar). Turbidity was monitored at 650 nm for 36 h. Autolytic enzymes were detected according to the method of Beliveau et al. (29Beliveau C. Potvin C. Trudel J. Asselin A. Bellmare G. J. Bacteriol. 1991; 173: 5619-5623Crossref PubMed Scopus (66) Google Scholar). 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. 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 (30Londono-Vallejo J.A. Fréhel C. Stragier P. Mol. Microbiol. 1997; 24: 29-39Crossref PubMed Scopus (105) Google Scholar). Ultrathin sections were contrasted with lead nitrate (30Londono-Vallejo J.A. Fréhel C. Stragier P. Mol. Microbiol. 1997; 24: 29-39Crossref PubMed Scopus (105) Google Scholar). We previously reported identification and quantitative comparison of 34 muropeptides from D344S and M512 by RP-HPLC, MS, and MS-MS (14Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 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-Ala4 →d-Asx-l-Lys3) versus l,d-transpeptidation (l-Lys3 →d-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 ampicillinStrains (MIC of ampicillin in μg/ml)Concentration of ampicillin in the culture mediumMuropeptides131-aDisaccharide-asparagine-tetrapeptide-asparagine-tripeptide-disaccharide.E1-bDisaccharide-asparagine-tripeptide-asparagine-tripeptide-disaccharide.G1-cDisaccharide-asparagine-tripeptide-asparagine-tetrapeptide-disaccharide.μg/mlD344S (0.06)096.91.21.90.0375.011.513.5M1 (0.5)088.68.43.00.1236.034.030.0M2 (1)088.19.22.70.2533.034.033.0M3 (2)088.39.42.30.527.440.432.2M4 (128)071.319.79.00.518.050.032.0M512 (>2000)028.542.928.60.56.650.542.932.01-dThese 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.0.069.430.610000.076.024.0Purified 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 (14Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) 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. Open table in a new tab 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). 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. 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) (21Coyette J. Perkins H.R. Polacheck I. Shockman G.D. Ghuysen J.M. Eur. J. Biochem. 1974; 44: 459-468Crossref PubMed Scopus (29) Google Scholar). l,d-Transpeptidase activity was detected in membrane preparations of D344S (Fig.2), which contained minor amounts of dimers with a l-Lys3 →d-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-Lys3 →d-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. 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• W2090927759 title "Balance between Two Transpeptidation Mechanisms Determines the Expression of β-Lactam Resistance in Enterococcus faecium" @default.
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