Aslfm, the D-Aspartate Ligase Responsible for the Addition of D-Aspartic Acid onto the Peptidoglycan Precursor of Enterococcus faecium*

d-Aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of d-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the β-carboxylate of d-Asp to the ϵ-amino group of l-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. d-iso-Asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the α-carboxyl of d-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by d-Asp instead of l-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of l-Ala2 by d-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.

Peptidoglycan is a macromolecule found on the outer face of the cytoplasmic membrane of all eubacteria except certain halophilic bacteria, such as Halobacterium halobium, and intracellular parasites, such as Mycoplasma pneumoniae (1). This structure is essential to protect bacteria against the internal osmotic pressure and plays a key role in cell division. The basic unit of peptidoglycan is a disaccharide peptide assembled by a series of cytoplasmic and membrane reactions (2). It is composed of GlcNAc linked to N-acetylmuramic acid (MurNAc) 2 substituted by a stem peptide. In pathogenic Gram-positive bacteria belonging to the genera Staphylococcus, Streptococcus, and Enterococcus, the stem peptide consists of a conserved pentapeptide (L-alanyl-␥-D-glutamyl-L-lysyl-D-alanyl-D-alanine) and a variable side chain linked to the ⑀-amino group of L-Lys 3 (1). The structure of the side chain is conserved in members of the same species but highly variable between species as it contains from 1 to 5 residues of the L and D configurations as well as glycine (1). L-Amino acids and glycine are activated as aminoacyl-tRNAs and transferred to the precursors by a family of non-ribosomal peptide bond-forming enzymes (Fem) (3,4). Members of the Fem family responsible for the addition of 5 glycines in Staphylococcus aureus (FmhB, FemA, FemB) (5, 6), 2 L-Ala in Enterococcus faecalis (BppA1, BppA2) (7), L-Ala in Weissella viridescens (FemX) (4,8), and L-Ser-L-Ala or L-Ala-L-Ala in Streptococcus pneumoniae (MurM, MurN) (9, 10) have been extensively characterized. In contrast, little is known about the incorporation of D-amino acids into peptidoglycan precursors. In 1972, a D-aspartate ligase of E. faecium, formerly designated as Streptococcus faecalis, has been partially purified (11,12). The enzyme activates D-aspartate as ␤-D-aspartyl-phosphate and links D-Asp to the ⑀-amino group of L-Lys 3 in the cytoplasmic precursor UDP-MurNAc-pentapeptide (11) (Fig. 1A).
The side chains of peptidoglycan precursors play a critical role since their N terminus is used by the D,D-transpeptidases in the last crosslinking step of peptidoglycan synthesis (Fig. 1B). In S. aureus, the transferase responsible for incorporation of the first residue in the pentaglycine side chain (FmhB) is essential presumably because the D,D-transpeptidases cannot catalyze cross-link formation with unsubstituted pentapeptide stems (5). In addition, FemA and FemB are essential for methicillin resistance mediated by a low affinity D,D-transpeptidase (PBP2a) (13,14). Because of their essential role in peptidoglycan synthesis and ␤-lactam resistance, Fem transferases are considered as promising targets for the development of novel antibiotics (15).
In this report, we have identified by reverse genetics a two-gene cluster responsible for incorporation of D-Asp into the peptidoglycan pre-* This work was supported by the NIAID National Institutes of Health (Grant R01 AI45626) and the program "ACI Microbiologie 2003" from the Fonds National de la Science (Grant ACIM-4-9). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  cursors of E. faecium. Heterospecific expression of the cluster in E. faecalis was used to demonstrate the function of the proteins in vivo and to study the consequences of substitution of L-Ala 2 by D-Asp on peptidoglycan cross-linking by the D,D-transpeptidases.
The specificity of the D-aspartate ligase was studied by replacing D-[ 14 C]Asp by D-Asp, L-Asp, D-iso-asparagine, D-Glu, D-Ala, and D-malic acid (5 mM; Sigma, Saint-Quentin Fallavier, France). The products of the addition reactions were detected by mass spectrometry (MS), and their structures were determined by tandem mass spectrometry (MS/MS) (7). Identification of the D-Aspartate Ligase by Reverse Genetics-The D-aspartate ligase (Asl fm ) of E. faecium D359 (17) was partially purified in three chromatographic steps and identified as a member of the ATPgrasp protein family by MS/MS amino acid sequencing. Briefly, E. fae-cium D359 was grown to an OD 650 nm of 0.7 in 20 liters of brain heart infusion broth (Difco, Elancourt, France), harvested by centrifugation (6,000 ϫ g for 20 min at 4°C), and washed twice in 50 mM sodium phosphate buffer (pH 7.0). Bacteria were disrupted with glass beads in a refrigerated cell disintegrator (Sartorius, Palaiseau, France) for 3 ϫ 30 s. The extract was centrifuged at 7,000 ϫ g for 10 min at 4°C to remove cell debris, and the supernatant was ultracentrifuged at 100,000 ϫ g for 1 h at 4°C. The supernatant was dialyzed against 50 mM phosphate buffer (pH 6.0) containing 200 mM NaCl (buffer A). Proteins (1.3 g) were loaded onto a cation exchange column equilibrated with buffer A (HiLoad 26/10 SP Sepharose HP; Amersham Biosciences, Saclay, France). Active fractions, eluting between 0.8 and 0.9 M NaCl, were pooled (12 mg of proteins), concentrated in a dialysis bag with solid polyethylene glycol, and loaded onto a gel filtration column (Superdex 75 HR26/60, Amersham Biosciences) equilibrated with buffer A. Active fractions (1.8 mg of proteins) were loaded onto a 1-ml cation exchange column (HiTrap SP Sepharose fast flow, Amersham Biosciences) equilibrated with buffer A. Proteins (200 g) eluting between 0.8 and 0.95 M NaCl were dialyzed against buffer A, concentrated by lyophilization, and analyzed by 12% SDS-PAGE. The native enzyme was stored at Ϫ20°C in 50 mM phosphate buffer (pH 6.0) containing 200 mM NaCl.
For amino acid sequencing, protein bands were excised from the SDS-PAGE gel, reduced with dithiothreitol (Sigma, Poole, UK), alkylated with iodoacetamide, and digested overnight at 37°C with trypsin (modified trypsin, sequencing grade, Roche Applied Science). Tryptic digests were dried under vacuum, resuspended in 4 l of 0.1% formic acid, and analyzed by HPLC (LC Packing system, LC Packing, San Francisco, CA) coupled to mass spectrometry. Chromatography was performed at a flow rate of 200 nl/min in 0.1% formic acid with three consecutive linear acetonitrile gradients (0 -2% for 1 min, 2-50% for 40 min, and 50 -90% for 10 min). The LC system was connected to an ion trap mass spectrometer (LCQ Deca, Finnigan Corp., San Jose, CA). The spray voltage was set at 2.1 kV, the temperature of the ion transfer tube The reaction proceeds through formation of a ␤-aspartyl-phosphate intermediate (11). D-Asp is produced by the Rac fm aspartate racemase. B, peptidoglycan is polymerized from a disaccharide-peptide subunit containing ␤-1-4linked GlcNAc and MurNAc, a pentapeptide stem (L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), and a side chain D-Asp residue linked to the ⑀-amino group of L-Lys was set at 180°C, and the normalized collision energies were set at 35% for MS/MS. The sequences of the uninterpreted spectra were identified by correlation with the peptide sequences from the National Center for Biotechnology Information (NCBI) non-redundant protein data base using the SpectrumMill program (Millenium Pharmaceuticals, Cambridge, MA). The D-aspartate ligase was identified as a member of the ATP-grasp protein family present in the major 49-KDa protein band.
Western Blot Analysis-Anti-Asl fm antiserum was obtained by three subcutaneous injections at 3-week intervals of 200 g of purified Asl fm in a New Zealand rabbit. Proteins were separated by SDS-PAGE, electrotransferred to a nitrocellulose membrane (Hybond, Amersham Biosciences, Little Chalfont, UK), and incubated with the anti-Asl fm antiserum at a 1/1000 dilution. Goat anti-rabbit IgG coupled to peroxidase (SouthernBiotech, Birmingham, AL) was used as a secondary antibody, and Asl fm was detected by chemiluminescence (ECL kit, Pierce and Amersham Biosciences). Tagged proteins were alternatively detected using polyclonal anti-His 6 rabbit antibodies (Ebiosciences, San Diego, CA).
Heterospecific Expression of the D-Aspartate Ligase Gene (asl fm ) in E. faecalis-The shuttle expression vector pJEH11 (our laboratory collection) is a derivative of pAT392 (20) that confers gentamicin resistance and replicates in E. coli and in Gram-positive hosts. The NcoI site used for cloning in pJEH11 is preceded by a constitutive promoter (20) and a ribosome binding site active in enterococci. The BamHI site is followed by in-frame codons specifying a His 6 tag. The NcoI-BamHI fragment of pSJL1 (above) containing the asl fm open reading frame was cloned into pJEH11. The resulting plasmid pSJL2(asl fm ) was introduced into E. faecalis JH2-2 by electroporation (20), and clones were selected and subcultured in brain heart infusion containing 128 g/ml gentamicin.
Co-expression of the D-Aspartate Ligase (asl fm ) and Aspartate Racemase (rac fm ) Genes in E. faecalis-The rac fm open reading frame was amplified with primers rac1 (5Ј-AAAGAAGGATCCTAGCCATG-GAGAATTTTTTCAGTATTTTAGGCGG-3Ј), containing BamHI (underlined) and a stop codon (bolded), and rac2 (5Ј-AAAGGATC-CCTTTTCCGATGCTGTATCCAATGCC-3Ј), containing BamHI. The PCR fragment was cloned into the BamHI restriction site of pSJL2(asl fm ) (above). Insertion of the PCR product introduced a stop codon at the end of asl fm (TAG present in primer rac1) and generated an in-frame fusion between the 3Ј end of rac fm and the sequence of the vector pJEH11 specifying the His 6 tag. The resulting plasmid, pSJL3(asl fm rac fm ), carried a bi-cistronic operon encoding Asl fm and E. faecium His 6 -tagged aspartate racemase (Rac fm ). To obtain inducible expression of asl fm and rac fm , the genes were subcloned with SacI and XbaI under the control of the IPTG-inducible promoter of vector pJEH4, generating pSJL4(asl fm rac fm ). The shuttle vector pJEH4 (our laboratory collection) confers resistance to spectinomycin (120 g/ml for selection) and carries the lacI gene and lac operator fused to transcription and translation signals that are functional in enterococci.
Peptidoglycan Structure Analysis-The structure of the peptidoglycan was determined by mass spectrometry as described previously (21).  Briefly, derivatives of E. faecalis JH2-2 harboring plasmids pJEH11, pSJL2(asl fm ), and pSJL4(asl fm ,rac fm ) were grown at 37°C to an optical density of 0.7 in 250 ml of brain heart infusion broth in the presence or absence of D-aspartate (50 mM). Peptidoglycan was extracted with boiling SDS and digested with mutanolysin and lysozyme (Sigma). The resulting muropeptides were cleaved under alkaline conditions to generate lactoyl-peptides, separated by rp-HPLC, and analyzed by MS and MS/MS using an electrospray time-of-flight mass spectrometer operating in positive mode (Qstar Pulsar I, Applied Biosystems, Courtaboeuf, France).

RESULTS
Asl fm Is a Member of the ATP-Grasp Protein Family-D-Aspartate ligase activity was detected in a cytoplasmic fraction of E. faecium D359 using a radioactive assay ( Fig. 2A) and partially purified in three chromatographic steps (Fig. 2B). Amino acid sequencing was performed by MS/MS on individual protein bands digested with trypsin. The 49-kDa protein (Fig. 2B) was encoded by the EfaeDRAFT_0086 nucleotide sequence in the E. faecium genome data base (U. S. Department of Energy (DOE) Joint Genome Institute). Sequence data obtained by MS/MS included the N terminus of the 49-kDa protein, revealing that the methionine specified by the proposed translation initiation codon had been cleaved. The protein was produced in E. coli with a C-terminal His 6 tag and purified by affinity and cation exchange chromatographies (Fig. 2C). The recombinant protein was active in the radioactive assay (Fig. 3A), indicating that the gene encoding the D-aspartate ligase (asl fm ) had been successfully identified. Asl fm belonged to the ATP-grasp protein superfamily composed of highly diverse enzymes that catalyze ATPdependent carboxylate-amine ligation reactions (22) and form acylphosphate intermediates (23,24).
Characterization of Asl fm Activity-To determine the structure of the reaction product, the assay was scaled up using non-radioactive D-Asp for mass spectrometry (Fig. 3B) and tandem mass spectrometry (Fig. 3,  C and D) analyses. The monoisotopic mass of the compound eluting  (lanes 3, 4, 5, and 6, respectively). The Asl fm antiserum was raised against an Asl fm -His 6 tagged protein and therefore contained anti-His 6 antibodies (data not shown). This accounts for detection of Rac fm -His 6 in panel C. , digested with muramidases, and treated with ammonium hydroxide, and the resulting lactoyl-peptides were separated by rp-HPLC. Peaks 1-10 correspond to the major muropeptides from E. faecalis (21). Peaks A-R correspond to additional muropeptides only found in JH2-2/pSJL2(asl fm ). mAU, absorbance unit ϫ 10 3 at 210 nm.
with the same retention time as radioactive peak II was determined to be 1,264. To gain insight in the specificity of Asl fm , D-[ 14 C]Asp was replaced by 2-amino and 2-hydroxy acids, and their addition to UDP-MurNAcpentapeptide was determined by mass spectrometry. L-Asp was not a substrate, indicating that Asl fm is stereospecific. Product formation was not observed with D-Glu, D-Ala, D-iso-asparagine, and D-malic acid, indicating that Asl fm is highly specific for D-Asp in vitro. Finally, the radioactive assay confirmed that the ligase activity of Asl fm was depend-ent upon the presence of ATP and Mg 2ϩ and independent from tRNA using RNase A (data not shown).
Asl fm Is Functional in a Heterologous Host-To assess the in vivo activity of the D-aspartate ligase, plasmid pSJL2(asl fm ) was introduced in E. faecalis JH2-2, which produces two transferases of the Fem family (BppA1 and BppA2) for the addition of an L-Ala 2 side chain to the peptidoglycan precursors. This approach was used as versatile tools for gene inactivation have not been developed for E. faecium. Expression of the asl fm D-aspartate ligase gene under the control of a constitutive promoter led to production of the protein as determined by Western blot analysis with anti-Asl fm (Fig. 4A) and anti-His 6 (Fig. 4B) antisera. D-Aspartate ligase activity was detected in extracts from E. faecalis JH2-2/pSJL2(asl fm ) but not in the control strain harboring the vector pJEH11 used to construct pSJL2 (data not shown).
To investigate the impact of Asl fm ligase activity on growth rate and peptidoglycan composition, E. faecalis JH2-2/pSJL2(asl fm ) and JH2-2/ pJEH11 were grown in the presence or absence of D-Asp. The addition of D-Asp (50 mM) to the culture medium had no significant impact on the growth rate of the control strain E. faecalis JH2-2/pJEH11. The rp-HPLC profile of the muropeptides (Fig. 5A) and their structure determined by MS (Table 1) were indistinguishable from those previously reported for E. faecalis JH2-2 (21). The muropeptides contained 2 L-alanyl residues in the free N-terminal side chains and in the cross-bridges.
In the absence of D-Asp in the culture medium, production of Asl fm in E. faecalis JH2-2/pSJL2(asl fm ) did not affect the growth rate and the peptidoglycan structure (data not shown). Upon the addition of 50 mM D-Asp, the generation time increased (ϳ3-fold), and the muropeptide

Muropeptides from E. faecalis JH2-2/pSJL2(asl fm ) and E. faecalis JH2-2/pJEH11 grown in the presence of D-aspartate (50 mM)
The relative abundance (%) of the material in the peaks was calculated by integration of the absorbance at 210 nm. The sequence of the side chain and of the cross-bridge was determined by tandem mass spectrometry for peaks A to G and 1 to 4. Assignment of amino acids to the side chain and to the cross-bridge(s) is arbitrary for other muropeptides. The abbreviations are as follows: Tri, tripeptide L-Ala-D-iGln-L-Lys; Tetra, tetrapeptide; L-Ala-D-iGln-L-Lys-D-Ala ; Penta, pentapeptide L-Ala-D-iGln-L-Lys-D-Ala -D-Ala; -, not detected; NA, not applicable. The D-Aspartate Ligase of Enterococcus faecium profile was drastically altered (Fig. 5B). The L-Ala 2 -containing muropeptides were only detected as minor peaks (peaks 1-6), and a novel series of peaks (peaks A-R) was detected. The main monomer (peak C) had a monoisotopic mass of 674.3, which matched the calculated value for a D-lactoyl-pentapeptide stem substituted by a side chain consisting of one D-aspartyl residue. The structure was confirmed by MS/MS (Fig. 6), indicating that Asl fm catalyzed the addition of D-Asp to the peptidoglycan precursor of E. faecalis JH2-2 in competition with the addition of L-Ala by the BppA1 and BppA2 transferases of the host. Incorporation of D-Asp by Asl fm was efficient since only 12% of the monomers contained the usual L-Ala 2 side chain (Table 1). Hybrid side chains containing both L-Ala and D-Asp were not detected. This indicates that Asl fm did not elongate L-alanyl-containing side chains to form the sequence L-Ala-D-Asp or L-Ala 2 -D-Asp. This also indicates that the host transferases did not add L-Ala to D-Asp-containing precursors to form D-Asp-L-Ala or D-Asp-L-Ala 2 side chains. D-Asp-Substituted Precursors Are Used by the E. faecalis D,D-Transpeptidases-Since Asl fm mediated incorporation of D-Asp into the precursor of E. faecalis, we investigated the participation of the modified side chain to cross-link formation. Sequencing of the crossbridges in muropeptide dimers indicated that the D,D-transpeptidases of E. faecalis cross-linked the D-aspartate-containing precursors ( Table 1). The peptide stems substituted by D-aspartate were used in the transpeptidation reaction both as acceptors and donors. However, the substitution of L-Ala 2 by D-Asp appeared to impair the cross-linking activity of the host D,D-transpeptidases since the trimers, tetramers, and pentamers were less abundant (Fig. 5). Besides these differences, the peptidoglycan of E. faecalis JH2-2/pSJL2(asl fm ) retained several characteristics of wild-type E. faecalis peptidoglycan (7). In particular, the relative abundance of stem peptides ending in L-Lys-D-Ala-D-Ala, (pentapeptide), L-Lys-D-Ala (tetrapeptide), and L-Lys (tripeptide) was conserved (Table 1).
Rac fm Produces the Substrate of Asl fm -Incorporation of D-Asp into the peptidoglycan of E. faecalis JH2-2/pSJL2(asl fm ) was only observed when the strain was grown in the presence of D-Asp. This implies that E. faecalis does not produce D-aspartic acid, which is not a component of the peptidoglycan of this species. The open reading frame located downstream from asl fm encoded a protein related to various pyridoxal 5Ј-phosphate-independent amino acid racemases (25)(26)(27)(28). The function of this likely candidate for D-Asp production was investigated based on co-expression with asl fm in E. faecalis. Since production of D-Aspsubstituted precursors led to impaired growth of E. faecalis JH2-2/ pSJL2(asl fm ), we used the IPTG-inducible expression vector pJEH4 for plasmid construction. A bi-cistronic operon encoding Asl fm (without a His 6 tag) and the His 6 -tagged candidate racemase (Rac fm ) was cloned under the control of the inducible promoter and introduced into E. faecalis JH2-2. Western blot analysis using anti-Asl fm and anti-His 6 antibodies indicated that production of Asl fm and Rac fm was inducible by IPTG, although the promoter was leaky (Fig. 4, C and D). Induction with IPTG in E. faecalis JH2-2/pSLJ4(asl fm rac fm ) led to production of peptidoglycan containing D-Asp in the absence of this amino acid in the culture medium (data not shown). Thus, the chromosomal asl fm -rac fm cluster encoded two enzymes for incorporation of D-Asp into the peptidoglycan precursors according to the pathway depicted in Fig. 1A.

DISCUSSION
Incorporation of L-amino acids and glycine into the side chains of peptidoglycan precursors by the Fem transferases involves activation of the substrate by the aminoacyl-tRNA synthetases of the translation machinery, a pathway that cannot be directly tailored for incorporation of D-amino acids. Instead, D-aspartate has been shown in 1972 to be activated as ␤-aspartyl-phosphate (Fig. 1) in an ATP-dependent reaction, although the identity of the enzyme remained unknown for the following 33 years. Using reverse genetics (Fig. 2), we have identified the gene encoding the D-aspartate ligase of E. faecium (Asl fm ). The UDP-MurNAc-pentapeptide:D-Asp ligase activity of Asl fm (Fig. 1) has been demonstrated in vitro based on purification of the protein produced in E. coli and determination of the structure of the hexapeptide product by tandem mass spectrometry (Fig. 3). The catalytic activity of Asl fm has also been demonstrated in vivo based on heterospecific expression of the asl fm gene in E. faecalis and detection of D-Asp in the peptidoglycan cross-bridges of the recombinant strain (Figs. 5 and 6).
Genes encoding close homologues of Asl fm (Fig. 7) are present in the 10 known genomes of bacteria producing precursors substituted by D-Asp. These genes were present at a single copy per genome except for the chromosome of Lactobacillus delbrueckii, which encodes two Asl fmrelated proteins (81% identity). Close homologues of Asl fm were not detected in bacteria producing directly cross-linked peptidoglycan or cross-bridges containing L-amino acids and glycine. Thus, incorporation of D-amino acids into peptidoglycan precursors appears to be mediated by a single family of closely related proteins that are specialized in this function.
Low level similarity was also detected with the ubiquitous carbamoyl phosphate synthases and D-alanine:D-alanine ligases, indicating that Asl fm is a novel member of the ATP-grasp superfamily. Members of the family, which also includes glutathione synthetases, biotin carboxylases, and succinyl-CoA synthases, form acylphosphate intermediates and catalyze ATP-dependent ligation of a carboxyl group carbon to an amino or imino nitrogen, a hydroxyl oxygen, or a thiol sulfur (22,23,29).   The D-Ala:D-Ala ligases (24) and closely related enzymes for synthesis of D-Ala-D-lactate (30) and D-Ala-D-Ser (31) in glycopeptide-resistant Gram-positive bacteria are the only members of the family known to be involved in peptidoglycan synthesis. Assembly of the linear pentapeptide stem is performed by a distinct family of enzymes, the Mur synthetases (MurC, MurD, MurE, MurF), which sequentially add L-Ala, D-Glu, L-Lys (or an alternate diaminoacid), and the dipeptide D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, respectively (32). A fifth member of the family, Mpl, ligates the recycled tripeptide L-Ala-D-iso-Glu-L-Lys to UDP-MurNAc (33). The Mur enzymes operate by carboxyl activation of the nucleotide substrate to an acylphosphate intermediate followed by nucleophilic attack by the amino group of the condensing amino acid (MurC, MurD, MurE), dipeptide (MurF), or tripeptide (Mpl) (EC 6.3.2). In contrast, the nucleotide substrate has the opposite role in the reaction catalyzed by Asl fm as D-Asp is activated as ␤-aspartyl-phosphate prior to nucleophilic attack by the amino group of L-lysine at the third position of UDP-MurNAc-pentapeptide (EC 6.3.1) (Fig. 1). In conclusion, the assembly of peptidoglycan precursors in eubacteria involves only three classes of evolutionary unrelated peptide (or amide) bond-forming enzymes, Asl fm and the D-Ala:D-Ala ligases, the Mur synthetases, and the tRNA-dependent transferases of the Fem family.
The rac fm gene, located downstream from asl fm , appeared as a likely candidate for D-Asp production in E. faecium since it encoded a protein related to pyridoxal-independent amino acid racemase (25)(26)(27)(28). The function of Rac fm in D-Asp production was confirmed based on coexpression of the rac fm and asl fm genes in E. faecalis JH2-2. Close homologues of Rac fm were detected in the 10 species listed in Fig. 7, and the linkage with asl fm -related genes was conserved in six genomes. This protein subfamily is related to the glutamate racemases that generate D-Glu present at the second position of the pentapeptide stem of peptidoglycan precursors (34). Since synthesis of modified precursor by Asl fm in E. faecalis required either expression of the rac fm gene or the addition of D-Asp in the culture medium, racemization of aspartate and glutamate is performed by distinct enzymes.
Analysis of the specificity of Asl fm for its amino acid substrate indicated that minor modifications of D-Asp were not tolerated by the enzyme, including an increase in the length of the side chain (D-Glu), substitution of the ␣-amino group by a hydroxyl group (D-malic acid), and substitution of the ␣-carboxyl group by CONH 2 (D-isoasparagine). The latter observation indicates that the presence of D-iso-asparagine in the cross-bridges of E. faecium exclusively originates from amidation of the ␣-carboxyl group of D-Asp after its incorporation into the precursors by Asl fm . The enzyme is also highly specific in vivo since the peptidoglycan cross-bridges exclusively contain D-Asp and its amidated form in E. faecium (35), and heterospecific expression of asl fm in E. faecalis JH2-2 led only to the incorporation of D-Asp (Fig. 5).
Heterospecific expression of asl fm revealed that the PBPs of E. faecalis catalyzed peptidoglycan cross-linking with acceptor and donor stem peptides substituted by D-Asp. Thus, the presence of a free carboxyl group on the ␣-carbon of D-Asp did not prevent recognition of the ␣-amino group of the acceptor, although the reacting group is normally located at the extremity of an L-Ala 2 side chain in this host. A similar observation has been previously reported for the low affinity PBPs responsible for ␤-lactam resistance in E. faecalis (PBP5) and in methicillin-resistant S. aureus (PBP2a) (21). Both low affinity PBPs conferred ␤-lactam resistance in E. faecium, indicating that these D,D-transpeptidases catalyzed the cross-linking of D-Asp-substituted stem peptides, although they function with L-Ala 2 -and Gly 5 -substituted precursors in their original hosts, respectively. Full expression of ␤-lactam resistance mediated by PBP5 and PBP2a in E. faecium required a mutation in an unknown locus of the host. In the current study, we designed two systems for conditional production of D-Asp-substituted precursors based on the addition of this amino acid in the culture medium of a recombinant E. faecalis strain expressing only the asl fm gene or inducible expression of a bi-cistronic operon comprising both asl fm and rac fm . These systems provided direct evidence that the peptidoglycan synthesis machinery tolerates the replacement of L-Ala 2 by D-Asp in the absence of any mutation.
Vancomycin-resistant enterococci have emerged as important nosocomial pathogens in the U. S. A. and more recently in Europe, where hospital outbreaks are being reported with increasing frequency (36). Co-resistance to glycopeptides and ␤-lactams, mostly in E. faecium, has complicated the management of enterococcal infections (37), and the optimal antimicrobial therapy is not yet defined since the use of the newest agents is limited by emerging resistance and toxicity. Thus, the Asl fm ligase appears as an attractive target for the design of new narrow spectrum antibacterial agents active against multiresistant E. faecium.