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Journal of Bacteriology, September 2001, p. 5122-5127, Vol. 183, No. 17
INSERM E0004-LRMA, UFR Broussais-Hôtel
Dieu, Université Paris VI, 75270 Paris,1
and Biochimie Moléculaire et Cellulaire, UMR 8619 CNRS,
Université Paris-Sud, 91405 Orsay Cedex,2
France
Received 20 February 2001/Accepted 13 June 2001
Many species of gram-positive bacteria produce branched
peptidoglycan precursors resulting from the transfer of various
L-amino acids or glycine from amino acyl-tRNA to the
In gram-positive bacteria, the
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5122-5127.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of the
UDP-MurNAc-Pentapeptide:L-Alanine Ligase for Synthesis of
Branched Peptidoglycan Precursors in Enterococcus
faecalis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-amino group of L-lysine. The
UDP-MurNAc-pentapeptide:L-alanine ligase and alanyl-tRNA synthetase genes from Enterococcus faecalis were
identified, cloned, and overexpressed in Escherichia coli.
The purified enzymes were necessary and sufficient for tRNA-dependent
addition of L-alanine to
UDP-MurNAc-pentapeptide in vitro. The ligase belonged to the Fem family
of proteins, which were initially identified genetically as factors
essential for methicillin resistance in Staphylococcus aureus.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-amino group of L-lysine in the pentapeptide stem of
peptidoglycan precursors is often replaced by amino acid chains of
various lengths and compositions (19) (Fig.
1). These amino acids form cross bridges
between L-Lys3 and
D-Ala4 following cross-linking of the
peptide stems from nascent chains of peptidoglycan by the
D,D-transpeptidases. The ligases for the
addition of glycine and L-amino acids to the -amino
group of L-lysine use aminoacyl-tRNAs as substrates,
whereas D-amino acids are added in a tRNA-independent
reaction (15, 20).
View larger version (12K):
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FIG. 1.
Structure of cross-linked peptidoglycan. (A) Fragment of
the primary structure of peptidoglycan. The position of the peptide
bond formed by the D,D-transpeptidases
(penicillin-binding proteins) between the acceptor and donor peptide
stems is indicated (cross-link). The C-terminal D-Ala
residue of the pentapeptide acceptor stem (shown in parentheses) is
generally cleaved by D,D-carboxypeptidases. (B)
Sequence of the cross bridges in various bacterial species. The amino
acid residues are added to the -amino group of L-Lys of
peptidoglycan precursors by various ligases discussed in the text.
D-Asp is incorporated into the peptidoglycan precursors of
E. faecium and is secondarily partially amidated
(D-Asx may be D-Asp or D-Asn),
according to Schleifer and Kandler (19). Arrows
indicate CO
NH orientation of peptide bonds.
In Staphylococcus aureus, the femA,
femB, and fmhB genes were shown to be essential
for incorporation of glycine into the side chain of peptidoglycan
precursors (18, 21). The femAB locus was
initially identified as a factor essential for methicillin resistance
(fem) based on random insertional inactivation of
chromosomal genes and a screen for reduced expression of resistance
mediated by the penicillin binding protein 2A (PBP2A) (2,
5). Inactivation of femA or femB was
subsequently reported to prevent incorporation of glycine residues at
positions 2 to 5 or positions 4 to 5 of the penta-glycine cross bridge
since muropeptides cross-linked by one or three glycine residues were
detected in the corresponding mutants (4, 21).
Inactivation of fmhB, formerly femX, is lethal,
but the construction of a mutant conditionally expressing fmhB under the control of a xylose-inducible promoter showed
that the gene was essential for synthesis of branched peptidoglycan precursors (18). These results indicated that the
fem gene products were required for incorporation of glycine
at positions 1 (FmhB), 2 and 3 (FemA), and 4 and 5 (FemB) of the cross
bridge, although the catalytic activity of the proteins was not
directly assessed (18). Similarly, inactivation of two
fmhB homologues in Streptococcus pneumoniae,
designated murM (fibA) and murN
(fibB), reduced addition of L-Ala or
L-Ser to the -amino group of
L-Lys and subsequent addition of a second
L-Ala residue, respectively (6, 7, 23). Overall, disruption of the murMN operon reduced
the proportion of branched peptide stems in the peptidoglycan from 89 to 33% (6). In contrast to what occurs in S. aureus (18), direct cross-linking of
L-Lys to D-Ala occurs in
S. pneumoniae, and the murMN operon was
accordingly reported to be unessential (6). However,
production of branched peptidoglycan precursors was necessary for
expression of penicillin resistance (6) and appeared to affect mainly the activity of mosaic PBP2x (23).
In enterococci, streptococci, and staphylococci, the ligation reactions are catalyzed by membrane-associated enzymes acting exclusively or preferentially on late peptidoglycan precursors linked to the undecaprenyl-phosphate lipid carrier, namely undecaprenyl-PP-MurNAc-pentapeptide (lipid I) and undecaprenyl-PP-MurNAc(pentapeptide)-GlcNAc (lipid II) (14). This conclusion is supported by the detection of only small amounts (<4%) of UDP-MurNAc-hexapeptide in Staphylococcus haemolyticus and S. aureus in spite of high-level accumulation (ca. 12-fold) of UDP-MurNAc-pentapeptide in bacteria treated with ramoplanin, which blocks lipid II synthesis (3). Similarly, moderate accumulation of L-Ala-containing UDP-MurNAc-hexapeptide was detected in penicillin-treated Enterococcus faecalis (12). Finally, UDP-MurNAc-pentapeptide was not a substrate of the ligases of S. aureus and Streptococcus pyogenes, suggesting that the enzymes act at a later step of peptidoglycan synthesis (8). In contrast, Weissella viridescens, formerly Lactobacillus viridescens, produces a soluble UDP-MurNAc-pentapeptide:L-alanine ligase (15). The enzyme was purified and characterized in 1970 (16), but the corresponding gene was identified only recently (8). In this study, the alanyl-tRNA synthetase, UDP-MurNAc-pentapeptide:L-alanine ligase, and tRNA from W. viridescens were partially purified to develop an assay for ligation of L-alanine to UDP-MurNAc-pentapeptide. This assay was used to identify the alanyl-tRNA synthetase and ligase genes for incorporation of the first L-alanine of the E. faecalis cross bridge.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In vitro UDP-MurNAc-hexapeptide synthesis. The assay was performed in a total volume of 15 µl containing Tris-HCl (50 mM, pH 7.2), MgCl2 (12.5 mM), ATP (2.5 mM), L-[14C]alanine (67 µM, 93 mCi/mmol; ICN, Costa Mesa, Calif.), UDP-MurNAc-pentapeptide (67 µM) purified from S. aureus as previously described (1), and fractions containing tRNA (4 µg), alanyl-tRNA synthetase, and UDP-MurNAc-pentapeptide:L-alanine ligase. The reaction mixture was incubated 30 min at 30°C and the reaction was stopped by heating at 96°C for 2 min. L-[14C]alanine was separated from [14C]UDP-MurNAc-hexapeptide by descending paper chromatography (Whatman no. 4 filter paper) with a mobile phase composed of isobutyric acid and 1 M ammonia (5:3, vol/vol). The radioactive spots were located and quantified with a radioactivity scanner (model Multi-Tracermaster LB285; EG and G Wallac/Berthold, Courtaboeuf, France). L-Alanine and UDP-MurNAc-hexapeptide were also separated by high-pressure liquid chromatography (HPLC) at 30°C on a Hypersil C18 column (3 µm, 4.6 by 250 mm; Interchrom, Montluçon, France) at a flow rate of 0.5 ml/min with a 0 to 4% acetonitrile gradient in 10 mM ammonium acetate, pH 5.0 (3). Products were detected by the absorbance at 262 nm and, when appropriate, by liquid scintillation with a Radioflow Detector (LB508; Perkin-Elmer, Courtaboeuf, France) coupled to the HPLC apparatus (L-62000A; Merck, Nogent-sur-Marne, France).
Partial purification of W.
viridescens
UDP-MurNAc-pentapeptide:L-alanine ligase, alanyl-tRNA
synthetase, and tRNA.
W. viridescens CIP 102810 T
(Institut Pasteur collection), formerly designated L. viridescens ATCC 12706 (15), was grown to late
exponential phase in lactobacillus MRS broth (Difco, Elancourt, France). Bacteria were ground with alumina in a chilled mortar at
4°C. Soluble proteins were loaded onto a DEAE Sepharose Fast Flow
column (XK26/70; Pharmacia, Saclay, France) and eluted with a linear (0 to 300 mM) KCl gradient as previously described (16). Fraction I contained
UDP-MurNAc-pentapeptide:L-alanine ligase and
alanyl-tRNA synthetase activity (elution between 170 and 200 mM KCl). A
10-ml fraction eluting at ca. 250 mM KCl was treated with phenol,
sodium acetate (pH 5.2) was added to a final concentration of 0.3 M,
and nucleic acids were precipitated with 3 volumes of ethanol at
20°C for 30 min. The preparation was centrifuged in aliquots in a
microcentrifuge at 4°C for 20 min. The pellets were washed
with ethanol, dried under vacuum, and resuspended in a total volume of
10 ml of water. The resulting preparation, designated fraction II,
contained tRNA (2 µg/µl), as judged by agarose gel electrophoresis
and absorbance measurements.
Plasmid construction. Three E. faecalis genes encoding proteins related to the Bacillus subtilis alanyl-tRNA synthetase (alaSEfa) and to the S. aureus FmhB protein (orf1 and orf2) were identified based on sequence comparison at The Institute for Genomic Research (TIGR) website (http://www.tigr.org). Genomic DNA from E. faecalis JH2-2 (9) was prepared (11), and the alaSEfa, orf1, and orf2 genes were amplified by PCR using the pwo DNA polymerase (Roche, Meylan, France). The PCR products were purified by agarose gel electrophoresis and cloned into vector pCRblunt (Invitrogen, Groningen, The Netherlands) generating plasmids pDA18(alaSEfa), pDA4(orf1), and pDA15(orf2).
The alanyl-tRNA synthetase gene was subcloned into vector pTrcHis60 (17) to generate a translational fusion with 6 codons specifying His at the 3' end of the gene. The resulting plasmid construct, designated pDA26, was obtained by cloning the BspHI-BamHI fragment of pDA18 between the NcoI and BglII sites of pTrcHis60. Plasmid pDA28(orf1) was generated by ligating the SacI-SmaI fragment of pDA4 with pTrc99A (Pharmacia) digested with the same enzymes placing orf1 under the control of the ptrc promoter of the vector. Similarly, pDA29(orf2) was obtained by cloning orf2 of pDA15 into pTrc99A using SacI and SmaI. Derivatives of pCRblunt were propagated in Escherichia coli Top10 (Invitrogen). E. coli JM83 (22) was the host for other plasmids. All cultures of E. coli strains were performed at 37°C in brain heart infusion (BHI) broth or agar (Difco) containing ampicillin (100 µg/ml) or kanamycin (50 µg/ml) for plasmid selection.Purification of the E. faecalis alanyl-tRNA
synthetase.
E. coli harboring recombinant plasmid
pDA26(alaSEfa) was grown to an optical
density at 600 nm of ca. 0.3 in 1 liter of BHI broth containing 100 µg of ampicillin per ml,
isopropyl-
-D-thiogalactopyranoside (IPTG) was
added to a final concentration of 1 mM, and incubation was continued
for 2 h. Bacteria were harvested by centrifugation (8,000 × g for 20 min at 4°C), washed in 100 ml of 50 mM Tris-HCl, pH 7.5, and resuspended in 5 ml of the same buffer containing 2 mM
2-mercaptoethanol and 300 mM KCl (buffer A). Bacteria were disrupted by
sonication with a Branson Sonifier 450 for 2 min with cooling, the
extract was centrifuged at 27,000 × g for 30 min at
4°C, and the supernatant (crude extract) was mixed with 4 ml of
Ni2+-nitrilotriacetate-agarose resin (Pharmacia)
previously equilibrated with buffer A. After 2 h of incubation at
4°C, the resin was recovered by centrifugation and washed with buffer
A containing increasing concentrations of imidazole (0, 10, 15, 20, 25, 30, 40, 100, and 200 mM). Proteins eluting at 200 mM imidazole were
loaded onto a Superdex 75 HR10/30 gel filtration column (Pharmacia)
equilibrated with 50 mM Tris-HCl, pH 7.5, containing 2 mM
2-mercaptoethanol.
Preparation of E. faecalis crude extracts containing ORF1 and ORF2. E. coli JM83/pDA28(orf1) and JM83/pDA29(orf2) were grown to an optical density at 600 nm of 0.7 in 1 liter of BHI broth containing 100 µg of ampicillin per ml, and induction was performed with 1 mM IPTG for 2 h. Bacteria were collected by centrifugation (8,000 × g for 20 min at 4°C), washed in 100 ml of 50 mM Tris-HCl, pH 7.2, and resuspended in 5 ml of the same buffer containing 2 mM 2-mercaptoethanol (buffer B). Bacteria were disrupted by sonication and centrifuged (27,000 × g for 30 min at 4°C), and the supernatants were collected (crude extracts).
Purification of E. faecalis ORF2. The crude extract from E. coli JM83/pDA29(orf2) was loaded onto a 10-ml Bio-Scale Q anion exchange column (Bio-Rad, Ivry sur Seine, France) equilibrated in buffer B at a flow rate of 1 ml/min, and elution was performed with a 0 to 2 M NaCl gradient in buffer B. A 5-ml fraction eluting at ca. 320 mM NaCl (fraction 1) was saved for further purification steps.
Solid ammonium sulfate was added to fraction 1 to a final concentration of 1.8 M, precipitated proteins were removed by centrifugation (27,000 × g for 30 min at 4°C), and the supernatant (fraction 2) was loaded at a flow rate of 1 ml/min onto an alkyl Superose HR5/5 column (Pharmacia) equilibrated with buffer C (50 mM potassium phosphate, pH 7.0, 2 mM 2-mercaptoethanol) containing 1.8 M ammonium sulfate. A 1.8 to 0 M ammonium sulfate gradient was applied in buffer C, and proteins eluting between 1.17 and 0.99 M were pooled (fraction 3). Eight milliliters of buffer C were added to fraction 3 (2 ml) and the proteins were loaded onto a HiTrap heparin affinity column (1 ml; Pharmacia) equilibrated in buffer C at a flow rate of 0.17 ml/min. Elution was performed with a 0 to 2 M NaCl gradient in buffer C, providing a 0.25-ml fraction eluting at ca. 900 mM NaCl (fraction 4). Gel filtration was performed on fraction 4 with a Superdex 75 HR10/30 column (Pharmacia) equilibrated with buffer C containing 200 mM NaCl at a flow rate 0.5 ml/min. Fraction 5 eluted between 8.5 and 9.0 ml.Mass spectrometry. Samples of UDP-MurNAc-hexapeptide produced by the W. viridescens and E. faecalis ligases were isolated by HPLC, lyophilized, and dissolved in H2O:CH3CN (50:50, vol/vol). A drop of 10% ammonium hydroxide was added to improve ionization, and the sample was injected at a flow rate of 20 µl/min into an electrospray Micromass LCT mass spectrometer in the negative mode.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Synthesis of UDP-MurNAc-hexapeptide by W.
viridescens extracts.
Three fractions containing partially
purified tRNA, alanyl-tRNA synthetase, and
UDP-MurNAc-pentapeptide:L-alanine ligase were obtained by
ion exchange and hydrophobic-interaction chromatography (Table
1; Materials and Methods). Addition of
L-[14C]Ala to
UDP-MurNAc-pentapeptide required these three fractions, Mg2+, and ATP, as previously described
(16). Formation of the radioactive UDP-MurNAc-hexapeptide
was abolished by RNase. The assay developed with partially purified
enzymes and tRNA from W. viridescens was subsequently used
to identify the alanyl-tRNA synthetase and
UDP-MurNAc-pentapeptide:L-alanine ligase genes of
E. faecalis based on cloning and expression in E. coli (Table 1). Such heterologous systems for branched
peptidoglycan synthesis were expected to be functional since
aminoacyl-tRNA synthetases and ligases are not strictly specific for
the tRNA issued from the same species (8, 13).
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Overproduction and purification of the E. faecalis alanyl-tRNA synthetase. The partial E. faecalis genome sequence available at the TIGR website contained a single open reading frame encoding a protein displaying high-level amino acid identity with the alanyl-tRNA synthetases from B. subtilis (61%) and E. coli (45%). The E. faecalis gene identified by sequence comparison (alaSEfa) was amplified by PCR and cloned into vector pTrcHis60 to generate a translational fusion with six histidine codons at the 3' end of the gene. The resulting plasmid construct, pDA26(alaSEfa), specified an active alanyl-tRNA synthetase since the crude E. coli extract harboring the plasmid could replace W. viridescens fraction IV in the assay for UDP-MurNAc-hexapeptide synthesis (Table 1). The E. faecalis alanyl-tRNA synthetase containing the C-terminal six-His tag was purified in two steps based on affinity chromatography on a nickel column and exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the expected ca. 100-kDa protein band estimated to be >95% pure (data not shown).
Identification of the E. faecalis
UDP-MurNAc-pentapeptide:L-alanine ligase gene.
The
partial E. faecalis genome sequence contained two open
reading frames (orf1 and orf2) encoding
polypeptides related to the S. aureus FmhB protein. These
open reading frames were amplified and cloned into the expression
vector pTrc99A. Both recombinant plasmids,
pDA28(orf1) and pDA29(orf2), directed the
synthesis of soluble proteins of ca. 46 kDa (Fig.
2 and data not shown) in agreement with
the calculated molecular mass of the deduced products of
orf1 (48,321 Da) and orf2 (46,023 Da). The crude
extract from E. coli pDA29(orf2) contained
UDP-MurNAc-pentapeptide:L-alanine ligase activity
(Table 1). Addition of
L-[14C]Ala to
UDP-MurNAc-pentapeptide was not observed with the crude extract from
E. coli JM83/pDA28(orf1).
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Purification of the E. faecalis UDP-MurNAc-pentapeptide:L-alanine ligase. The ligase was purified by anion exchange, hydrophobic-interaction, affinity, and exclusion chromatographies (Fig. 2; Materials and Methods). Precipitation of the protein was the major difficulty in the elaboration of the purification procedures. This difficulty was overcome by avoiding dialysis and maintaining the ionic strength above 250 mM.
Characterization of the product of the E. faecalis
and W. viridescens ligases.
The
[14C]UDP-MurNAc-hexapeptides produced by the
W. viridescens and E. faecalis enzymes were
analyzed by HPLC coupled to a radioactive flow detector (Fig.
3A and B). The HPLC profiles revealed a
single radioactive peak in addition to
L-[14C]alanine. The
products of the W. viridescens and E. faecalis ligases had the same retention time and eluted after
UDP-MurNAc-pentapeptide as expected (Fig. 3A and B).
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and
[M-2H]2 ions, respectively (Fig.
4A). The molecular mass of the product of
the reaction catalyzed by the W. viridescens ligase was
found to be 1,220 Da from peaks at m/z 1,219.8 and 609.3, which were assigned to be [M-H] and
[M-2H]2 ions, respectively (Fig. 4B). The
same peaks (1,219.7 and 609.3) were detected in the mass spectrum for
the product obtained with the E. faecalis ORF2 (Fig. 4C).
These molecular mass assignments were confirmed by the presence of
[M+Na-2H] and
[M+Na-3H]2 adduct ions. These molecular
masses, 1,149 and 1,220 Da, obtained by electrospray mass spectrometry,
match the predicted values of 1,149 and 1,220 Da for
UDP-MurNAc-pentapeptide and UDP-MurNAc-hexapeptide, respectively.
Together, these results indicate that the W. viridescens and
E. faecalis ligases catalyze the same reaction.
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Comparison of the UDP-MurNAc-pentapeptide:L-alanine
ligases of W. viridescens and E.
faecalis
Synthesis of UDP-MurNAc-hexapeptide by the
W. viridescens enzyme was independent of the
concentration of UDP-MurNAc-pentapeptide in the 70 to 1,120 µM range
(Fig. 5) in agreement with the reported Km value of 0.2 µM
(16). In contrast, the amount of UDP-MurNAc-hexapeptide
produced by the E. faecalis ligase increased with the
concentration of UDP-MurNAc-pentapeptide up to the highest concentration tested (1,120 µM) (Fig. 5), implying a
Km greater than 200 µM. Overall, the
E. faecalis enzyme appeared catalytically less active
than the W. viridescens enzyme since similar amounts
of proteins were used to generate the data in Fig. 5, although the
W. viridescens enzyme was not extensively purified. The
difference in the activities of the two enzymes could be at least
partially due to the different Km for UDP-MurNAc-pentapeptide. Impurities present in the W.
viridescens enzyme preparation may also be involved since an
unknown high-molecular-mass factor (>200 kDa) was reported to
accelerate the reaction catalyzed by the W. viridescens enzyme four- to eightfold (8).
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Assay for UDP-MurNAc-heptapeptide synthesis. HPLC chromatograms revealed only two radioactive peaks corresponding to UDP-MurNAc-hexapeptide and L-alanine (Fig. 3A and B). This observation suggests that the W. viridescens and E. faecalis ligases added a single L-alanine to UDP-MurNAc-pentapeptide. To confirm this result, unlabeled UDP-MurNAc-hexapeptide was prepared as described above (Fig. 3C and D) and incubated with various extracts containing ligase activity under the standard assay conditions described in Materials and Methods. [14C]UDP-MurNAc-heptapeptide was not detected with the purified E. faecalis ligase or with the partially purified W. viridescens ligase (fraction III), confirming that these enzymes do not add more than one L-alanine to UDP-MurNAc-pentapeptide.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The E. faecalis gene encoding the ligase for addition of the first amino acid in the side chain of peptidoglycan precursors (Fig. 1) was identified on the basis of sequence homology with the S. aureus fmhB gene, purification of the encoded protein (Fig. 2), and demonstration of UDP-MurNAc-pentapeptide:L-alanine ligase activity (Fig. 3 and 4). Synthesis of UDP-MurNAc-hexapeptide was obtained in vitro by using extensively purified alanyl-tRNA synthetase and ligase, both produced in E. coli, which does not possess the synthesis pathway for branched peptidoglycan precursors. Thus, incorporation of the first L-alanine into the side chain of peptidoglycan precursors does not require any additional enzyme in E. faecalis. In contrast, previous identification of the proteins involved in similar pathways in S. aureus (18) and S. pneumoniae (7) were based only on gene inactivation and determination of the structure of the cross bridge in mature peptidoglycan. In agreement with this, synthesis of the UDP-MurNAc-hexapeptide has been recently obtained by using purified ligase from W. viridescens (8).
The assay for UDP-MurNAc-hexapeptide synthesis proved useful in identifying the E. faecalis ligase (Table 1), although this enzyme is likely to preferentially act in vivo on the lipid intermediates, as reported for several membrane-associated ligases of gram-positive cocci (enterococci, staphylococci, and streptococci) (3, 8, 14). W. viridescens is an exception since it produces a soluble ligase for addition of L-alanine to cytoplasmic UDP-MurNAc-pentapeptide (8, 15). The difference in the apparent Km values of the E. faecalis and W. viridescens ligases for UDP-MurNAc-pentapeptide is consistent with this notion since it implies that the two enzymes may preferentially use different substrates in vivo (Fig. 5; see also Results). Localization of the ligase from gram-positive cocci at the inner surface of the membrane may optimize interaction with the lipid intermediates. The nature of the association with the membrane remains unknown. Binding to other peptidoglycan biosynthesis enzymes may be involved since the E. faecalis ligase does not appear to contain a membrane anchor composed of clustered hydrophobic amino acids. In addition, the ligase, as overproduced in E. coli, was soluble, indicating that the enzyme was not strongly associated with the lipid bilayer in this host (Fig. 2 and data not shown).
Comparison of pairs of sequences and phylogenetic analysis (data not shown) indicated that the E. faecalis ligase is closely related (39% identity) to MurM, which adds L-Ala or L-Ser at the first position of the cross bridge in S. pneumoniae (7). FmhB belonged to the same lineage, suggesting that the ligases adding the first amino acid in the cross bridges form a cluster of related sequences and may therefore be orthologs. ORF1 from E. faecalis and MurN from S. pneumoniae may also be orthologs (37% identity) and belong to a distinct lineage also including FemA and FemB. These observations are clearly limited and should be critically evaluated since the structure and mode of synthesis of the cross bridge can be extremely variable in related bacteria (Fig. 1) (19). For example, ligases from species belonging to the same genus, such as E. faecalis and E. faecium, may incorporate amino acids of the L or D configuration (Fig. 1) by distinct pathways involving mechanistically unrelated ligases (15, 20). This observation is surprising since it implies that the essential D,D-transpeptidases from related species use different acceptors bearing L- or D-amino acids in the cross-linking reaction (Fig. 1).
The ligases for branched peptidoglycan synthesis are attractive targets
for the design of new antibiotics (10). The spectra of
such antibiotics would be expected to be narrow since the pathway is
not present in many bacteria, including Pseudomonas
aeruginosa and members of the family
Enterobacteriaceae, but this may be advantageous by limiting
the overall spread of resistance. Inhibitors of the ligases for
branched peptidoglycan synthesis may also be useful to restore
-lactam activity against resistant strains since the integrity of
the side chain of the acceptor appears to be essential for the
activities of PBP2x in S. pneumoniae (7) and of
PBP2A in S. aureus (18), although
murMN and femAB are not essential operons for
these two organisms, respectively.
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ACKNOWLEDGMENTS |
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This work was supported by Wyeth-Ayerst Research and by the Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires (MENRT).
E. faecalis genome sequence data were kindly provided by The Institute for Genomic Research (TIGR) as publicly released at http: //www.tigr.org. We thank K. Tabei and M. M. Siegel for MS analysis.
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FOOTNOTES |
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* Corresponding author. Mailing address: LRMA, Université Paris VI, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France. Phone: 33 (0)1 43 25 00 33. Fax: 33 (0)1 43 25 68 12. E-mail: michel.arthur{at}bhdc.jussieu.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, September 2001, p. 5122-5127, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5122-5127.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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