JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JB.00087-07v1
189/11/3987    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hervé, M.
Right arrow Articles by Mengin-Lecreulx, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hervé, M.
Right arrow Articles by Mengin-Lecreulx, D.
Journal of Bacteriology, June 2007, p. 3987-3995, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00087-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:L-Alanyl-{gamma}-D-Glutamyl-meso- Diaminopimelate Ligase{triangledown}

Mireille Hervé,1,2* Audrey Boniface,2 Stanislav Gobec,3 Didier Blanot,1,2 and Dominique Mengin-Lecreulx1,2

CNRS, Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR8619, Orsay, France 91405,1 Université Paris-Sud, Orsay, France 91405,2 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia3

Received 17 January 2007/ Accepted 13 March 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The UDP-N-acetylmuramate:L-alanyl-{gamma}-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-{gamma}-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biosynthesis of bacterial cell wall peptidoglycan (murein) is a complex process involving many cytoplasmic and membrane steps (for a review, see reference 54). The main cytoplasmic precursors are a series of seven nucleotide compounds, ranging from UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylmuramyl-L-Ala-{gamma}-D-Glu-X-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) (where X is meso-diaminopimelic acid [meso-A2pm] in Escherichia coli), whose sequential formation is catalyzed by a set of highly specific enzymes designated the Mur synthetases MurA to MurF (54). Subsequent steps, which occur in the membrane, consist of the transfer of the MurNAc-pentapeptide and GlcNAc motifs to the undecaprenyl phosphate carrier lipid, generating lipid II, which is then translocated to the outer side of the membrane and used for polymerization reactions catalyzed by the penicillin-binding proteins.

The cell wall should not be considered a static structure since permanent remodeling necessarily occurs during cell growth and division. This is believed to a result of balanced functioning of murein-hydrolyzing and murein-synthesizing activities, with the former degrading the old peptidoglycan structure to allow insertion of new material. In E. coli, as many as 18 murein hydrolases have been identified, and these enzymes belong to six different families and include lytic transglycosylases, amidases, and endopeptidases; there is a specific hydrolase for almost every covalent bond in the murein (24). The remodeling of the murein by these different "autolysins" results in dramatic turnover, estimated at 40 to 50% per generation time. Some cell wall peptides are released into the growth medium during this process (21, 22). However, the main turnover products, which have been identified as GlcNAc-MurNAc(anhydro)-tetra- and tripeptides, are imported into the cytoplasm by a specific permease (AmpG) (26) and are efficiently reused in a process that has been termed the recycling pathway (41). This process involves a large set of enzymes (the amidase AmpD, the LD-carboxypeptidase LdcA, and the ß-N-acetylglucosaminidase NagZ) that catalyze the stepwise breakdown of anhydro-muropeptides, yielding GlcNAc, anhydro-MurNAc, D-Ala, and the tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm (9, 10, 27, 42, 49). The murein tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm is then directly ligated to UDP-MurNAc by a dedicated Mur synthetase, UDP-N-acetylmuramate:L-alanyl-{gamma}-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]), and thus can reenter the biosynthesis pathway for de novo peptidoglycan synthesis (39). The two amino sugars are also reused and reinjected into the general metabolism (51-53).

The mpl gene was identified previously by a search of databases for proteins exhibiting significant homology with the UDP-MurNAc:L-alanine ligase MurC, the enzyme that adds the first amino acid residue to the nucleotide precursor in the peptidoglycan biosynthesis pathway (39). Overexpression of mpl by using plasmid vectors was correlated with increased L-Ala-{gamma}-D-Glu-meso-A2pm ligase activity, and, conversely, disruption of this gene in the chromosome of E. coli resulted in a complete loss of tripeptide ligase activity (39). The ldcA gene encoding the cytoplasmic LD-carboxypeptidase which releases the terminal D-alanine residue from the tetrapeptide recycling product L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala was then identified by Templin et al. (49). Interestingly, an ldcA mutant was shown to accumulate UDP-MurNAc-tetrapeptide, an unusual compound that normally is not present at significant levels in E. coli cells (15, 49) as the biosynthesis pathway involves direct conversion of UDP-MurNAc-tripeptide to UDP-MurNAc-pentapeptide by the D-Ala-D-Ala-adding enzyme MurF (54). This suggested that the accumulated tetrapeptide was efficiently transferred to UDP-MurNAc, most likely by the Mpl enzyme, clearly indicating that the specificity of this enzyme is not restricted to the tripeptide substrate.

In the present study, the Mpl protein from E. coli was purified to near homogeneity, and its kinetic parameters and substrate specificity were determined. An analysis of the pools of nucleotide peptidoglycan precursors and of the main peptides released during the recycling of the peptidoglycan material was performed using mpl and ldcA mutant strains. The biochemical properties of Mpl determined in vitro and the physiological properties that this enzyme exhibits in normally growing cells were correlated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. DNA restriction enzymes were purchased from New England Biolabs, oligonucleotides were produced and DNA sequencing was performed by MWG-Biotech, and DNA purification kits were obtained from Promega. UDP-[U-14C]GlcNAc (9.85 to 11.1 GBq·mmol–1) was purchased from Amersham Biosciences, and L-[14C]Ala (5.55 GBq·mmol–1) was purchased from Perkin-Elmer. Purified MurC (31), MurD (1), MurE (23), and MurF (13) synthetases, penicillin-binding protein 5 (PBP5) DD-carboxypeptidase (17, 49), and meso-A2pm (55) were prepared by using previously described procedures. The pentapeptide L-Ala-{gamma}-D-Glu-L-Lys-D-Ala-D-Ala was obtained from Bachem. Proteases, mutanolysin, antibiotics, amino acids, and reagents were obtained from Sigma.

Bacterial strains, plasmids, and growth conditions. E. coli strain DH5{alpha} (Life Technologies, Inc.) was used as the host for plasmids and for overproduction of the Mpl enzyme. Plasmid pAM1005 carrying the murC gene cloned into the expression vector pTrc99A has been described previously (31). The pTrcHis60 vector for expression of proteins with a C-terminal His6 tag also has been described previously (43). Plasmids and strains used for gene disruption experiments were kindly provided by B. Wanner via the E. coli Genetic Stock Center (Yale University, New Haven, CT) (12). 2YT and LB media and minimal medium M63 were used to grow cells (40), and growth was monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. For strains carrying drug resistance genes, ampicillin, chloramphenicol, and kanamycin were used at concentrations of 100, 25, and 40 µg·ml–1, respectively.

General DNA techniques and E. coli cell transformation. Small-scale plasmid isolation and large-scale plasmid isolation were carried out by the alkaline lysis method (46). Standard procedures for endonuclease digestion, ligation, and agarose electrophoresis were used (46). E. coli cells were made competent for transformation with plasmid DNA by the method of Dagert and Ehrlich (11) or by electroporation.

Construction of plasmids. A plasmid allowing high-level expression of the E. coli Mpl protein (C-terminally His6-tagged form) under control of the trc promoter was constructed as follows. PCR primers Mpl-O1 and Mpl-O2 (Table 1) were designed to incorporate a BspHI site 5' of the initiation codon of the gene and a BglII site 3' of the gene to replace the stop codon, respectively. These primers were used to amplify the mpl gene from the E. coli chromosome. The resulting material was treated with BspHI and BglII and was ligated between the compatible NcoI and BglII sites of plasmid vector pTrcHis60 (43), generating plasmid pMLD131. The pET2130 vector, which was derived from pET21d (Novagen), was constructed by removal (filling in) of the unique BglII site and replacement of the NcoI-BamHI region of the polylinker with the corresponding region from the pTrcHis30 vector (43). A plasmid for expression of a soluble N-terminal His6-tagged form of E. coli PBP5 was constructed as follows: an appropriate truncated form of the PBP5 gene (dacA) (44) was amplified from the chromosome by PCR using oligonucleotides Pbp5-O1 and Pbp5-O2 (Table 1), and the resulting material was treated with BamHI and HindIII and ligated between the same sites of plasmid vector pET2130, generating plasmid pET-PBP5s.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Oligonucleotides and bacterial strains used in this study

 
Disruption of the chromosomal ldcA and mpl genes. An ldcA null mutant strain, MLD2501 ({Delta}ldcA::Cmr), was constructed by the one-step inactivation method of Datsenko and Wanner (12). This technique uses the highly efficient phage {lambda} Red recombination system and direct transformation of PCR products consisting of the cat marker gene flanked by approximately 50-bp homology extensions of the gene to be deleted. Strain BW25113(pKD46) was transformed with the PCR fragment amplified by using plasmid pKD3 as the template and primers LdcA-inact1 and LdcA-inact2 (Table 1). Chloramphenicol-resistant clones were isolated, and deletion of the chromosomal ldcA gene was confirmed by PCR, using primers LdcA-ext1 and LdcA-ext2 (Table 1). A {Delta}mpl::Cmr null mutant strain (MLD2502) was generated similarly, using primers Mpl-inact1 and Mpl-inact2 for the inactivation procedure and primers Mpl-ext1 and Mpl-ext2 for confirmation of the gene deletion (Table 1). The Cmr cassette was then excised from the chromosome of the {Delta}ldcA::Cmr strain by transformation with the pCP20 plasmid, which expresses the FLP recombinase (12). Transformants were selected at 30°C and then purified once at 43°C, yielding {Delta}ldcA strain MLD2503 that had lost all antibiotic resistance markers. A {Delta}ldcA {Delta}mpl::Cmr double mutant (MLD2504) was generated by phage P1 transduction (40) of the {Delta}mpl::Cmr allele into the {Delta}ldcA strain.

Isolation of sacculi and quantitation of peptidoglycan. Cells (0.8-liter cultures) were grown exponentially at 37°C in 2YT medium to an optical density (OD) of 0.7 (ca. 250 mg of bacteria [dry weight] per liter of culture). Then the cells were rapidly chilled to 0°C, harvested under cold conditions, and washed with a cold 0.85% NaCl solution. The bacteria were resuspended in 2 ml of NaCl solution and then injected with vigorous stirring into 40 ml of a hot (95 to 100°C) aqueous 4% sodium dodecyl sulfate (SDS) solution. Each mixture was incubated at 95 to 100°C for 30 min. After incubation overnight at room temperature, the suspensions were centrifuged for 30 min at 200,000 x g, and the pellets were washed several times with water. Final suspensions in 5 ml of water were homogenized by brief sonication. Aliquots were hydrolyzed (16 h at 95°C in 6 M HCl) and analyzed with a Hitachi model 8800 amino acid analyzer. The peptidoglycan content of the sacculi was expressed in terms of the muramic acid content (35, 38).

Further purification of the peptidoglycan material was performed essentially as described previously (18, 35). Briefly, crude peptidoglycan preparations were treated with proteases (pancreatin, pronase, and trypsin) to remove covalently linked Braun lipoprotein and other contaminating proteins. The purity of the resulting material was confirmed by analysis of its amino acid and hexosamine contents, as described above. The peptidoglycan was then treated with mutanolysin, and the fragments released (muropeptides) were reduced with sodium borohydride and separated by high-performance liquid chromatography (HPLC) on a 3-µm octyldecyl silane-Hypersil column (4.6 by 250 mm), using a gradient of acetonitrile (from 0 to 20% in 100 min) in 0.05% trifluoroacetic acid at a flow rate of 0.6 ml·min–1. Peaks were collected, and muropeptides were identified by amino acid, hexosamine, and matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry analyses.

Levels of peptidoglycan precursors. Cells (0.8-liter cultures) were grown exponentially at 37°C in 2YT medium and harvested at an OD of 0.7. The conditions used for extraction of the peptidoglycan nucleotide precursors, as well as the analytical procedure used for separation and quantitation of these precursors, have been described previously (16, 35, 36).

Functional complementation of a murC mutation. The E. coli murC temperature-sensitive mutant H-1119 (33) was transformed with plasmids. Transformants were grown at the permissive temperature (30°C) before they were shifted to the restrictive temperature (42°C). Cultures were incubated with and without 1 mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG) for 5 min before the temperature shift. The growth rate was determined by recording the OD at 600 nm at suitable times.

Overproduction and purification of the Mpl protein. Cells of strain DH5{alpha} harboring the pMLD131 plasmid were grown exponentially at 37°C in 2YT-ampicillin medium (1-liter cultures). Expression of the Mpl protein was induced with 1 mM IPTG when the OD of the culture reached 0.8. Cells were harvested 3 h later and were washed with 40 ml of cold 20 mM potassium phosphate buffer (pH 7.4) containing 0.5 mM MgCl2 and 0.1% 2-mercaptoethanol (buffer A). The wet cell pellet was suspended in 10 ml of the same buffer and disrupted by sonication (model 72412 Bioblock Vibracell sonicator) for 10 min with cooling. The resulting suspension was centrifuged at 4°C for 30 min at 200,000 x g with a Beckman TL100 centrifuge, and the supernatant was stored at –20°C.

One-step purification of the His6-tagged Mpl protein was carried out under native conditions, basically using the steps recommended by the manufacturer (QIAGEN, Santa Clarita, CA), as follows: binding of the tagged protein on Ni2+-nitrilotriacetate agarose (Ni2+-NTA agarose), washing with buffer A containing 200 mM KCl and 20 mM imidazole to remove impurities, and elution with increasing concentrations of imidazole (40 to 300 mM) added to buffer A (the His6-tagged Mpl protein eluted in the fractions containing 50 and 100 mM imidazole). The purified protein was then dialyzed overnight at 4°C against buffer A, and 15% glycerol was added for storage of the protein at –20°C.

SDS-polyacrylamide gel electrophoresis (PAGE) analysis of proteins was performed as previously described using 13% polyacrylamide gels (29). Protein concentrations were determined by the method of Bradford, using bovine serum albumin as a standard (8).

Preparation of nucleotide precursors and peptides. Chemical synthesis of UDP-MurNAc has been described previously (3). UDP-MurNAc-peptides, ranging from UDP-MurNAc-L-Ala to UDP-MurNAc-L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala-D-Ala, were prepared by enzymatic synthesis using the purified Mur synthetases MurC, MurD, MurE, and MurF from E. coli. UDP-MurNAc-tetrapeptides were generated by treatment of UDP-MurNAc-pentapeptides with PBP5 from E. coli. The latter enzyme was overexpressed in the soluble His6-tagged form from the pET-PBP5s plasmid and was purified on Ni2+-NTA agarose by using classical procedures. Radiolabeled UDP-[14C]MurNAc was prepared from UDP-[14C]GlcNAc, phosphoenolpyruvate, NADPH, and partially purified MurA and MurB as described previously (7). 14C-labeled UDP- MurNAc-peptides labeled either in the first (L-Ala) or third (A2pm) amino acid residue were generated as previously described (6, 14).

MurNAc-peptides were obtained by mild acid hydrolysis (10 min at 100°C in 0.1 M HCl) of UDP-MurNAc-peptides, and they were purified as a mixture of the two anomers ({alpha} and ß) by HPLC (4). The dipeptide L-Ala-D-Glu was synthesized chemically as described by Sachs and Brand (45). The tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm, the tetrapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala, and the pentapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala-D-Ala were generated by treatment of the corresponding MurNAc-peptides with partially purified N-acetylmuramyl-L-alanine amidase, as described by van Heijenoort et al. (56). The tripeptide L-Ala-{gamma}-D-Glu-L-Lys was synthesized by a modified procedure of Schmidt et al. (48). The tetrapeptide L-Ala-{gamma}-D-Glu-L-Lys-D-Ala was generated by treatment of the pentapeptide L-Ala-{gamma}-D-Glu-L-Lys-D-Ala-D-Ala (Bachem) with purified PBP5, as described above. These different peptides were purified by HPLC on a Vydac C18 column (10 by 250 mm) using 0.1% trifluoroacetic acid as the eluent.

The identities of all these compounds were confirmed by amino acid, hexosamine, and MALDI-TOF mass spectrometry analyses.

Assay for tripeptide ligase activity. The standard assay mixture (40 µl) contained 100 mM Tris-HCl buffer (pH 8.4), 5 mM ATP, 15 mM MgCl2, 0.3 mM L-Ala-{gamma}-D-Glu-meso-A2pm, 0.4 mM UDP-[14C]MurNAc (500 Bq), and purified Mpl (20 ng of protein). Mixtures were incubated at 37°C for 30 min, and the reactions were stopped by addition of 10 µl of acetic acid, followed by lyophilization. Each residue was dissolved in 100 µl of 50 mM ammonium formate (pH 3.6), and 80 µl was injected onto a Nucleosil 100 C18 5-µm column (4.6 by 150 mm; Alltech-France) using the same buffer at a flow rate of 0.6 ml·min–1 as the mobile phase. Detection was performed with a radioactive flow detector (model LB506-C1; Berthold) using a Quicksafe Flow 2 scintillator (Zinsser Analytic) at a flow rate of 0.6 ml·min–1. Quantitation was carried out with the Winflow software (Berthold).

For determination of the kinetic constants, the same assay was used with various concentrations of one substrate and fixed concentrations of the other substrates. In all cases, the substrate consumption was <20%, and linearity was observed within the interval even at the lowest substrate concentration. The data were fitted to the equation v = VmaxS/(Km + S) using the MDFitt software developed by M. Desmadril (IBBMC, Orsay, France).

Identical assay conditions were used when other peptides were tested as substrates. The buffer and pH used for separation of the radiolabeled substrate and product varied slightly, however, depending on the substrate used: ammonium formate at pH 4.3 was used for L-Ala, ammonium formate at pH 4.7 was used for L-Ala-D-Glu, L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala, L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala-D-Ala, and L-Ala-{gamma}-D-Glu-L-Lys, and ammonium acetate at pH 6 was used for L-Ala-{gamma}-D-Glu-L-Lys-D-Ala and L-Ala-{gamma}-D-Glu-L-Lys-D-Ala-D-Ala.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression and purification of Mpl. A plasmid that allowed high-level expression of the mpl gene product with a His6 tag at the C terminus was constructed. In this plasmid, designated pMLD131, gene expression was under the control of the strong IPTG-dependent trc promoter. SDS-PAGE analysis of cell extracts showed that induced DH5{alpha}(pMLD131) cells accumulated a protein species with a molecular mass of about 50 kDa (Fig. 1), a value consistent with the molecular mass (including the Arg-Ser-His6 extension) calculated from the gene sequence (50,940 Da). In contrast to the untagged Mpl protein which was previously shown to frequently aggregate and form inclusion bodies in induced cells (39), here the overproduced His6-tagged enzyme was detected almost exclusively in the supernatant fraction following cell disruption and ultracentrifugation. The soluble fraction was purified further by affinity chromatography on Ni2+-NTA agarose, and pure fractions (Mpl was eluted with 50 to 100 mM imidazole) were dialyzed against buffer A, concentrated to about 5 mg·ml–1 of protein, and then conserved at –20°C in the presence of 15% glycerol. The final preparation of the His6-tagged Mpl enzyme was at least 95% pure, as judged by SDS-PAGE (Fig. 1), and the yield was about 100 mg of purified protein per liter of culture. MALDI-TOF mass spectrometry analyses confirmed the purity and integrity of the Mpl preparation; peaks at m/z 50,961 and 25,490 corresponding to the [M + H]+ and [M + 2H]2+ ions, respectively, were observed (data not shown), in agreement with the calculated molecular mass (50,940 Da).


Figure 1
View larger version (113K):
[in this window]
[in a new window]

  		FIG. 1. Overproduction and purification of the Mpl protein (His6-tagged form), as determined by SDS-PAGE. Lane MW, molecular mass markers; lane A, total extract from DH5{alpha} cells; lane B, total extract from IPTG-induced DH5{alpha} cells carrying the pMLD131 plasmid; lane C, Mpl protein purified by chromatography on Ni2+-NTA agarose (fraction containing 100 mM imidazole). Staining was performed with Coomassie brilliant blue R250.

 
Kinetic parameters and substrate specificity of Mpl. We showed previously that cells producing high levels of the wild-type (untagged) Mpl protein had tripeptide ligase activity of about 120 to 200 nmol·min–1·mg–1 (39). The Vmax determined here for the pure His6-tagged enzyme (5,800 nmol·min–1·mg–1) clearly showed that the presence of the tag and the purification procedure did not have deleterious effects on the enzyme activity. Preliminary assays were carried out to determine the optimal conditions for enzyme activity. Variation of the activity as a function of pH resulted in a classical bell shape with an optimal pH of 8.4 (Fig. 2A). As observed previously for the other members of the Mur synthetase family (32), Mpl activity had an absolute requirement for a divalent cation. The optimal concentration determined for Mg2+ was 15 mM (Fig. 2B).


Figure 2
View larger version (6K):
[in this window]
[in a new window]

  		FIG. 2. UDP-MurNAc:L-Ala-{gamma}-D-Glu-meso-A2pm-adding activity of Mpl as a function of pH (A) and Mg2+ concentration (B). The assays were performed in 100 mM Tris buffer, and the concentrations of ATP, radiolabeled UDP-MurNAc, and L-Ala-{gamma}-D-Glu-meso-A2pm were 5 mM, 400 µM, and 300 µM, respectively. The activity is expressed as a percentage of the radioactivity incorporated into the UDP-MurNAc-tripeptide product during the assay. The data are means of duplicate determinations. The standard deviations were within ±5% of the values indicated.

 
The kinetic parameters of the Mpl enzyme with its three substrates were determined (Table 2). The values obtained (kcat = 290 ± 70 min–1; Km = 0.25 ± 0.02 mM for UDP-MurNAc, Km = 0.19 ± 0.04 mM for ATP, and Km = 0.10 ± 0.02 mM for L-Ala-{gamma}-D-Glu-meso-A2pm) were the same order of magnitude as the values determined previously with MurC (kcat = 318 min–1; Km = 0.1 mM for UDP-MurNAc, Km = 0.45 mM for ATP, and Km = 0.02 mM for L-Ala) (31), a Mur synthetase which shares two substrates with Mpl (39).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Kinetic parameters of Mpl with different substratesa

 
The constant maturation and remodeling of the peptidoglycan structure that occur during cell growth and division result in the release of free peptides, mainly tetra- and tripeptides, into the cell (21, 22). The conversion of the tetrapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala into the Mpl substrate L-Ala-{gamma}-D-Glu-meso-A2pm is catalyzed by the LD-carboxypeptidase LdcA. The accumulation of UDP-MurNAc-tetrapeptide in E. coli ldcA mutant cells strongly suggested that Mpl could transfer both tetra- and tripeptides to the UDP-MurNAc nucleotide precursor (49). The substrate specificity of the purified Mpl enzyme was therefore analyzed in detail in vitro. As shown in Table 2, the tri- and tetrapeptides, as well as the pentapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala-D-Ala, were accepted as substrates with quite similar efficiencies (kcat/Km, 1,900 to 3,300 min–1·mM–1). Surprisingly, Mpl could also catalyze addition of the dipeptide L-Ala-D-Glu and addition of a single L-Ala residue (a MurC-type activity) to UDP-MurNAc, although at a much lower rate (Table 2). Since one of the main differences observed between the peptidoglycan structures in bacteria is the presence of either meso-A2pm or L-Lys at the third position of the peptide (47), the activity of Mpl was also tested with L-Lys-containing peptides. Changing A2pm to L-Lys did not eliminate the activity with tri- to pentapeptides but resulted in significant decrease in the catalytic efficiency (Table 2). The ability of the Mpl enzyme to add Lys-containing tri-, tetra-, and pentapeptides to UDP-MurNAc has been reported previously and was used to generate the corresponding UDP-MurNAc-peptides (2).

Complementation analysis of Mpl in an E. coli temperature-sensitive murC mutant. The very low but detectable L-alanine-adding activity of the purified Mpl protein raised the question of whether traces of MurC enzyme contaminated the Mpl preparation. In order to confirm the MurC-like activity of Mpl, the temperature-sensitive E. coli strain H-1119 (33) harboring a defective murC gene was transformed with the mpl-expressing plasmid pMLD131 and the control vector pTrcHis30. Growth of H-1119(pTrcHis30) transformants to the early logarithmic phase at 30°C, followed by a shift to 42°C, led to rapid lysis of the cell culture (Fig. 3). The same results were observed with cultures of H-1119(pMLD131) to which no IPTG was added. In contrast, cultures of H-1119(pMLD131) to which IPTG was added 5 min before the temperature shift continued growing at 42°C (Fig. 3). Thus, high-level (IPTG-induced) expression of Mpl from the pMLD131 plasmid permitted complementation of the temperature-dependent UDP-MurNAc:L- alanine ligase (MurC) defect in E. coli, demonstrating that Mpl could effectively exhibit such an activity, although at a very low level, both in vitro and in vivo.


Figure 3
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 3. Overexpression of the mpl gene restores growth of a murC mutant. Cells of the thermosensitive E. coli mutant strain H-1119 carrying either the pMLD131 plasmid (open symbols) or the pTrcHis30 control vector (solid symbols) were grown in 2YT-ampicillin medium at 30°C. At the time indicated by the arrow, the temperature of the cultures was either maintained at 30°C (circles) or shifted to 42°C (squares). In some cases, IPTG (1 mM) was added to cultures at 42°C (diamonds).

 
Effects of Mpl and LdcA deficiencies on E. coli peptidoglycan metabolism. Both cytoplasmic enzymes, Mpl and LdcA LD-carboxypeptidase, play a role in the recycling of the peptidoglycan degradation products. We constructed E. coli single and double mutant strains that had deletions of the two genes, and the effects of these mutations on cell growth and peptidoglycan metabolism were investigated. As reported previously (39), disruption of the mpl gene did not alter the growth of E. coli. Templin et al. (49) observed previously that an ldcA null mutant strain often lysed when it entered the stationary phase in LB medium. However, with our ldcA mutant we observed a precocious decrease in the growth rate, and the mutant entered the stationary phase at a lower cell density (Fig. 4), but partial lysis of the population sometimes also occurred. The same behavior was observed with the ldcA mpl double mutant (Fig. 4).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. Effects of mpl and ldcA mutations on the growth (A) and peptidoglycan structure (B) of E. coli. (A) Growth at 37°C in LB medium of wild-type strain BW25113 ({square}), {Delta}mpl ({blacklozenge}) and {Delta}ldcA ({blacktriangleup}) single mutants, and {Delta}mpl {Delta}ldcA double mutant ({circ}). (B) The peptidoglycan from the wild-type and mutant strains was purified and digested with muramidase M1 (mutanolysin), and the resulting compounds (muropeptides) were separated by HPLC as described in Materials and Methods. Compounds under each peak were identified by quantitative amino acid and hexosamine analyses, as well as by MALDI-TOF mass spectrometry. The main monomers were as follows: peak 1, GlcNAc-MurNAc-L-Ala-{gamma}-D-Glu-meso-A2pm; peak 2, GlcNAc-MurNAc-L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala; peak 3, GlcNAc-MurNAc(anhydro)-L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala. The main dimers were as follows: peak 4, cross-linked peak 1 and 2 monomers; peak 5, two cross-linked peak 2 monomers; peak 6, cross-linked peak 2 and 3 monomers; peak 7, peak 6 dimer carrying an additional Lys-Arg dipeptide originating from Braun's lipoprotein (18).

 
The cell peptidoglycan content and the peptidoglycan composition were determined for the wild-type and each mutant strain after exponential growth in rich medium. None of the mutations significantly modified the amount of peptidoglycan per cell (data not shown) or the peptidoglycan structure (Fig. 4). Höltje and coworkers also previously reported that the fine peptidoglycan structure was not modified following inactivation of the ldcA gene (49).

The compositions of the pools of the main peptidoglycan nucleotide precursors, as well as the pools of the free peptides resulting from peptidoglycan recycling, were determined for the wild-type and the mpl and ldcA mutant strains (Table 3). In the mpl mutant, a 17-fold increase in the level of the tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm, the Mpl substrate, and a 1.7-fold decrease in the level of the UDP-MurNAc-pentapeptide pool were observed compared to the levels in the wild-type strain. There was also significant accumulation of the pentapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala-D-Ala (Table 3). In the ldcA mutant, a decrease in the level of the UDP-MurNAc-pentapeptide was also observed (2.9-fold), but the most striking feature was the large increases in the levels of the UDP-MurNAc-tetrapeptide and the tetrapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala, consistent with the in vivo defect of LD-carboxypeptidase activity (49). The UDP-MurNAc-tetrapeptide, which normally is not present or is very poorly represented in a wild-type E. coli strain (15, 28, 49), is a dead end product in an ldcA mutant strain. In the mpl ldcA double mutant, the level of UDP-MurNAc-pentapeptide was slightly decreased (ratio, 1.3), but no UDP-MurNAc-tetrapeptide was detected. Instead, high levels of tri-, tetra- (mainly), and pentapeptides were present (Table 3). In particular, the huge amount of tetrapeptide that accumulated in the mpl ldcA double mutant more or less corresponded to the amounts of UDP-MurNAc-tetrapeptide plus tetrapeptide that accumulated in the ldcA single mutant. These findings clearly indicated that the Mpl enzyme was responsible for the formation of the UDP-MurNAc-tetrapeptide that accumulated in the ldcA genetic background, demonstrating that this enzyme can transfer the tetrapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala to UDP-MurNAc both in vitro and in vivo. It is noteworthy that the level of tetrapeptide that accumulated in the mpl ldcA mutant was much higher than the level of tripeptide that accumulated in the mpl mutant (Table 3). This was certainly due to the presence in the E. coli cells of the recycling MpaA amidase that is known to hydrolyze the tripeptide into L-Ala-D-Glu and meso-A2pm (50). Since the amount of tetrapeptide present in the double mutant is huge, we predict that the tetrapeptide must be a poor substrate for the MpaA amidase.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Levels of UDP-MurNAc-peptides and free peptides in parental and mutant strains

 
Attempts to incorporate L-Ala-{gamma}-D-Glu-L-Lys into the peptidoglycan via Mpl. Incorporation of L-Lys into the peptidoglycan of E. coli was previously shown to be deleterious for cell growth (34). Cell lysis was observed when more than 50 to 60% of meso-A2pm residues were replaced by L-Lys. In particular, this replacement could be induced by overexpression in E. coli of murE gene products (from Staphylococcus aureus, Thermotoga maritima, etc.) that exhibit UDP-MurNAc-L-Ala-D-Glu:L-Lys-adding activity (5, 34). Since Mpl could also use L-Ala-{gamma}-D-Glu-L-Lys as a substrate, we wondered whether this peptide could have any antibacterial activity, i.e., whether it could be taken up by E. coli cells and subsequently incorporated by this enzyme into peptidoglycan. However, addition of large amounts of this peptide (up to 1 mg·ml–1) to the culture medium of E. coli cells (rich 2YT medium or glucose minimal medium) did not have any apparent effect on cell growth and cell morphology (data not shown). The recycling amidase MpaA, which hydrolyzes the tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm into L-Ala-D-Glu and meso-A2pm in the cytoplasm, was shown previously to be inactive on the Lys-containing peptide L-Ala-{gamma}-D-Glu-L-Lys (48), suggesting that the absence of toxic effects for this peptide was not due to rapid degradation inside cells; rather, it could have been due to poor uptake or to a low rate of incorporation into peptidoglycan precursors.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The peptidoglycan structure is subjected to a constant remodeling during bacterial cell growth and division. This maturation process involves specific periplasmic lytic transglycosylase, endopeptidase, and DD-carboxypeptidase activities (for a review, see references 24 and 57) which degrade the cell wall polymer, allowing insertion of newly synthesized material. One of the main degradation products generated in this way is the disaccharide-tetrapeptide GlcNAc-MurNAc(1,6-anhydro)-L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala, a compound previously designated the tracheal cytotoxin since it was shown to be released by Bordetella pertussis growing cells and to be responsible for the epithelial cytopathology associated with pertussis infection (19). In E. coli, the disaccharide-tetrapeptide has been shown to be transported into the cytoplasm by the permease AmpG (26), where it is further degraded into anhydro-MurNAc, GlcNAc, D-Ala, and the tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm by the actions of three enzymes, the 1,6-anhydro-N-acetylmuramoyl-L-alanine amidase AmpD (25, 27), the ß-N-acetylglucosaminidase NagZ (9, 58), and the LD-carboxypeptidase LdcA (49). As the substrate specificity of the latter three enzymes is not strict, the order in which they act has not really been defined. The released tripeptide was shown to be directly reinjected into the biosynthetic pathway via the Mpl enzyme activity (39), but it can also be degraded to the individual amino acids (48, 50). GlcNAc and anhydro-MurNAc are also reused (52). This recycling process represents a major cell metabolic pathway as 30 to 40% of the cell wall peptidoglycan is degraded each generation, which is followed by uptake and reuse of most of its components (more than 106 molecules of murein tripeptide are recycled per generation).

In contrast to its homolog MurC that is required for peptidoglycan biosynthesis, Mpl is not essential for bacterial growth, although it could be required for growth under certain circumstances (for instance, when murein tripeptide instead of A2pm is provided as a source of A2pm for an A2pm-auxotrophic strain) (39). In fact, none of the other gene products participating in peptidoglycan recycling has been shown to be essential. Thus, this pathway is generally simply considered a dispensable economic process. However, the fact that the AmpG and AmpD proteins, which are essential components of the inducible ß-lactamase system of many bacteria, are present in E. coli, a bacterium which lacks an inducible ß-lactamase, has led to speculation that recycling may be a way by which the cell monitors the condition of its cell wall structure and metabolism. For instance, fluctuations in the pools of recycling intermediates may signal whether the cell should modify the rate of peptidoglycan synthesis, continue growing, or commit to the stationary phase. Mpl would also be part of this hypothetical regulatory circuit. As observed for the multiple signals determining whether bacilli continue to grow or sporulate, the regulatory sensors are not essential. Interestingly, mpl, along with ampD and pbpG, was recently identified in a search for genes defining intrinsic resistance to antibiotics in Acinetobacter baylyi (20). Disruption of these genes in the latter bacterial species indeed resulted in at least a 10-fold reduction in the MIC of ß-lactam antibiotics. However, inactivation of these genes in E. coli had little or no significant effect on the susceptibility of E. coli cells to these antibiotics (20). The fact that inhibition of the nonessential recycling pathway could, at least in some classes of pathogens, potentiate the effects of classical antibiotics targeting peptidoglycan biosynthesis is very interesting. Mpl and the other recycling enzymes could thus be considered potential targets whose exploitation could open the possibility of expanding the spectrum of available drugs to specific classes of pathogens and lead to the development of narrow-spectrum potentiators dedicated to particular infections.

In the present work, the E. coli Mpl protein was purified to homogeneity, and its kinetic properties were investigated in detail. This enzyme was found to have relatively broad substrate specificity as it could accept tri-, tetra-, and pentapeptides and either A2pm or L-Lys at position 3. Recently, the ability of Mpl to accept Lys-containing peptides was also reported by other workers and used to generate MurF UDP-MurNAc-tripeptide substrates more easily (2). The broad enzyme specificity explains why an ldcA mutant strain, which could not convert the tetrapeptide L-Ala-{gamma}-D-Glu-meso-A2pm-D-Ala into the tripeptide L-Ala-{gamma}-D-Glu-meso-A2pm, accumulates UDP-MurNAc-tetrapeptide (49) (Table 3). Mpl is clearly responsible for the observed in vivo addition of the tetrapeptide to UDP-MurNAc as no accumulation of UDP-MurNAc-tetrapeptide was observed in the ldcA mpl double mutant. As mentioned above, the order in which the AmpD, NagZ, LdcA, and Mpl enzymes act is not well defined because these enzymes do not exhibit very strict substrate specificity. This is most likely the reason why trace amounts of UDP-MurNAc-tetrapeptide are reproducibly detected in E. coli cells following labeling of peptidoglycan precursors with radioactive A2pm (15, 28, 49); it is possible that Mpl acts before LdcA acts in vivo.

As mentioned above, the Mpl recycling enzyme, although not essential for cell growth, could be considered an interesting target in the search for antibacterial compounds as inhibition of Mpl could result in increased susceptibility of some pathogenic strains to antibiotics. Alternatively, the broad substrate specificity of Mpl indicates that this enzyme potentially could be used for incorporation of toxic peptides into the peptidoglycan network. As the incorporation of L-Lys into peptidoglycan is known to be deleterious for E. coli cells, we wondered whether the tripeptide L-Ala-{gamma}-D-Glu-L-Lys synthesized in the present work could affect cell growth. No growth inhibition was observed, however, indicating that there was either poor uptake of the peptide or a low rate of incorporation into peptidoglycan precursors (compared with the endogenous synthesis of the meso-A2pm-containing precursors catalyzed by the MurC, MurD, and MurE synthetase activities). Lys-containing peptides targeting the meso-A2pm biosynthetic pathway were shown previously to be internalized poorly by E. coli cells (30). Combinatorial peptide synthesis analyses and further analyses of the substrate specificity and structure of the Mpl enzyme are now in progress to identify Mpl substrate analogs with antibacterial activity.


   ACKNOWLEDGMENTS
 
This work was supported by CNRS and by European Union grant LSHM-CT-2004-512138 for the EUR-INTAFAR project.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire des Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Université Paris-Sud, Bât. 430, 91405 Orsay Cedex, France. Phone: 33-1-69-15-61-34. Fax: 33-1-69-85-37-15. E-mail: mireille.herve{at}u-psud.fr Back

{triangledown} Published ahead of print on 23 March 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Auger, G., L. Martin, J. Bertrand, P. Ferrari, E. Fanchon, S. Vaganay, Y. Petillot, J. van Heijenoort, D. Blanot, and O. Dideberg. 1998. Large-scale preparation, purification, and crystallization of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli. Protein Expr. Purif. 13:23-29.[CrossRef][Medline]
  2. Baum, E. Z., S. M. Crespo-Carbone, D. Abbanat, B. Foleno, A. Maden, R. Goldschmidt, and K. Bush. 2006. Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF. Antimicrob. Agents Chemother. 50:230-236.[Abstract/Free Full Text]
  3. Blanot, D., G. Auger, D. Liger, and J. van Heijenoort. 1994. Synthesis of {alpha} and ß anomers of UDP-N-acetylmuramic acid. Carbohydr. Res. 252:107-115.
  4. Blanot, D., A. Kretsovali, M. Abo-Ghalia, D. Mengin-Lecreulx, and J. van Heijenoort. 1983. Synthesis of analogues of precursors of bacterial peptidoglycan, p. 311-314. In K. Bláha and P. Malon (ed.), Peptides 1982. Walter de Gruyter, Berlin, Germany.
  5. Boniface, A., A. Bouhss, D. Mengin-Lecreulx, and D. Blanot. 2006. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity. J. Biol. Chem. 281:15680-15686.[Abstract/Free Full Text]
  6. Bouhss, A., M. Crouvoisier, D. Blanot, and D. Mengin-Lecreulx. 2004. Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis. J. Biol. Chem. 279:29974-29980.[Abstract/Free Full Text]
  7. Bouhss, A., S. Dementin, J. van Heijenoort, C. Parquet, and D. Blanot. 2002. MurC and MurD synthetases of peptidoglycan biosynthesis: borohydride trapping of acyl-phosphate intermediates. Methods Enzymol. 354:189-196.[Medline]
  8. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
  9. Cheng, Q., H. Li, K. Merdek, and J. T. Park. 2000. Molecular characterization of the ß-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling. J. Bacteriol. 182:4836-4840.[Abstract/Free Full Text]
  10. Cheng, Q., and J. T. Park. 2002. Substrate specificity of the AmpG permease required for recycling of cell wall anhydro-muropeptides. J. Bacteriol. 184:6434-6436.[Abstract/Free Full Text]
  11. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23-28.[CrossRef][Medline]
  12. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.[Abstract/Free Full Text]
  13. Dementin, S., A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx, O. Dideberg, J. van Heijenoort, and D. Blanot. 2001. Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. Eur. J. Biochem. 268:5800-5807.[Medline]
  14. Duncan, K., J. van Heijenoort, and C. T. Walsh. 1990. Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli. Biochemistry 29:2379-2386.[CrossRef][Medline]
  15. El Ghachi, M., A. Bouhss, H. Barreteau, T. Touze, G. Auger, D. Blanot, and D. Mengin-Lecreulx. 2006. Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors. J. Biol. Chem. 281:22761-22772.[Abstract/Free Full Text]
  16. Flouret, B., D. Mengin-Lecreulx, and J. van Heijenoort. 1981. Reverse-phase high-pressure liquid chromatography of uridine diphosphate N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan. Anal. Biochem. 114:59-63.[CrossRef][Medline]
  17. Girardin, S. E., L. H. Travassos, M. Hervé, D. Blanot, I. G. Boneca, D. J. Philpott, P. J. Sansonetti, and D. Mengin-Lecreulx. 2003. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278:41702-41708.[Abstract/Free Full Text]
  18. Glauner, B. 1988. Separation and quantification of muropeptides with high-performance liquid chromatography. Anal. Biochem. 172:451-464.[CrossRef][Medline]
  19. Goldman, W. E., D. G. Klapper, and J. B. Baseman. 1982. Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells. Infect. Immun. 36:782-794.[Abstract/Free Full Text]
  20. Gomez, M. J., and A. A. Neyfakh. 2006. Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrob. Agents Chemother. 50:3562-3567.[Abstract/Free Full Text]
  21. Goodell, E. W. 1985. Recycling of murein by Escherichia coli. J. Bacteriol. 163:305-310.[Abstract/Free Full Text]
  22. Goodell, E. W., and U. Schwarz. 1985. Release of cell wall peptides into culture medium by exponentially growing Escherichia coli. J. Bacteriol. 162:391-397.[Abstract/Free Full Text]
  23. Gordon, E., B. Flouret, L. Chantalat, J. van Heijenoort, D. Mengin-Lecreulx, and O. Dideberg. 2001. Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase from Escherichia coli. J. Biol. Chem. 276:10999-11006.[Abstract/Free Full Text]
  24. Höltje, J. V. 1998. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62:181-203.[Abstract/Free Full Text]
  25. Höltje, J. V., U. Kopp, A. Ursinus, and B. Wiedemann. 1994. The negative regulator of ß-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L-alanine amidase. FEMS Microbiol. Lett. 122:159-164.[CrossRef][Medline]
  26. Jacobs, C., L. J. Huang, E. Bartowsky, S. Normark, and J. T. Park. 1994. Bacterial cell wall recycling provides cytosolic muropeptides as effectors for ß-lactamase induction. EMBO J. 13:4684-4694.[Medline]
  27. Jacobs, C., B. Joris, M. Jamin, K. Klarsov, J. van Beeumen, D. Mengin-Lecreulx, J. van Heijenoort, J. T. Park, S. Normark, and J. M. Frère. 1995. AmpD, essential for both ß-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-L-alanine amidase. Mol. Microbiol. 15:553-559.[Medline]
  28. Kohlrausch, U., F. B. Wientjes, and J. V. Höltje. 1989. Determination of murein precursors during the cell cycle of Escherichia coli. J. Gen. Microbiol. 135:1499-1506.[Medline]
  29. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:575-599.[CrossRef][Medline]
  30. Le Roux, P., D. Blanot, D. Mengin-Lecreulx, and J. van Heijenoort. 1991. Peptides containing 2-aminopimelic acid. Synthesis and study of in vitro effects on bacterial cells. Int. J. Peptide Protein Res. 37:103-111.[Medline]
  31. Liger, D., A. Masson, D. Blanot, J. van Heijenoort, and C. Parquet. 1995. Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli. Eur. J. Biochem. 230:80-87.[Medline]
  32. Lugtenberg, E. J. 1972. Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide. J. Bacteriol. 110:26-34.[Abstract/Free Full Text]
  33. Lugtenberg, E. J., and A. van Schijndel-van Dam. 1972. Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme. J. Bacteriol. 110:35-40.[Abstract/Free Full Text]
  34. Mengin-Lecreulx, D., T. Falla, D. Blanot, J. van Heijenoort, D. J. Adams, and I. Chopra. 1999. Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth. J. Bacteriol. 181:5909-5914.[Abstract/Free Full Text]
  35. Mengin-Lecreulx, D., B. Flouret, and J. van Heijenoort. 1982. Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J. Bacteriol. 151:1109-1117.[Abstract/Free Full Text]
  36. Mengin-Lecreulx, D., B. Flouret, and J. van Heijenoort. 1983. Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis. J. Bacteriol. 154:1284-1290.[Abstract/Free Full Text]
  37. Mengin-Lecreulx, D., E. Siegel, and J. van Heijenoort. 1989. Variations in UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-pentapeptide pools in Escherichia coli after inhibition of protein synthesis. J. Bacteriol. 171:3282-3287.[Abstract/Free Full Text]
  38. Mengin-Lecreulx, D., and J. van Heijenoort. 1985. Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli. J. Bacteriol. 163:208-212.[Abstract/Free Full Text]
  39. Mengin-Lecreulx, D., J. van Heijenoort, and J. T. Park. 1996. Identification of the mpl gene encoding UDP-N-acetylmuramate:L-alanyl-{gamma}-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan. J. Bacteriol. 178:5347-5352.[Abstract/Free Full Text]
  40. Miller, J. H. 1972. Experiments in molecular genetics, p. 431-435. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  41. Park, J. T. 1995. Why does Escherichia coli recycle its cell wall peptides? Mol. Microbiol. 17:421-426.[Medline]
  42. Park, J. T., D. Raychaudhuri, H. Li, S. Normark, and D. Mengin-Lecreulx. 1998. MppA, a periplasmic binding protein essential for import of the bacterial cell wall peptide L-alanyl-{gamma}-D-glutamyl-meso-diaminopimelate. J. Bacteriol. 180:1215-1223.[Abstract/Free Full Text]
  43. Pompeo, F., J. van Heijenoort, and D. Mengin-Lecreulx. 1998. Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes. J. Bacteriol. 180:4799-4803.[Abstract/Free Full Text]
  44. Pratt, J. M., M. E. Jackson, and I. B. Holland. 1986. The C terminus of penicillin-binding protein 5 is essential for localisation to the E. coli inner membrane. EMBO J. 5:2399-2405.[Medline]
  45. Sachs, H., and E. Brand. 1953. Optical rotation of peptides. VII. {alpha}- and {gamma}-dipeptides of glutamic acid and alanine. J. Am. Chem. Soc. 75:4608-4610.[CrossRef]
  46. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  47. Schleifer, K. H., and O. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477.[Free Full Text]
  48. Schmidt, D. M., B. K. Hubbard, and J. A. Gerlt. 2001. Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. Biochemistry 40:15707-15715.[CrossRef][Medline]
  49. Templin, M. F., A. Ursinus, and J. V. Höltje. 1999. A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli. EMBO J. 18:4108-4117.[CrossRef][Medline]
  50. Uehara, T., and J. T. Park. 2003. Identification of MpaA, an amidase in Escherichia coli that hydrolyzes the {gamma}-D-glutamyl-meso-diaminopimelate bond in murein peptides. J. Bacteriol. 185:679-682.[Abstract/Free Full Text]
  51. Uehara, T., and J. T. Park. 2004. The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. J. Bacteriol. 186:7273-7279.[Abstract/Free Full Text]
  52. Uehara, T., K. Suefuji, T. Jaeger, C. Mayer, and J. T. Park. 2006. MurQ etherase is required by Escherichia coli in order to metabolize anhydro-N-acetylmuramic acid obtained either from the environment or from its own cell wall. J. Bacteriol. 188:1660-1662.[Abstract/Free Full Text]
  53. Uehara, T., K. Suefuji, N. Valbuena, B. Meehan, M. Donegan, and J. T. Park. 2005. Recycling of the anhydro-N-acetylmuramic acid derived from cell wall murein involves a two-step conversion to N-acetylglucosamine-phosphate. J. Bacteriol. 187:3643-3649.[Abstract/Free Full Text]
  54. van Heijenoort, J. 2001. Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat. Prod. Rep. 18:503-519.[CrossRef][Medline]
  55. van Heijenoort, J., and E. Bricas. 1968. Contribution à l'étude des isomères de l'acide {alpha},{alpha}'-diaminopimélique. Bull. Soc. Chim. Fr. 7:2828-2831.
  56. van Heijenoort, J., C. Parquet, B. Flouret, and Y. van Heijenoort. 1975. Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K 12. Purification and properties of the enzyme. Eur. J. Biochem. 58:611-619.[Medline]
  57. Vollmer, W., and J. V. Höltje. 2001. Morphogenesis of Escherichia coli. Curr. Opin. Microbiol. 4:625-633.[CrossRef][Medline]
  58. Votsch, W., and M. F. Templin. 2000. Characterization of a ß-N-acetylglucosaminidase of Escherichia coli and elucidation of its role in muropeptide recycling and ß-lactamase induction. J. Biol. Chem. 275:39032-39038.[Abstract/Free Full Text]

Journal of Bacteriology, June 2007, p. 3987-3995, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00087-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JB.00087-07v1
189/11/3987    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow