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Originally published In Press as doi:10.1074/jbc.M600114200 on February 28, 2006

J. Biol. Chem., Vol. 281, Issue 17, 11586-11594, April 28, 2006
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Aslfm, the D-Aspartate Ligase Responsible for the Addition of D-Aspartic Acid onto the Peptidoglycan Precursor of Enterococcus faecium*

Samuel Bellais{ddagger}, Michel Arthur{ddagger}, Lionnel Dubost§, Jean-Emmanuel Hugonnet{ddagger}, Laurent Gutmann{ddagger}, Jean van Heijenoort||, Raymond Legrand**, Jean-Paul Brouard§, Louis Rice{ddagger}{ddagger}, and Jean-Luc Mainardi{ddagger}1

From the {ddagger}INSERM, U655-LRMA; Université Pierre et Marie Curie–Paris 6; Université Paris-Descartes, Faculté deMédecine, Centre de Recherches Biomédicales des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France, the §Muséum National d'Histoire Naturelle, USM0502, CNRS, UMR8041, Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département Recherche Développement et Diversité Moléculaire, 75005 Paris, France, the Université Paris-Descartes, Faculté de Médecine, AP-HP Hôpital Européen Georges Pompidou, 20 rue Leblanc 75908 Paris Cedex 15, France, the ||CNRS, UMR 8619, Université de Paris-Sud, Enveloppes Bactériennes et Antibiotiques, Bâtiment 430, 91405 Orsay, France, the **Aventis Pharma France, 93235 Romainville, France, and the {ddagger}{ddagger}Medical and Research Services, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106

Received for publication, January 5, 2006 , and in revised form, February 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 beta-carboxylate of D-Asp to the {epsilon}-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 {alpha}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{gamma}-D-glutamyl-L-lysyl-D-alanyl-D-alanine) and a variable side chain linked to the {epsilon}-amino group of L-Lys3 (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 beta-D-aspartyl-phosphate and links D-Asp to the {epsilon}-amino group of L-Lys3 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 cross-linking 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 beta-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 precursors 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-Ala2 by D-Asp on peptidoglycan cross-linking by the D,D-transpeptidases.


Figure 1
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  		FIGURE 1.
Incorporation of D-Asp into the peptidoglycan precursor and cross-bridge formation in E. faecium. A, D-Asp is added to the {epsilon}amino group of L-Lys3 by the Aslfm D-aspartate ligase. The reaction proceeds through formation of a beta-aspartyl-phosphate intermediate (11). D-Asp is produced by the Racfm aspartate racemase. B, peptidoglycan is polymerized from a disaccharide-peptide subunit containing beta-1-4-linked 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 {epsilon}-amino group of L-Lys3. The {alpha}-carboxyl group of D-Asp is partially amidated in mature peptidoglycan (D-Asx). The {alpha}-carboxyl group of D-iso-Glu is fully amidated (D-iGln).

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
D-Aspartate Ligase Assays—The standard assay was performed in a total volume of 25 µl containing Tris-HCl (100 mM, pH 8.5), MgCl2 (50 mM), ATP (20 mM), D-[14C]aspartic acid (0.11 mM, 2 GBq/mmol, Isobio, Fleurus, Belgium), and UDP-MurNAc-pentapeptide (0.15 mM) prepared from S. aureus (16). The reaction was incubated for 15–120 min at 37 °C and stopped by boiling for 3 min. UDP-MurNAc-pentapeptide-D-[14C]Asp was separated from D-[14C]Asp by descending paper chromatography (Whatman 4MM, Elancourt, France) with a mobile phase composed of isobutyric acid and 1 M ammonia (5:3, v/v). Alternatively, the products of the reaction were separated by reverse phase high-pressure liquid chromatography (rp-HPLC) on a Hypersil C18 column (3 µm, 4.6 x 250 nm, Interchrom, Montluçon, France) using isocratic elution (10 mM ammonium acetate, pH 5.0) at a flow rate of 0.5 ml/min. The products were detected by the absorbance at 262 nm and liquid scintillation with a Radioflow Detector (LB508; PerkinElmer Life Sciences, Courtaboeuf, France) coupled to the HPLC apparatus (L-62000A; Merck, Nogent-Sur-Marne, France).

The specificity of the D-aspartate ligase was studied by replacing D-[14C]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 (Aslfm) of E. faecium D359 (17) was partially purified in three chromatographic steps and identified as a member of the ATP-grasp protein family by MS/MS amino acid sequencing. Briefly, E. faecium D359 was grown to an OD650 nm of 0.7 in 20 liters of brain heart infusion broth (Difco, Elancourt, France), harvested by centrifugation (6,000 x 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 x 30 s. The extract was centrifuged at 7,000 x g for 10 min at 4 °C to remove cell debris, and the supernatant was ultracentrifuged at 100,000 x 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 µg) 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 –20 °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 µl 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 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.

Production of the D-Aspartate Ligase in Escherichia coli and Purification of the Protein—DNA of E. faecium D359 was amplified with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) using primers Asl1 (5'-GAGAGACCATGGTGAACAGTATTGAAAATGAAG-3') and Asl2 (5'-CTCCATGGCTAGGATCCTTCTTTCACATGAAAATACTTTTTG-3'). The PCR product was digested with NcoI and BamHI (underlined) and cloned into pET2818 (18), and the resulting plasmid (pSJL1) was introduced into E. coli BL21 (DE3) harboring the pREP4 plasmid (19). For protein production, bacteria were grown at 37 °C to an optical density at 600 nm of 0.7 in 2 liters of brain heart infusion broth containing kanamycin (50 µg/ml) and ampicillin (100 µg/ml). Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added (0.5 mM), and incubation was continued for 3.5 h at 37 °C. Aslfm was purified from a clarified lysate by affinity chromatography on Ni2+-nitrilotriacetateagarose resin (Qiagen GmbH, Hilden, Germany) and cation exchange chromatography (HiTrap SP-Sepharose fast flow, Amersham Biosciences). The protein was concentrated by ultrafiltration and stored at –20 °C in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 50% glycerol.

Western Blot Analysis—Anti-Aslfm antiserum was obtained by three subcutaneous injections at 3-week intervals of 200 µg of purified Aslfm 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-Aslfm antiserum at a 1/1000 dilution. Goat anti-rabbit IgG coupled to peroxidase (SouthernBiotech, Birmingham, AL) was used as a secondary antibody, and Aslfm was detected by chemiluminescence (ECL kit, Pierce and Amersham Biosciences). Tagged proteins were alternatively detected using polyclonal anti-His6 rabbit antibodies (Ebiosciences, San Diego, CA).

Heterospecific Expression of the D-Aspartate Ligase Gene (aslfm) 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 His6 tag. The NcoI-BamHI fragment of pSJL1 (above) containing the aslfm open reading frame was cloned into pJEH11. The resulting plasmid pSJL2(aslfm) was introduced into E. faecalis JH2-2 by electroporation (20), and clones were selected and subcultured in brain heart infusion containing 128 µg/ml gentamicin.

Co-expression of the D-Aspartate Ligase (aslfm) and Aspartate Racemase (racfm) Genes in E. faecalis—The racfm open reading frame was amplified with primers rac1 (5'-AAAGAAGGATCCTAGCCATGGAGAATTTTTTCAGTATTTTAGGCGG-3'), containing BamHI (underlined) and a stop codon (bolded), and rac2 (5'-AAAGGATCCCTTTTCCGATGCTGTATCCAATGCC-3'), containing BamHI. The PCR fragment was cloned into the BamHI restriction site of pSJL2(aslfm) (above). Insertion of the PCR product introduced a stop codon at the end of aslfm (TAG present in primer rac1) and generated an in-frame fusion between the 3' end of racfm and the sequence of the vector pJEH11 specifying the His6 tag. The resulting plasmid, pSJL3(aslfm racfm), carried a bi-cistronic operon encoding Aslfm and E. faecium His6-tagged aspartate racemase (Racfm). To obtain inducible expression of aslfm and racfm, the genes were subcloned with SacI and XbaI under the control of the IPTG-inducible promoter of vector pJEH4, generating pSJL4(aslfm racfm). The shuttle vector pJEH4 (our laboratory collection) confers resistance to spectinomycin (120 µg/ml for selection) and carries the lacI gene and lac operator fused to transcription and translation signals that are functional in enterococci.


Figure 2
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  		FIGURE 2.
Identification and purification of Aslfm. A, the detection of D-aspartate ligase activity in cytoplasmic extracts from E. faecium D359. UDP-MurNAc-pentapeptide (0.15 mM), D-[14C]aspartic acid (0.11 mM, 2 GBq/mmol), ATP (20 mM), and MgCl2 (50 mM) were incubated in the absence (–) or presence (+) of cytoplasmic extract (60 µg of protein) for 2 h at 37 °C.The products were separated by descending paper chromatography and revealed by autoradiography. B, active fraction obtained by three chromatographic steps. Sequencing of a trypsin digest by tandem mass spectrometry identified the 49-kDa protein band as the aslfm gene product (EfaeDRAFT_0086, U. S. DOE Joint Genome Institute). C, purification of Aslfm produced in E. coli.

 


Figure 3
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  		FIGURE 3.
Structure of the UDP-MurNAc-pentapeptide-D-Asp product of Aslfm. A, separation by rp-HPLC of D-[14C]Asp (peak I) and UDP-MurNAc-pentapeptide-D-[14C]Asp (peak II) formed by Aslfm purified from E. coli. CTS, counts. B, the assay was repeated with unlabeled D-Asp, and the material eluting with the same retention time as peak II was analyzed by MS. C and D, MS/MS analysis of peaks at m/z 1,265.4 and 676.3, respectively.

 
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(aslfm), and pSJL4(aslfm,racfm) 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).


Figure 4
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  		FIGURE 4.
Heterospecific expression of the aslfm and racfm genes in E. faecalis JH2-2. Western blot analysis was performed with polyclonal anti-Aslfm (A and C) or anti-His6 (B and D) antibodies. Proteins (10 µg) from crude extracts of derivatives of E. faecalis JH2-2 harboring the vector pJEH11 (lane 1), pSJL2(aslfm) (lane 2) and pSLJ4(aslfm racfm) (lanes 3–6) were separated by SDS-PAGE. Induction of the aslfm and racfm genes of pSLJ4 was performed with IPTG at 0, 0.003, 0.03, and 0.3 mM (lanes 3, 4, 5, and 6, respectively). The Aslfm antiserum was raised against an Aslfm-His6 tagged protein and therefore contained anti-His6 antibodies (data not shown). This accounts for detection of Racfm-His6 in panel C.

 


Figure 5
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  		FIGURE 5.
rp-HPLC muropeptide profiles of JH2-2/pJEH11 (A) and JH2-2/pSJL2(aslfm)(B). Peptidoglycan was extracted from bacteria grown in the presence of D-Asp (50 mM), 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(aslfm). mAU, absorbance unit x 103 at 210 nm.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Aslfm 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 His6 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 (aslfm) had been successfully identified. Aslfm belonged to the ATP-grasp protein superfamily composed of highly diverse enzymes that catalyze ATP-dependent carboxylate-amine ligation reactions (22) and form acylphosphate intermediates (23, 24).

Characterization of Aslfm 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 with the same retention time as radioactive peak II was determined to be 1,264.4 Da from peaks at m/z 1,265.4, 633.2, 644.2, and 652.2, which were assigned to be [M+H]1+, [M+2H]2+, [M+H+Na]2+, and [M+H+K]2+ ions, respectively (Fig. 3B). This monoisotopic mass matches the predicted value of 1,264.4 Da for UDP-MurNAc-pentapeptide-D-Asp. Fragmentation of the peak at m/z 1,265.4 gave ions at m/z 861.4 and 676.3 corresponding to the MurNAc-pentapeptide-D-Asp and lactoyl-pentapeptide-D-Asp moieties of the molecule (Fig. 3C). An additional stage of MS-MS was performed on the peak at m/z 676.3 (lactoyl-pentapeptide-D-Asp) to sequence the peptide portion of the molecule (Fig. 3D). The peak at m/z 561.3 matched the predicted value for loss of one D-aspartate residue. Loss one or two C-terminal D-Ala from the ion at m/z 676.3 gave ions at m/z 587.3 and 516.3, respectively. The peak at m/z 533.2 and 404.2 matched the expected mass of {gamma}-D-Glu-L-Lys-(N{epsilon}-D-Asp)-D-Ala-D-Ala and L-Lys-(N{epsilon}-D-Asp)-D-Ala-D-Ala. These ions confirmed that one D-aspartate residue is branched to the L-lysyl residue. The remaining peaks can be accounted for by combinations of the fragmentations described above. For example, loss of one or two D-Ala from peak at m/z 533.2 resulted in the peaks at m/z 444.2 and 373.2, respectively.

To gain insight in the specificity of Aslfm, D-[14C]Asp was replaced by 2-amino and 2-hydroxy acids, and their addition to UDP-MurNAc-pentapeptide was determined by mass spectrometry. L-Asp was not a substrate, indicating that Aslfm is stereospecific. Product formation was not observed with D-Glu, D-Ala, D-iso-asparagine, and D-malic acid, indicating that Aslfm is highly specific for D-Asp in vitro. Finally, the radioactive assay confirmed that the ligase activity of Aslfm was dependent upon the presence of ATP and Mg2+ and independent from tRNA using RNase A (data not shown).

Aslfm Is Functional in a Heterologous Host—To assess the in vivo activity of the D-aspartate ligase, plasmid pSJL2(aslfm) was introduced in E. faecalis JH2-2, which produces two transferases of the Fem family (BppA1 and BppA2) for the addition of an L-Ala2 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 aslfm 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-Aslfm (Fig. 4A) and anti-His6 (Fig. 4B) antisera. D-Aspartate ligase activity was detected in extracts from E. faecalis JH2-2/pSJL2(aslfm) but not in the control strain harboring the vector pJEH11 used to construct pSJL2 (data not shown).

To investigate the impact of Aslfm ligase activity on growth rate and peptidoglycan composition, E. faecalis JH2-2/pSJL2(aslfm) 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.


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  		TABLE 1
Muropeptides from E. faecalis JH2–2/pSJL2(aslfm) 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.

 
In the absence of D-Asp in the culture medium, production of Aslfm in E. faecalis JH2-2/pSJL2(aslfm) 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 (~3-fold), and the muropeptide profile was drastically altered (Fig. 5B). The L-Ala2-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 Aslfm 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 Aslfm was efficient since only 12% of the monomers contained the usual L-Ala2 side chain (Table 1). Hybrid side chains containing both L-Ala and D-Asp were not detected. This indicates that Aslfm did not elongate L-alanyl-containing side chains to form the sequence L-Ala-D-Asp or L-Ala2-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-Ala2 side chains.

D-Asp-Substituted Precursors Are Used by the E. faecalis D,D-Transpeptidases—Since Aslfm 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 cross-bridges 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-Ala2 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(aslfm) 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).

Racfm Produces the Substrate of Aslfm—Incorporation of D-Asp into the peptidoglycan of E. faecalis JH2-2/pSJL2(aslfm) 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 aslfm encoded a protein related to various pyridoxal 5'-phosphate-independent amino acid racemases (25–28). The function of this likely candidate for D-Asp production was investigated based on co-expression with aslfm in E. faecalis. Since production of D-Asp-substituted precursors led to impaired growth of E. faecalis JH2-2/pSJL2(aslfm), we used the IPTG-inducible expression vector pJEH4 for plasmid construction. A bi-cistronic operon encoding Aslfm (without a His6 tag) and the His6-tagged candidate racemase (Racfm) was cloned under the control of the inducible promoter and introduced into E. faecalis JH2-2. Western blot analysis using anti-Aslfm and anti-His6 antibodies indicated that production of Aslfm and Racfm was inducible by IPTG, although the promoter was leaky (Fig. 4, C and D). Induction with IPTG in E. faecalis JH2-2/pSLJ4(aslfm racfm) 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 aslfm-racfm cluster encoded two enzymes for incorporation of D-Asp into the peptidoglycan precursors according to the pathway depicted in Fig. 1A.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 beta-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 (Aslfm). The UDP-MurNAc-pentapeptide:D-Asp ligase activity of Aslfm (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 Aslfm has also been demonstrated in vivo based on heterospecific expression of the aslfm gene in E. faecalis and detection of D-Asp in the peptidoglycan cross-bridges of the recombinant strain (Figs. 5 and 6).


Figure 6
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  		FIGURE 6.
Structure of the main muropeptide monomer (peak C) from JH2-2/pSJL2(Aslfm). A and B, fragmentation of the ion at m/z 675.3 (A) and inferred structure (B). Peaks at m/z 560.3 matched the predicted value for loss of one N-terminal D-Asp residue. Loss of one and two D-Ala from the C terminus of the pentapeptide stem gave ions at m/z 586.2 and 515.2. The peak at m/z 532.2 matched the predicted value for loss of D-Lac-L-Ala (where D-Lac is D-lactoyl). Additional loss of one and two D-Ala from the C terminus gave ions at m/z 443.2 and 372.1 (indicated in parentheses in B). The peak at m/z 144.0 corresponded to the D-Lac-L-Ala moiety of the molecule. Fragmentation at the D-iGln-L-Lys peptide bond produced ions at 272.1 and 404.2. Additional ions could be accounted for by combinations of the fragmentation described above.

 
Genes encoding close homologues of Aslfm (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 Aslfm-related proteins (81% identity). Close homologues of Aslfm 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 Aslfm 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 Aslfm as D-Asp is activated as beta-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, Aslfm and the D-Ala:D-Ala ligases, the Mur synthetases, and the tRNA-dependent transferases of the Fem family.


Figure 7
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  		FIGURE 7.
Alignment of Aslfm with putative D-aspartate ligases from lactobacilli, lactococci, and pediococci. Conserved residues in the ATP-grasp protein family that interact with ATP are indicated by an asterisk. Ent. faeci, E. faecium; Ltc lacti, Lactococcus lactis subsp. Lactis IL1403; Ltc cremo, Lactococcus lactis subsp cremoris SK11; Ltb gasse, Lactobacillus gasseri ATCC 333323; Ltb johns, Lactobacillus johnsonii NCC 533; Ltb delbr, L delbrueckii subsp bulgaricus ATCC BAA-365; Ltb brevi, Lactobacillus brevis ATCC 367; Ltb casei, Lactobacillus casei ATCC 334; Ltb acido, Lactobacillus acidophilus NCFM; Ltb sakei, Lactobacillus sakei subsp sakei 23K; Ped pento, Pediococcus pentosaceus ATCC 24745.

 
The racfm gene, located downstream from aslfm, appeared as a likely candidate for D-Asp production in E. faecium since it encoded a protein related to pyridoxal-independent amino acid racemase (25–28). The function of Racfm in D-Asp production was confirmed based on co-expression of the racfm and aslfm genes in E. faecalis JH2-2. Close homologues of Racfm were detected in the 10 species listed in Fig. 7, and the linkage with aslfm-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 Aslfm in E. faecalis required either expression of the racfm 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 Aslfm 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 {alpha}-amino group by a hydroxyl group (D-malic acid), and substitution of the {alpha}-carboxyl group by CONH2 (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 {alpha}-carboxyl group of D-Asp after its incorporation into the precursors by Aslfm. 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 aslfm in E. faecalis JH2-2 led only to the incorporation of D-Asp (Fig. 5).

Heterospecific expression of aslfm 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 {alpha}-carbon of D-Asp did not prevent recognition of the {alpha}-amino group of the acceptor, although the reacting group is normally located at the extremity of an L-Ala2 side chain in this host. A similar observation has been previously reported for the low affinity PBPs responsible for beta-lactam resistance in E. faecalis (PBP5) and in methicillin-resistant S. aureus (PBP2a) (21). Both low affinity PBPs conferred beta-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-Ala2- and Gly5-substituted precursors in their original hosts, respectively. Full expression of beta-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 aslfm gene or inducible expression of a bi-cistronic operon comprising both aslfm and racfm. These systems provided direct evidence that the peptidoglycan synthesis machinery tolerates the replacement of L-Ala2 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 beta-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 Aslfm ligase appears as an attractive target for the design of new narrow spectrum antibacterial agents active against multiresistant E. faecium.


   FOOTNOTES
 
* 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. Back

1 To whom correspondence should be addressed: INSERM U655-LRMA, Centre de Recherches Biomédicales des Cordeliers, Université Paris 6; Université Paris-Descartes, 15 rue de l'Ecole de Médecine 75270 Paris Cedex 06, France. Tel.: 33-1-42-34-68-62; Fax: 33-1-43-25-68-12; E-mail: jlmainar{at}bhdc.jussieu.fr.

2 The abbreviations used are: MurNAc, N-acetylmuramic acid; Asl, beta-D-aspartate:UDP-N-acetylmuramoyl-L-alanyl-{gamma}-D-glutamyl-L-lysyl-D-alanyl-D-alanine ligase (ADP-forming) (EC.6.3.1.x); Rac, aspartate racemase; IPTG, isopropyl-beta-D-thiogalactopyranoside; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high-pressure liquid chromatography; rp-HPLC, reverse-phase HPLC; PBP, penicillin-binding protein; iGln, iso-Gln. Back


   ACKNOWLEDGMENTS
 
We thank S. Mesnage for helpful discussion and M. Fourgeaud for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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