Synthesis of Mosaic Peptidoglycan Cross-bridges by Hybrid Peptidoglycan Assembly Pathways in Gram-positive Bacteria

The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly5, l-Ala2, and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred β-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs. The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly5, l-Ala2, and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred β-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs. The bacterial cell wall peptidoglycan is a net-like macromolecule that completely surrounds the cytoplasmic membrane and supplies the cell with mechanical protection against its own osmotic pressure (1Höltje J. Microbiol. Mol. Biol. Rev. 1998; 62: 181-203Crossref PubMed Google Scholar). The stress-bearing peptidoglycan is polymerized from a subunit containing β-1,4-linked GlcNAc and N-acetylmuramic acid (MurNAc) 1The abbreviations used are: MurNAc, N-acetylmuramic acid; d-Lac, d-lactate or d-lactoyl; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography; PBP, penicillin-binding protein; BHI, brain heart infusion.1The abbreviations used are: MurNAc, N-acetylmuramic acid; d-Lac, d-lactate or d-lactoyl; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography; PBP, penicillin-binding protein; BHI, brain heart infusion. substituted by a peptide stem (2van Heijenoort J. Nat. Prod. Rep. 2001; 18: 503-519Crossref PubMed Scopus (355) Google Scholar). In the pathogenic Gram-positive bacteria belonging to the genera Enterococcus, Streptococcus, and Staphylococcus, the stem peptide consists of a conserved pentapeptide (l-Ala1-d-iGln2-l-Lys3-d-Ala4-d-Ala5) and a variable side chain (see Fig. 1) (3Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar).Polymerization of the peptidoglycan subunit at the cell surface is performed by glycosyltransferases that catalyze elongation of the glycan strands by formation of β-1,4 bonds and d,d-transpeptidases that cross-link glycan strands (4van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar). The latter reaction is catalyzed by essential high molecular weight penicillin-binding proteins (PBPs) that cleave the C-terminal residue (d-Ala5) of a donor stem peptide and link the carboxyl group of the penultimate residue (d-Ala4) to the side chain amino group of an acceptor stem peptide (5Jamin M. Wilkin J.M. Frère J.-M. Essays Biochem. 1995; 29: 1-24PubMed Google Scholar) (see Fig. 1). This two-step reaction involves formation of a covalent adduct between the β-hydroxyl of the active site serine of the PBPs and the carboxylate of d-Ala4 of the donor stem (acyl-enzyme) (5Jamin M. Wilkin J.M. Frère J.-M. Essays Biochem. 1995; 29: 1-24PubMed Google Scholar). β-Lactam antibiotics are structural analogues of the d-Ala4-d-Ala5 extremity of peptidoglycan precursors that irreversibly inactivate the PBPs in a similar acylation reaction. Most bacterial species produce multiple PBPs that have partially overlapping functions (6Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar). Multimodular PBPs associate a C-terminal d,d-transpeptidase module to N-terminal glycosyltransferase (class A) or non catalytic (class B) modules. Clinically relevant β-lactam resistance phenotypes in staphylococci and enterococci involve production of class B d,d-transpeptidases that are inefficiently acylated by β-lactams (commonly referred to as low affinity PBPs). Methicillin-resistant Staphylococcus aureus has acquired an additional pbp gene (mecA encoding low affinity PBP2a) presumably from a related staphylococcal species (7Wu S.W. de Lencastre H. Tomasz A. J. Bacteriol. 2001; 183: 2417-2424Crossref PubMed Scopus (136) Google Scholar). Resistance is an intrinsic property of Enterococcus faecalis and Enterococcus faecium because virtually all isolates are resistant to moderate (e.g. ampicillin) or high (e.g. ceftriaxone) levels of β-lactams and produce species-specific low affinity PBPs designated PBP5fs and PBP5fm, respectively (8Signoretto C. Boaretti M. Canepari P. FEMS Microbiol. Lett. 1994; 123: 99-106Crossref PubMed Scopus (43) Google Scholar, 9Rice L.B. Carias L.L. Hutton-Thomas R. Sifaoui F. Gutmann L. Rudin S.D. Antimicrob. Agents Chemother. 2001; 45: 1480-1486Crossref PubMed Scopus (74) Google Scholar, 10Sauvage E. Kerff F. Fonzé E. Herman R. Schoot B. Marquette J.P. Taburet Y. Prevost D. Dumas J. Leonard G. Stefanic P. Coyette J. Charlier P. Cell. Mol. Life Sci. 2002; 59: 1223-1232Crossref PubMed Scopus (86) Google Scholar).Diversification of the side chain structure during speciation (see Fig. 1) is potentially associated with diversification of the substrate specificity of the d,d-transpeptidases. In S. aureus, the FmhB transferase for incorporation of the first residue of the pentaglycine side chain (see Fig. 1) is an essential enzyme. This indicates that unsubstituted pentapeptide stems cannot be cross-linked (11Rohrer S. Ehlert K. Tschierske M. Labischinski H. Berger-Bächi B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9351-9356Crossref PubMed Scopus (114) Google Scholar). In addition, femA and femB mutants of methicillin-resistant S. aureus are susceptible to methicillin. This suggests that a complete pentaglycine side chain is essential for the d,d-transpeptidase activity of PBP2a (12Strandén A.M. Ehlert K. Labischinski H. Berger-Bächi B. J. Bacteriol. 1997; 179: 9-16Crossref PubMed Scopus (164) Google Scholar). Similarly, the side chain is essential for penicillin resistance in Streptococcus pneumoniae (13Filipe S.R. Pinho M.G. Tomasz A. J. Biol. Chem. 2000; 275: 27768-27774Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Inactivation of bppA1 encoding the transferase for incorporation of the first residue of the l-Ala-l-Ala side chain in E. faecalis has not been obtained (14Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Deletion of bppA2 led to production of precursors substituted by a single l-Ala and to impaired expression of intrinsic β-lactam resistance (14Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For these reasons, transferases of the Fem family are considered to be potential targets for the development of novel antibiotics active against β-lactam-resistant Gram-positive cocci (15Kopp U. Roos M. Wecke J. Labischinski H. Microb. Drug Resist. 1996; 2: 29-41Crossref PubMed Scopus (77) Google Scholar).In this study, we have further investigated the synthesis of the side chains of peptidoglycan precursors by transferases of the Fem family and their use by the PBPs in the cross-linking reaction. The study was designed to test the hypothesis that a narrow specificity of the PBPs could account for the essential role of transferases of the Fem family in peptidoglycan synthesis and β-lactam resistance. Heterospecific expression of genes encoding transferases and PBPs was used to manipulate the structure of the side chain of the peptidoglycan precursors and the PBPs responsible for their polymerization. We first determined whether heterospecific expression of the S. aureus fmhB, femA, and femB genes in E. faecalis leads to synthesis of mosaic side chains. Mass spectrometry and tandem mass spectrometry (MS/MS) analyses of uncross-linked muropeptide monomers revealed that the Fem transferases of S. aureus were functional in E. faecalis and retained their substrate specificity as displayed in the original host. The participation of the mosaic precursors in the cross-linking reaction was then deduced from the structure of cross-linked muropeptide dimers. Mosaic side chains were detected both at the donor and acceptor positions of the dimers, indicating that the transpeptidases of E. faecalis had a broad substrate specificity. In a second set of experiments, the mecA, pbp5fs, and pbp5fm were heterologously expressed in E. faecalis and E. faecium to evaluate the capacity of the corresponding low affinity PBPs to confer β-lactam resistance. PBP2a and PBP5fm were found to be functional in the heterologous hosts producing precursors with l-Ala-l-Ala and d-Asx side chains, reflecting again a broad substrate specificity. Thus, analysis of the different hybrid peptidoglycan assembly pathways revealed that the high specificity of the enzymes for side chain synthesis accounts for conservation of the structure of the peptidoglycan cross-bridges in staphylococci and enterococci, although the conservation of substrate structure is not essential for the transpeptidase activity of the PBPs.EXPERIMENTAL PROCEDURESBacterial Strains and Growth Conditions—Bacterial strains were grown in brain heart infusion (BHI) broth or agar (Becton Dickinson, le Pont de Claix, France) at 37 °C. Population analysis profiles were performed as previously described (16Tomasz A. Nachman S. Leaf H. Antimicrob. Agents Chemother. 1991; 35: 124-129Crossref PubMed Scopus (232) Google Scholar). Briefly, the bacteria were grown overnight at 37 °C in BHI broth, and serial 10-fold dilutions were plated on BHI agar containing increasing concentrations of ceftriaxone (Roche Applied Science). Colony forming units were determined after 48 h of incubation at 37 °C. To study the stability of ceftriaxone resistance phenotypes, the bacteria were sequentially subcultured for 5 days in 10 ml of BHI broth by using 0.1 ml of the preceding overnight culture as the innoculum.Derivatives of expression vector pNJ2 harboring fem and pbp genes (see below) were introduced into E. faecalis JH2Sm::Tn916 (17Arbeloa A. Segal H. Hugonnet J.-E. Josseaume N. Dubost L. Brouard J.-P. Gutmann L. Mengin-Lecreulx D. Arthur M. J. Bacteriol. 2004; 186: 1221-1228Crossref PubMed Scopus (82) Google Scholar) by electroporation and transferred by conjugation to E. faecalis JH2–2 (18Jacob A.E. Hobbs S.J. J. Bacteriol. 1974; 117: 360-372Crossref PubMed Google Scholar), E. faecalis JH2–2ΔbppA2 (14Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), E. faecalis JH2–2Δpbp5 (17Arbeloa A. Segal H. Hugonnet J.-E. Josseaume N. Dubost L. Brouard J.-P. Gutmann L. Mengin-Lecreulx D. Arthur M. J. Bacteriol. 2004; 186: 1221-1228Crossref PubMed Scopus (82) Google Scholar), E. faecium D344S (19Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and E. faecium BM4107 (20Sifaoui F. Arthur M. Rice L. Gutmann L. Antimicrob. Agents Chemother. 2001; 45: 2594-2597Crossref PubMed Scopus (70) Google Scholar), as previously described (17Arbeloa A. Segal H. Hugonnet J.-E. Josseaume N. Dubost L. Brouard J.-P. Gutmann L. Mengin-Lecreulx D. Arthur M. J. Bacteriol. 2004; 186: 1221-1228Crossref PubMed Scopus (82) Google Scholar). Spectinomycin (60 μg/ml) was used in all experiments to counter select loss of the plasmids.Plasmid Construction—The open reading frame and ribosome-binding site of the fem and mecA genes of S. aureus and of the pbp5fm gene of E. faecium were amplified and cloned under the control of the aph-A-3p promoter of the shuttle expression vector pNJ2 (17Arbeloa A. Segal H. Hugonnet J.-E. Josseaume N. Dubost L. Brouard J.-P. Gutmann L. Mengin-Lecreulx D. Arthur M. J. Bacteriol. 2004; 186: 1221-1228Crossref PubMed Scopus (82) Google Scholar). Briefly, the mecA and fem genes of S. aureus Mu50 (21Hiramatsu K. Hanaki H. Ino T. Yabuta K. Oguri T. Tenover F. J. Antimicrob. Chemother. 1997; 40: 135-146Crossref PubMed Scopus (1655) Google Scholar) were amplified with the following primers that contained SacI or XbaI restriction sites (underlined): mecA, 5′-TTGAGCTCATATAAGGAGGATATTGATG-3′ and 5′-TTTCTAGACGGATTGCTTCACTGTTTTG-3′; fmhB, 5′-TTGAGCT-CAGGTATTGTTAAATAGAAGG-3′ and 5′-TTTCTAGAGAGCGTTCA-GATTTCAGTCG-3′; femA,5′-TTGAGCTCATTAACGAGAGACAAATA-GG-3′ and 5′-TTTCTAGACCTTCCTAAAAAATTCTGTC-3′; and femB, 5′-TTGAGCTCACAGAATTTTTTAGGAAGGG-3′ and 5′-TTTCTAGAG-CCCTAACATCATTTACATC-3′. The pbp5fm gene of E. faecium D344 (22Mainardi 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 (112) Google Scholar) was amplified with 5′-TTGAGCTCTTCCTCAAAGACATATGT-3′ and 5′-TTTCTAGATTATTGATAATTTTGGTTG-3′. The amplicons were cloned into pCR-Blunt (Invitrogen) and subcloned into pNJ2 by using SacI and XbaI. The nucleotide sequence of the inserts in the resulting recombinant plasmids was determined. The construction of the pNJ2 derivative for expression of the pbp5fs gene of E. faecalis JH2–2 has been previously described (17Arbeloa A. Segal H. Hugonnet J.-E. Josseaume N. Dubost L. Brouard J.-P. Gutmann L. Mengin-Lecreulx D. Arthur M. J. Bacteriol. 2004; 186: 1221-1228Crossref PubMed Scopus (82) Google Scholar).The fmhB, femA, and femB genes were cloned together into pNJ2 to obtain a policistrionic operon under control of the aphA-3p promoter in two steps. A SacI restriction fragment containing fmhB was recovered from pCR-BluntΩfmhB and cloned into the SacI site located upstream from femB in pNJ2ΩfemB. The resulting plasmid, pNJ2ΩfmhB femB, contained a unique SpeI restriction site located between fmhB and femB that was used to introduce the XbaI fragment of pCR-BluntΩfemA containing femA. The orientation of the three genes in the resulting plasmid, pNJ2ΩfmhB femA femB, was determined by restriction analysis and partial nucleotide sequencing.Preparation of Disaccharide Peptides—Bacteria were grown in 500 ml of BHI broth at 37 °C to an optical density at 650 nm of 0.7. Peptidoglycan was extracted by treating the bacterial pellet with 14 ml of 4% SDS at 100 °C for 30 min. Peptidoglycan was washed five times by centrifugation (12,000 × g for 10 min at 20 °C) with 20 ml of water. Peptidoglycan was serially treated overnight at 37 °C with Pronase (200 μg/ml) in 1 ml of Tris-HCl (10 mm, pH 7.4) and with trypsin (200 μg/ml) in 1 ml of phosphate buffer (20 mm, pH 7.8). Peptidoglycan was washed twice with 20 ml of water and digested overnight with mutanolysin (200 μg/ml; Sigma-Aldrich) and lysozyme (200 μg/ml; Sigma-Aldrich) at 37 °C in 1 ml of phosphate buffer (25 mm, pH 6.0) containing MgCl2 (0.1 mm). Mutanolysin and lysozyme were inactivated for 3 min at 100 °C, and soluble disaccharide peptides were recovered by ultracentrifugation (100,000 × g for 30 min at 20 °C).Reduction of Disaccharide Peptides—For reduction of MurNAc to N-acetylmuramitol, equal volumes (200 μl) of the solution of disaccharide peptides and of borate buffer (250 mm, pH 9.0) were mixed. Two mg of sodium borohydride were added, and the solution was incubated for 20 min at room temperature. The pH of the solution was adjusted to 4.0 with 20% orthophosphoric acid.Preparation of Lactoyl Peptides—The ether link internal to MurNAc was cleaved under alkaline conditions (23Klaic B. Carbohydr. Res. 1985; 138: 65-72Crossref Scopus (5) Google Scholar) to produce lactoyl peptide peptidoglycan fragments. To the solution of unreduced disaccharide peptides (200 μl), 32% ammonium hydroxide (64 μl) was added, and the mixture was incubated for 5 h at 37 °C. It was neutralized with acetic acid (61 μl), lyophilized, and dissolved in 200 μl of water containing 0.05% trifluoroacetic acid.Purification of Peptidoglycan Fragments—Reduced disaccharide peptides (200 μl) or lactoyl peptides (200 μl) were separated by reversed-phase high pressure liquid chromatography (HPLC) on a C18 column (3 μm, 4.6 × 250 mm; Interchrom, Montluçon, France) at a flow rate of 0.5 ml/min. A 0 –20% gradient was applied between 10 and 90 min (solvent A: 0.05% trifluoroacetic acid in water; solvent B: 0.035% trifluoroacetic acid in acetonitrile). UV detection was performed at 210 nm. The relative abundance of peptidoglycan fragments was estimated as the percentage of the total integrated area. The peaks were individually collected, lyophilized, and dissolved in 100 μl of water.Determination of the Mass of Peptidoglycan Fragments—The mass spectral data were collected with an electrospray time-of-flight mass spectrometer operating in the positive mode (Qstar Pulsar I, Applied Biosystems, Courtabœuf, France). Purified fractions (3 μl) of reduced disaccharide peptides or lactoyl peptides were directly injected into the mass spectrometer using HPLC pumps at a flow rate of 0.2 ml/min (acetonitrile 50%, water 49.5%, formic acid 0.5%, per volume). The data were acquired with a capillary voltage of 5,200 V and a declustering potential of 20 V. The mass scan range was from m/z 400 to 2,500, and the scan cycle was 1 s. Structure assignment of muropeptides based on mass was performed as previously described (14Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).Determination of the Structure of Monomers—Fragmentation performed on reduced disaccharide peptides and lactoyl peptides monomers gave essentially the same information on the structure of the peptide moieties of the molecules. Typical MS/MS experiments were performed by injecting 3–20 μl of the purified fractions, depending on the abundance of the peptidoglycan fragments, at a flow rate of 0.2 ml/min, as described above. The declustering potential was set to 60 V, the ions were selected based on the m/z value ([M+H]1+) in the high resolution mode, and fragmentation was performed with nitrogen as the collision gas. Collision energy was typically of 25–28 and 36–40 eV for reduced disaccharide peptide and lactoyl peptide monomers, respectively.Raising the declustering potential to 120 V led to formation of fragments of the lactoyl peptides that were analyzed by MS/MS. Such an analysis was used to confirm the structure assignment of the fragments obtained by tandem mass spectrometry.Determination of the Structure of Multimers—Tandem mass spectrometry performed on reduced disaccharide peptide dimers provided little information on the peptide moiety of the molecules because fragmentation occurred mainly at the β-1,4-GlcNAc-MurNAc bonds. In contrast, the entire peptide sequence could be determined by analyzing lactoyl peptide peptidoglycan fragments. In a first set of experiments, fragmentation was performed on the doubly charged ions of the molecules ([M+2H]2+), which gave a higher current intensity than singly charged ions ([M+H]1+). The declustering potential was set to 60 V, and the collision energy was typically of 26–35 eV. Fragments resulting from loss of d-Lac-l-Ala1 moieties of the molecules (loss of 143 atomic mass units) and additional NH3 (17 atomic mass units) or NH3 + CONH3 (17 + 45 atomic mass units) in various combinations gave ions of high current intensities. Fragments generated by cleavage within the cross-bridges gave ions of lower intensity. The fragmentation pattern was also complicated by the presence of singly and doubly charged forms of certain fragments.Optimization of the fragmentation conditions for lactoyl peptide dimers was achieved by raising the declustering potential to 100 V, which increased the current intensity of the singly charged form of the molecules ([M+H]1+). Fragmentation performed with a declustering potential of 100 V on singly charged ions with a collision energy of 57–65 eV exclusively produced singly charged fragments. The highest intensities were observed for cleavage within the cross-bridges. The data reported under "Results" were performed under these conditions by using 7–35 μl of the purified fractions.RESULTSStructure of the Main Monomer Resulting from Heterospecific Expression of femA in E. faecalis JH2–2ΔbppA2—The femA gene, encoding the transferase required for incorporation of glycyl residues at the second and third positions of the pentaglycine side chain in S. aureus (Fig. 1C), was cloned into the expression vector pNJ2 to generate plasmid pNJ2ΩfemA and introduced into E. faecalis JH2–2ΔbppA2. The latter host produces precursors substituted by a single l-alanyl residue following deletion of the bppA2 gene (Fig. 1A). The most abundant monomers of JH2–2ΔbppA2/pNJ2ΩfemA (Fig. 2, Peak 6) had a monoisotopic mass of 744.6, which matched the calculated value for a d-lactoyl-pentapeptide stem substituted by a side chain consisting of one l-alanyl and two glycyl residues. The structure of this branched peptide was solved by MS/MS, based on the detection of specific ions generated by loss of residues from the N terminus of the side chain (l-Ala-Gly-Gly-Nter) and from the carboxyl (d-Ala4-d-Ala5-Cter) or hydroxyl (OH-d-Lac-l-Ala1-d-iGln) extremities of the lactoyl-pentapeptide stem (Fig. 3). The interpretation of the fragmentation pattern (Fig. 3B) was confirmed by MS/MS performed on fragments of the molecule, as exemplified by fragmentation of the ion at m/z 474.3 (Fig. 4). The structure of the main monomer determined by these approaches (Figs. 3 and 4) indicates that FemA of S. aureus is functional in E. faecalis because it catalyzed the addition of two glycyl residues after the l-alanyl residue incorporated by BppA1.Fig. 2Structural analysis of peptidoglycan from JH2–2ΔbppA2/pNJ2ΩfemA.A, purified peptidoglycan was digested with muramidases and treated with ammonium hydroxide producing d-lactoyl peptide fragments that were separated by reversed-phase HPLC. Absorbance was monitored at 210 nm (absorbance unit × 103). B, model d-lactoyl peptide showing the main variations (boxed) in the free C terminus of the acceptor peptide stem, the cross-bridge, and the free N-terminal side chain. C, peaks 1–16 were individually collected, lyophilized, and analyzed by mass spectrometry. The relative abundance (%) of material in the 16 peaks was calculated by integration of the absorbance at 210 nm. The structure was deduced from the observed monoisotopic mass and, for most lactoyl peptides (indicated by stars), directly determined by tandem mass spectrometry. The most abundant d-lactoyl peptides, based on the relative absorbance at 210 nm and current intensity, are indicated in bold type. Tri, tripeptide l-Ala1-d-iGln2-l-Lys3; Tetra, tetrapeptide l-Ala1-d-iGln2-l-Lys3-d-Ala4; Penta, pentapeptide, l-Ala1-d-iGln2-l-Lys3-d-Ala4-d-Ala5; d-iGlu, the α-carboxyl group of the second residue was not amidated. Gly C-ter, Gly instead of d-Ala at the 5th (C-terminal position) of pentapeptide stems.View Large Image Figure ViewerDownload (PPT)Fig. 3Analysis of the main monomer from JH2–2ΔbppA2/pNJ2ΩfemA by tandem mass spectrometry.A, fragmentation was performed on the ion at m/z 745.6 corresponding to the [M+H]1+ from the major monomer. B, structure of the major monomer and inferred fragmentation pattern. The boxed m/z values in A originate from cleavage at single peptide bonds as represented in B. Peaks at m/z 631.5 and 560.4 matched the predicted values for loss of two N-terminal glycyl residues and of an additional l-alanyl residue, respectively. Loss of one and two d-Ala from the C terminus of the pentapeptide stem gave ions at m/z 656.5 and 585.4. Further loss of NH3 gave peaks at m/z 639.4 and 568.4. Fragmentation of the d-Lac-l-Ala1 amide bond was not observed. The peak at m/z 602.4 matched the predicted value for loss of d-Lac-l-Ala1. Further loss of NH3 and additional CONH3 led to peaks at m/z 585.4 and 540.4. Cleavage of the same peptide bond also produced peaks at m/z 144.0 and 116.0 corresponding to the d-Lac-l-Ala1 moiety of the molecule and loss of CO, respectively. Fragmentation at the d-iGln2-l-Lys3 peptide bond produced ions at 272.1 and 474.3. Additional loss of NH3 from ion at 474.3 gave an ion at 457.3. Additional ions could be accounted for by combinations of the fragmentations described above. In particular, loss of d-Ala4-d-Ala5 and d-Lac-l-Ala1 gave an ion at m/z 442.3. Further loss of NH3 and CONH3 led to peaks at m/z 425.3 and 380.2, respectively. Similarly, loss of d-Ala5 and d-Lac-l-Ala1 gave ions at m/z 513.3, 496.3, and 451.3. Peaks at m/z 471.3 matched the predicted value for loss of d-Ala5 and the l-Ala-Gly-Gly side chain. Peaks at m/z 289.2 and 314.2 matched the expected mass of l-Lys3 substituted by d-Ala4-d-Ala5 and l-Ala-Gly-Gly, respectively. Further fragmentation of the ion at m/z 314.2 led to ion at m/z 269.1 following loss of CONH3. Finally, ions at m/z 257.1 and 84.0 could correspond to d-iGln2-l-Lys3 and the immonium derivative of l-Lys. Loss of NH3 and additional CONH3 from peak at m/z 257.1 gave ions at 240.1 and 195.1, respectively. Together, these fragmentations account for the 29 ions with the highest relative current intensity. Ions of lower intensity could be accounted for by additional combinations of fragmentations (unlabeled peaks).View Large Image Figure ViewerDownload (PPT)Fig. 4Tandem mass spectrometry analysis of ion at m/z 474.3 corresponding to a fragment of the main monomer from JH2–2ΔbppA2/pNJ2ΩfemA. Tandem mass spectrometry (A) and inferred fragmentation pattern (B). The boxed m/z values in A originate from cleavage at single peptide bonds as represented in B. The peak at m/z 456.2 matched the predicted value for loss of H2O. The peaks at m/z 417.2, 360.2, and 289.2 matched the predicted value for loss of N-terminal Gly, Gly-Gly, and l-Ala-Gly-Gly from the side chain. Loss of the C-terminal d-Ala5 and additional CO gave ions at m/z 385.2 and 357.2, respectively. Loss of d-Ala4-d-Ala5 and additional CO or CONH3 gave ions at m/z 314.2, 286.2, and 269.2, respectively. Peak at m/z 328.2 matched the predicted value for loss of one Gly from the side chain and d-Ala5. The additional loss of one Gly resulted in peak at m/z at 271.2 and further loss of CO resulted in peak at 243.2. Peak at m/z 200.1 could correspond to the mass of l-Lys3 substituted by d-Ala4 or l-Ala, and further loss of CONH3 would lead to peak at m/z 155.1. The peak at m/z 186.1 matched the predicted value for the side chain l-Ala-Gly-Gly. Finally, peaks at m/z 129.1 and 84.1 could correspond to the l-Lys residue and its immonium ion, respectively.View Large Image Figure ViewerDownload (PPT)Structure of Secondary Monomers from JH2–2ΔbppA2/pNJ2ΩfemA—Muropeptide diversity was thoroughly investigated based on MS/MS analysis of the monomers (Fig. 2). In the order of decreased abundance, the first polymorphism was generated by the presence of side chains consisting of a single l-Ala (peak 4, 15.7%) instead of the sequence l-Ala-Gly-Gly (peak 6, 18.5%; see above). Side chains generated by BppA1 alone (l-Ala) or BppA1 and FemA (l-Ala-Gly-Gly) were therefore both produced by JH2–2ΔbppA2/pNJ2ΩfemA. Lactoyl peptides with side chains consisting of the sequence l-Ala-Gly were present in small amounts. Residues other than l-Ala were not detected at the first position of the side chains. Residues other than Gly were not detected at the second and third positions. Side chains containing glycyl residues were not detected in JH2–2 and JH2–2ΔbppA2 (14Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-