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 Gly(5), l-Ala(2), 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 beta-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.

Polymerization of the peptidoglycan subunit at the cell surface is performed by glycosyltransferases that catalyze elongation of the glycan strands by formation of ␤-1,4 bonds and D,D-transpeptidases that cross-link glycan strands (4). The latter reaction is catalyzed by essential high molecular weight penicillin-binding proteins (PBPs) that cleave the C-terminal residue (D-Ala 5 ) of a donor stem peptide and link the carboxyl group of the penultimate residue (D-Ala 4 ) to the side chain amino group of an acceptor stem peptide (5) (see Fig. 1). This two-step reaction involves formation of a covalent adduct between the ␤-hydroxyl of the active site serine of the PBPs and the carboxylate of D-Ala 4 of the donor stem (acyl-enzyme) (5). ␤-Lactam antibiotics are structural analogues of the D-Ala 4 -D-Ala 5 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 (6). Multimodular PBPs associate a C-terminal D,D-transpeptidase module to N-terminal glycosyltransferase (class A) or non catalytic (class B) modules. Clinically relevant ␤-lactam resistance phenotypes in staphylococci and enterococci involve production of class B D,D-transpeptidases that are inefficiently acylated by ␤-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 (7). 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 ␤-lactams and produce species-specific low affinity PBPs designated PBP5 fs and PBP5 fm , respectively (8 -10).
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 (11). 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 (12). Similarly, the side chain is essential for penicillin resistance in Streptococcus pneumoniae (13). 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 (14). Deletion of bppA2 led to production of precursors substituted by a single L-Ala and to impaired expression of intrinsic ␤-lactam resistance (14). For these reasons, transferases of the Fem family are considered to be potential targets for the development of novel antibiotics active against ␤-lactam-resistant Gram-positive cocci (15).
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 ␤-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, pbp5 fs , and pbp5 fm were heterologously expressed in E. faecalis and E. faecium to evaluate the capacity of the corresponding low affinity PBPs to confer ␤-lactam resistance. PBP2a and PBP5 fm 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 PROCEDURES
Bacterial 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 (16). 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.
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-Blunt⍀fmhB and cloned into the SacI site located upstream from femB in pNJ2⍀femB. The resulting plasmid, pNJ2⍀fmhB femB, contained a unique SpeI restriction site located between fmhB and femB that was used to introduce the XbaI fragment of pCR-Blunt⍀femA containing femA. The orientation of the three genes in the resulting plasmid, pNJ2⍀fmhB 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 ϫ g for 10 min at 20°C) with 20 ml of water. Peptidoglycan was serially treated overnight at 37°C with Pronase (200 g/ml) in 1 ml of Tris-HCl (10 mM, pH 7.4) and with trypsin (200 g/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 g/ml; Sigma-Aldrich) and lysozyme (200 g/ml; Sigma-Aldrich) at 37°C in 1 ml of phosphate buffer (25 mM, pH 6.0) containing MgCl 2 (0.1 mM). Mutanolysin and lysozyme were inactivated for 3 min at 100°C, and soluble disaccharide peptides were recovered by ultracentrifugation (100,000 ϫ g for 30 min at 20°C).
Reduction of Disaccharide Peptides-For reduction of MurNAc to N-acetylmuramitol, equal volumes (200 l) 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 (23) to produce lactoyl peptide peptidoglycan fragments. To the solution of unreduced disaccharide peptides (200 l), 32% ammonium hydroxide (64 l) was added, and the mixture was incubated for 5 h at 37°C. It was neutralized with acetic acid (61 l), lyophilized, and dissolved in 200 l of water containing 0.05% trifluoroacetic acid.
Purification of Peptidoglycan Fragments-Reduced disaccharide peptides (200 l) or lactoyl peptides (200 l) were separated by reversed-phase high pressure liquid chromatography (HPLC) on a C 18 column (3 m, 4.6 ϫ 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 l 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, Courtaboeuf, France). Purified fractions (3 l) 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 (14).
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 l 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 ([MϩH] 1ϩ ) 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 frag-ments 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 ␤-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 mole-cules ([Mϩ2H] 2ϩ ), which gave a higher current intensity than singly charged ions ([MϩH] 1ϩ ). 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-Ala 1 moieties of the molecules (loss of 143 atomic mass units) and additional NH 3 (17 atomic mass units) or NH 3 ϩ CONH 3 (17 ϩ 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.

FIG. 1. Schematic representation of peptidoglycan cross-linking in E. faecalis (A), E. faecium (B), and S. aureus (C).
Peptidoglycan is polymerized from a subunit comprising three elements: (i) a disaccharide composed of ␤-1,4-linked N-acetylglucosamine (GlcNAc) and MurNAc, (ii) a conserved pentapeptide stem (L-Ala 1 -D-iGln 2 -L-Lys 3 -D-Ala 4 -D-Ala 5 ) linked to the D-lactoyl residue of MurNAc, and (iii) a variable side chain linked to the ⑀-amino group of the third residue (L-Lys 3 ) of the pentapeptide stem. The structure of the side chain is generally highly conserved in members of the same species but variable in different species (3). Incorporation of glycine and of L-amino acids into the side chain is performed by a unique family of nonribosomal peptide bond-forming enzymes that use aminoacyl-tRNA as the substrate (Fem and Bpp transferases in S. aureus and E. faecalis, respectively) (25). In E. faecium, an unidentified ATP-dependent ligase catalyzes peptide bond formation between the ␤-carboxyl group of D-Asp and the ⑀-amino group of L-Lys 3 (33). The ␣-carboxyl group of D-Asp is secondarily partially amidated (D-Asx, D-Asn, or D-Asp). The two disaccharide peptide substrates participating in the D,D-transpeptidation reaction are referred to as carbonyl acceptor and donor. The leaving groups are circled. The amino acids of the cross-bridge are boxed. The orientation of the peptide bonds is indicated by arrows.
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 ([MϩH] 1ϩ ). 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 l of the purified fractions.

Structure of the Main Monomer Resulting from Heterospecific
Expression of femA in E. faecalis JH2-2⌬bppA2-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 pNJ2⍀femA and introduced into E. faecalis JH2-2⌬bppA2. 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 JH2-2⌬bppA2/pNJ2⍀femA (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 FIG. 2. Structural analysis of peptidoglycan from JH2-2⌬bppA2/pNJ2⍀femA. 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 ϫ 10 3 ). 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-Ala 1 -D-iGln 2 -L-Lys 3 ; Tetra, tetrapeptide L-Ala 1 -D-iGln 2 -L-Lys 3 -D-Ala 4 ; Penta, pentapeptide, L-Ala 1 -D-iGln 2 -L-Lys 3 -D-Ala 4 -D-Ala 5 ; D-iGlu, the ␣-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.  (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.
Structure of Secondary Monomers from JH2-2⌬bppA2/ pNJ2⍀femA-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 JH2-2⌬bppA2/pNJ2⍀femA. 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 JH2-2⌬bppA2 (14). Thus, BppA1 and FemA were highly specific both for the type (L-Ala versus Gly) and position (first versus second and third) of the residues incorporated into the side chains.
A second polymorphism was generated by the presence of stem peptides lacking one (tetrapeptide) or both (tripeptide) C-terminal D-alanyl residues (Fig. 2). As previously described (14), these stem peptides could be generated by D,D-carboxypeptidases and L,D-carboxypeptidases that cleave the D-Ala 4 -D-Ala 5 and L-Lys 3 -D-Ala 4 peptide bonds, respectively.
A third polymorphism was generated by the presence of Gly instead of D-Ala at the fifth position of lactoyl-pentapeptide stems. Pentapeptide stems with a C-terminal glycyl residue were also detected in E. faecalis JH2-2 and JH2-2⌬bppA2 and are therefore unrelated to the activity of FemA. The PBPs are likely to be responsible for exchange of the C-terminal D-Ala by Gly because cytoplasmic UDP-MurNAc-pentapeptide ending in a glycyl residue were not detected in the cytoplasm (data not shown).
A fourth polymorphism was generated by the lack of amidation of the ␣-carboxyl group of the D-isoglutaminyl residue at the second position of the stem peptide. This polymorphism is unrelated to the activity of FemA because it was also detected in the control strains JH2-2 and JH2-2⌬bppA2.
Structures of Multimers of E. faecalis JH2-2⌬bppA2/pNJ2⍀ femA-Expression of femA in JH2-2⌬bppA2 led to production of novel dimers differing from those present in preparations from the JH2-2⌬bppA2 host by an increment of 228 atomic mass units, as expected for the presence of a total of four additional glycyl residues (Fig. 2). Tandem mass spectrometry provided the sequence of the cross-bridge and of the free Nterminal side chain, which were both found to contain the sequence L-Ala-Gly-Gly (see Fig. 5 for the analysis of the main dimer in peak 11). As described for the monomers, MS/MS was also performed on fragments of the molecules to confirm the interpretation of the fragmentation patterns. The structure of the main dimer (Fig. 5B) indicates that the D,D-transpeptidases of E. faecalis cross-linked glycyl-containing precursors. Peptide stems substituted by L-Ala-Gly-Gly had participated in the transpeptidation reaction both as an acceptor and a donor.
Polymorphism in the multimers of JH2-2⌬bppA2/pNJ2⍀ femA was generated by the presence of dimers, trimers, and tetramers containing pentapeptide (major form) and tripeptide (of lesser abundance) acceptor stems (Fig. 2). Other polymorphisms could be accounted for by variations already described in detail for the monomers.
Expression of femA in E. faecalis JH2-2-Analysis of the monomers of E. faecalis JH2-2/pNJ2⍀femA indicated the participation of BppA1, BppA2, and FemA in side chain synthesis in various combinations (Table I). The wild-type sequence (L-Ala-L-Ala) was generated by BppA1 and BppA2. The sequence L-Ala-Gly-Gly, also observed in JH2-2⌬bppA2/pNJ2⍀femA (see above), was generated by BppA1 and FemA. The sequence L-Ala-L-Ala-Gly involved the participation of BppA1, BppA2, and FemA. Thus, FemA and BppA2 competitively added Gly and L-Ala, respectively, at the second position of the side chain. The sequence L-Ala-L-Ala-Gly also indicated that FemA can add a single residue. The three types of sequences (L-Ala-Gly-Gly, L-Ala-L-Ala, and L-Ala-L-Ala-Gly) were present both in the cross-bridges and in the free side chains of multimers.
FmhB-mediated Incorporation of Gly at the First Position of the Side Chain-Co-expression of the fmhB and bpp genes led to the production of monomers substituted by Gly or L-Ala in JH2-2⌬bppA2 and by the sequence Gly-L-Ala or L-Ala-L-Ala in JH2-2 (Table I). Thus, FmhB and BppA1 competitively added glycyl and L-alanyl residues at the first position of the side chain, respectively. Detection of the sequence Gly-L-Ala indicates the sequential participation of FmhB and BppA2 to side chain synthesis. BppA2 was therefore able to elongate a side chain consisting of Gly, although its natural substrate in E. faecalis contains L-Ala. The structure of the dimers indicated that glycyl-containing precursors were used as donors and acceptors in the transpeptidation reaction.
Expression of femB-The FemB transferase is required for incorporation of glycyl residues at the fourth and fifth positions of the pentaglycine side chain in S. aureus (Fig. 1). Expression of femB did not lead to incorporation of glycyl residues in the peptidoglycan of JH2-2 or JH2-2⌬bppA2 (Table I). Thus, FemB was unable to elongate side chains comprising one or two L-alanyl residues.
Synthesis of Pentaglycine Side Chains by FmhB, FemA, and FemB-Co-expression of the fmhB, femA, and femB genes in JH2-2⌬bppA2 led to production of muropeptides monomers substituted by five glycyl residues (Table I). Thus, synthesis of the complete pentaglycine side chain of S. aureus was obtained in E. faecalis. Side chains consisting of one L-alanyl and four glycyl residues were also present because of competitive incorporation of L-Ala and Gly by BppA1 and FmhB at the first position. Amino acid residues other than Gly were not observed at positions 2-5. Incomplete side chains were also present. Analysis of multimers indicated that precursors containing pentaglycine side chains were used as donors and acceptors in the cross-linking reaction. Expression of fmhB, femA, and femB in JH2-2 gave essentially the same results except that Gly and L-Ala were competitively incorporated also at the second position of the side chain (Table I).
Heterospecific Expression of Genes Encoding Low Affinity PBPs-Resistance to ␤-lactams mediated by heterospecific expression of mecA, pbp5 fs , and pbp5 fm was used as a screen to evaluate the capacity of the corresponding PBPs to catalyze cross-linking of pentapeptide stems substituted by L-Ala-L-Ala in E. faecalis and by D-Asx in E. faecium. mecA, pbp5 fs , and pbp5 fm were cloned into the expression vector pNJ2 and introduced into E. faecalis JH2-2⌬pbp5 and E. faecium D344S. The latter hosts are previously characterized mutants susceptible to ␤-lactams because of deletion of their respective species specific pbp5 genes (17,19). Ceftriaxone, a third generation cephalosporin, was used to monitor expression of ␤-lactam resistance, because deletion of the pbp5 genes produced large decreases (Ͼ1000-fold) in the minimal inhibitory concentration of this antibiotic both in E. faecium and E. faecalis.
Resistance Phenotype Mediated by mecA in E. faecalis and E. faecium-Population analysis profiles indicated that the mecA gene of S. aureus can confer resistance to ceftriaxone both in E. faecalis JH2-2⌬pbp5 (Fig. 6A) and E. faecium D344S (Fig.  6B). The resistance phenotypes were characterized in both hosts by the presence of subpopulations of bacteria highly resistant to the drug. Representatives of the resistant subpopulations obtained on agar containing ceftriaxone were homogeneously resistant to high levels of ceftriaxone. The resistance trait remained stable after five serial subcultures in the absence of the drug. Sequencing of the mecA open reading frame and upstream sequences comprising the relevant promoter and ribosome-binding site did not reveal any mutation. Moreover, introduction of pNJ2⍀mecA isolated from representatives of the resistant subpopulations into E. faecalis JH2-2⌬plp5 or E. faecium D344S resulted in expression of high level ceftriaxone resistance only in subpopulations of the bacteria, as initially observed for the wild-type plasmid. Thus, plasmid pNJ2⍀mecA of homogeneously resistant variants did not harbor any mutation affecting mecA or cis-acting sequences required for its expression. Together, these observations indicate that expression of wild-type mecA of S. aureus can confer high level resistance to ceftriaxone in E. faecalis and E. faecium. Modification of an unknown host factor was required for full expression of resistance, as also observed for the introduction of mecA in methicillin susceptible S. aureus (24).
Analysis of Peptidoglycan Structure of E. faecalis and E. faecium Strains Expressing mecA-To determine the contribution of PBP2a to peptidoglycan cross-linking, the resistant variants of E. faecalis JH2-2⌬plp5 harboring pNJ2⍀mecA was grown in the presence of ceftriaxone (100 g/ml) to inactivate the host PBPs, and the peptidoglycan was analyzed by mass spectrometry and tandem mass spectrometry. Complementation of the chromosomal pbp5 fs deletion of E. faecalis JH2-2⌬pbp5 by mecA or by pbp5 fs led to synthesis of similarly cross-linked peptidoglycan in the presence of ceftriaxone (Fig.  7). Likewise, no significant difference in the peptidoglycan structure was observed for complementation of the chromosomal pbp5 fm deletion of E. faecium D344S by mecA or by pbp5 fm (data not shown). Thus, production of low affinity PBP2a allowed ␤-lactam-insensitive cross-linking of side chains consisting of L-Ala-L-Ala in E. faecalis and D-Asx in E. faecium.
Heterospecific Expression of pbp5 fs and pbp5 fm -Derivatives of E. faecalis JH2-2⌬pbp5 harboring the expression vector pNJ2 or plasmid pNJ2⍀pbp5 fm were uniformly susceptible to ceftriaxone (Fig. 6A). Thus, the pbp5 fm gene of E. faecium did not confer ␤-lactam resistance in E. faecalis. In contrast, the pbp5 fs gene of E. faecalis conferred resistance to ceftriaxone in E. faecium (Fig. 6C). As found for mecA, expression of high level resistance was only detected in a fraction of the bacteria. Derivatives of the resistant subpopulation stably expressed homogenous resistance in the absence of mutational alteration of pNJ2⍀pbp5 fs .

DISCUSSION
The Fem proteins form a family of highly diverse tRNA-dependent aminoacyl-transferases with respect to the type, number, and position of the amino acids added to the side chain of peptidoglycan precursors (Fig. 1) (25). The catalytic activity of the transferases has been assessed only of purified FemX Wv from Weissella viridescens (26,27) and the Bpp transferases from E. faecalis (14,28). In S. aureus, transferases essential for incorporation of residues at specific positions of the pentaglycine side chain (Fig. 1) have been identified based on gene inactivation (femA and femB) and conditional gene expression (fmhB) (11,12). Individual inactivation of two additional femrelated genes had little impact on viability and amino acid composition of the peptidoglycan (29). We show here that FmhB competes with BppA1 for the incorporation of Gly at the first position of the side chain in E. faecalis JH2-2 and JH2-2⌬bppA2. FemA was sufficient for addition of two residues because expression of femA in JH2-2⌬bppA2 led to the incorporation of two Gly in the absence of the other four fem genes present in the chromosome of S. aureus (Figs. 2 and 3). The two reactions can occur independently because FemA mediated incorporation of one or two Gly to generate the sequence L-Ala-L-Ala-Gly and L-Ala-Gly-Gly in E. faecalis JH2-2 because of competition between FemA and BppA2 at the second position (Table I). FemB added two Gly and required a side chain composed of three residues, indicating that this transferase was highly specific for the fourth and fifth positions. Co-expression of fmhB, femA, and femB led to the production of stem peptides substituted by a pentaglycyl moiety, showing for the first time that synthesis of the complete side chain of S. aureus can be reproduced in a heterologous host.
The Fem transferases of S. aureus incorporated glycyl residues in E. faecalis, indicating that the enzymes retained the specificity displayed in the original host ( Fig. 2 and Table I). This implies that E. faecalis produces glycyl-tRNAs, which were efficiently used by the Fem transferases. Fractionation of the tRNAs isoacceptor of glycine and sequence analysis have raised the possibility that staphylococci produce unusual tRNAs dedicated to peptidoglycan synthesis (30,31). Our analysis indicates that these putative tRNAs are unlikely to play a key role in the specificity of the Fem transferases for the amino acids incorporated into the side chains of peptidoglycan precursors.
A narrow substrate specificity of the D,D-transpeptidases for the acceptor side chain was proposed to account for the essential role of the transferases in viability (e.g. FmhB) and ␤-lactam resistance (e.g. FemA and FemB) (11,12). However, this has not been directly established by kinetic analyses of purified PBPs, because the enzymes were not active in vitro, except in very special cases involving highly reactive substrates (e.g. thioester) or atypical enzymes (e.g. the soluble R61 D,D-peptidase from Streptomyces spp.) (see Ref. 32 for a recent discussion). For this reason, heterospecific expression of pbp and fem genes was used in the current study to gain insight into the specificity of the PBPs in vivo. The D,D-transpeptidases of E. faecalis tolerated variations in the number of residues in the side chains (from 1 to 5) and substitution of L-Ala by Gly at the N-terminal position both in the donor and the acceptor (Fig. 2 and Table I). The substrate specificity of the D,D-transpeptidases was also explored based on heterospecific expression of pbp genes encoding low affinity PBPs. The mecA gene of S. aureus conferred resistance to ceftriaxone in E. faecalis JH2-2⌬pbp5 and E. faecium D344S (Fig. 6). In neither host was the expression of resistance associated with a modification of peptidoglycan structure ( Fig. 7 and data not shown). Because the D,D-transpeptidases are the essential target of ␤-lactams (5, 6), PBP2a acted as a surrogate of the host D,D-transpeptidases and therefore catalyzed peptidoglycan cross-linking. This implies a low substrate specificity of PBP2a, because the amino group of the acceptor participating in the transpeptidation reaction was located on side chains consisting of five Gly, L-Ala-L-Ala, and D-Asx in S. aureus, E. faecalis, and E. faecium, respectively. These observations establish for the first time that mecA of S. aureus can confer ␤-lactam resistance in distantly related hosts belonging to the genus Enterococcus, despite substantial diversity in the structure of peptidoglycan precursors. Similarly, PBP5 fs from E. faecalis conferred resist-ance to ceftriaxone in E. faecium D344S (Fig. 6C), indicating that substitution of L-Ala-L-Ala by D-Asx was also tolerated by this D,D-transpeptidase. Horizontal gene transfer in natural conditions has been documented for mecA between related species belonging to the genus Staphylococcus. Our data indicate that there is no barrier to heterospecific expression of ␤-lactam Purified peptidoglycan was digested with muramidases and treated with ammonium hydroxide producing D-lactoyl peptide fragments that were separated by reversed-phase HPLC. A, E. faecalis JH2-2⌬pbp5/pNJ2⍀pbp5 fs . B, E. faecalis JH2-2⌬pbp5/pNJ2⍀mecA (representative of the subpopulation highly resistant to ceftriaxone). C, mass spectrometry was performed on individually collected peaks. The calculated mass (not shown) of the proposed structure for the D-lactoyl peptides differed from the observed monoisotopic mass by less than 0.2 atomic mass unit. The structure of the monomers and dimers was determined by tandem mass spectrometry. The relative abundance (%) of material in peaks 1-10 was calculated by integration of the absorbance at 210 nm. NA, not applicable. resistance mediated by the low affinity PBP2a and PBP5 fs in more distantly related bacterial species, and thus, intergeneric transfers of low affinity PBPs should be anticipated.
Among the three low affinity PBPs that were tested in the current study (PBP2a, PBP5 fs , and PBP5 fm ), only PBP5 fm from E. faecium did not confer ceftriaxone resistance in a heterologous host (Fig. 6). To explore the basis for the lack of expression of pbp5 fm -mediated resistance in E. faecalis, the bppA1 gene of E. faecalis was expressed in wild-type E. faecium BM4107 harboring pbp5 fm . The BppA1 transferase mediated incorporation of L-Ala instead of D-Asp in the E. faecium host, and the modified precursors were used as donors and acceptors in the cross-linking reaction (data not shown). Ceftriaxone did not inhibit the formation of the L-alanyl-containing cross-bridges. Thus, the lack of heterospecific expression of resistance to ceftriaxone, which inhibits all PBPs except PBP5 fm , did not appear to be due to the incapacity of E. faecium PBP5 fm to cross-link L-alanyl-containing peptidoglycan precursors. This observation implies, unexpectedly, that the requirements for expression of PBP5 fm -mediated ␤-lactam resistance in terms of side chain structure vary in different hosts. A similar conclusion can be drawn for PBP2a from S. aureus because this PBP conferred ␤-lactam resistance in hosts producing precursors substituted by L-Ala-L-Ala (E. faecalis) and D-Asx (E. faecium) (Fig. 6), whereas the complete pentaglycine side chain is essential for peptidoglycan cross-linking by PBP2a in its original host (12). The different requirements for PBP2a-and PBP5 fmmediated ␤-lactam resistance in different hosts may indicate that an as yet unknown component of the peptidoglycan polymerization complexes participates in the recognition of the acceptor side chain. It is clear that a narrow substrate specificity of low affinity PBPs cannot, in itself, account for the essential role of specific side chains in ␤-lactam resistance.
In conclusion, we have established that diversification of the structure of the side chains of peptidoglycan precursors associated with speciation in Gram-positive bacteria (Fig. 1) did not correlate with diversification of the substrate specificity of the PBPs. The cross-bridges of wild-type S. aureus, E. faecalis, and E. faecium were found to exclusively contain Gly, L-Ala, and D-Asx, respectively. This reflects the high in vivo efficacy and specificity of the enzymes for side chain synthesis rather than a narrow specificity of the D,D-transpeptidases.