Characterization of a β-N-acetylglucosaminidase ofEscherichia coli and Elucidation of Its Role in Muropeptide Recycling and β-Lactamase Induction*

Using the known mapping position the gene encoding a β-1,4-N-acetylglucosaminidase needed for the degradation of muropeptides could be identified. nagZencodes a cytosolic enzyme active onN-actylglucosamyl-β-1,4-(1,6)-anhydromuramic acid containing muropeptides. These degradation products of the peptidoglycan are formed during the enlargement of the murein sacculus as a consequence of a growth mechanism, which couples the controlled degradation of the cell wall polymer with the insertion of new material. NagZ is needed for the formation of monosaccharides from the released disaccharides during the cytosolic steps of the muropeptide-recycling pathway. The formation of intracellular 1,6-anhydro-N-acetylmuramyl-peptides is important for the expression control of the inducible β-lactamases of the AmpC type. A mutant lacking active NagZ cannot establish AmpC mediated β-lactam resistance. The biochemical characterization of the enzyme showed its activity on different muropeptides and inhibitors of enzyme activity could be identified. This observation might be important for designing inhibitors of NagZ that could prevent the establishment of β-lactam resistance of Enterobacteria possessing inducible β-lactamases.

Using the known mapping position the gene encoding a ␤-1,4-N-acetylglucosaminidase needed for the degradation of muropeptides could be identified. nagZ encodes a cytosolic enzyme active on N-actylglucosamyl-␤-1,4-(1,6)anhydromuramic acid containing muropeptides. These degradation products of the peptidoglycan are formed during the enlargement of the murein sacculus as a consequence of a growth mechanism, which couples the controlled degradation of the cell wall polymer with the insertion of new material. NagZ is needed for the formation of monosaccharides from the released disaccharides during the cytosolic steps of the muropeptide-recycling pathway. The formation of intracellular 1,6anhydro-N-acetylmuramyl-peptides is important for the expression control of the inducible ␤-lactamases of the AmpC type. A mutant lacking active NagZ cannot establish AmpC mediated ␤-lactam resistance. The biochemical characterization of the enzyme showed its activity on different muropeptides and inhibitors of enzyme activity could be identified. This observation might be important for designing inhibitors of NagZ that could prevent the establishment of ␤-lactam resistance of Enterobacteria possessing inducible ␤-lactamases.
Bacteria are enclosed by an exoskeletal structure found in the cell envelope (1). In most cases the bag-shaped macromolecule murein (peptidoglycan) stabilizes the cell mechanically and determines the shape of the bacterium (2). It is a heteropolymer, made up of glycan strands composed of an alternating sequence of two amino-sugars (N-acetylmuramic acid and Nacetylglucosamine) linked by ␤-1,4 glycosidic bonds (reviewed by Höltje in Ref. 3). The glycans are interlinked by short peptide bridges, and a covalently closed network completely surrounding the cell is formed. To enlarge this structure not only does new material have to be synthesized, it has to be integrated into the existing murein. This is accomplished by the concerted action of murein synthases and murein hydrolases (3)(4)(5)(6). During the controlled enlargement of the exoskeleton, old material from the sacculus gets released as turnover products while the polymer grows by the insertion of new murein glycan strands (7)(8)(9). Different enzymatic activities have been described to take part in this turning over of the murein sacculus. Lytic transglycosylases are muramidases that are degrading the glycan strands to anhydro-disaccharides (10), endopeptidases cleave peptide cross-links (11)(12)(13) and N-acetylmuramyl-L-alanine amidases release oligopeptides from the murein or from muropeptides (7). As a result of the action of these enzymes, disaccharides (N-acetylglucosamyl-␤-1,4-(1,6)-anhydro-N-acetylmuramic acid) are formed that carry a characteristic 1,6-anhydro ring structure at the muramic acid.
For Escherichia coli the turnover process has been well studied, and it could been shown that during one generation up to 50% of the cell wall gets degraded as a result of normal growth processes (9,14). These breakdown products are efficiently reutilized by E. coli in a recycling process; it could be shown that this reuse is an important source for the formation of new murein precursors (15). A major metabolic pathway, describing the fate of the peptide moiety originating from the breakdown of the murein could be elucidated. During the early steps of the recycling, the periplasmic degradation products get taken up by membrane transporters. Oligopeptides are internalized by the MppA/Opp system (16,17), whereas the membrane permease AmpG transports sugar containing muropeptides (18). In the cytosol the muropeptides get further degraded by the action of an N-acetylmuramyl-L-alanine amidase (AmpD; Refs. 19 and 20) and an essential LD-carboxypeptidase (LdcA; Ref. 21) and L-alanyl-D-glutamyl-meso-diaminopimelic acid gets released. The tripeptide is recognized by the muropeptide ligase Mpl (22) and added to UDP-N-acetylmuramic acid (UDP-Mur-NAc), 1 and UDP-MurNAc-tripeptide, a precursor for murein synthesis, is formed.
The fate of the sugar moiety is not clear despite the fact that the presence of a ␤-1,4-N-acetylglucosaminidase active on muropeptides had been reported over 20 years ago (23,24). Here the identification of the gene encoding NagZ, a cytosolic glucosaminidase involved in muropeptide recycling, is shown. Additionally, its need for the expression control of certain ␤-lactamases is described.

Agar Diffusion Test
LB agar plates (1.5% agar) were overlaid with 3 ml of LB top-agar (0.7% agar) containing 10 l of a bacterial culture grown overnight at 37°C. Filter discs (5 mm diameter) were loaded with 10 l of cefoxitin (20 mg/ml), dried, and placed on the agar plates. After incubation overnight at 37°C, the diameter of the inhibition zone was measured.

Determination of the Minimal Inhibitory Concentration of Cefoxitin
The MIC was determined by plating several hundred cells on LB agar plates containing a range of concentrations of cefoxitin that varied by a factor of 2.

␤-Lactamase Induction
To induce ␤-lactamase production 20 ml of culture of exponentially growing bacteria (A 578 ϭ 0.4) carrying pJP1 were diluted with 20 ml of prewarmed medium containing 5 g/ml cefoxitin. To measure ␤-lactamase activity, aliquots of the culture (0.5-4 ml) were removed and cells were harvested by centrifugation (15,000 ϫ g for 2 min). After washing with 2 ml of sodium phosphate buffer (50 mM, pH 7.0) cells were centrifuged again and frozen.

DNA Manipulations and PCR
Standard techniques were used for manipulating the plasmid DNA (26); E. coli was transformed as described by Inoue et al. (27). Restriction endonucleases were purchased from Roche Molecular Biochemicals; oligonucleotides came from MWG-Biotech (Ebersberg, Germany). PCR (28) was performed using PowerScript polymerase (PAN Systems, Nü rnberg, Germany) in a volume of 50 l of the supplied buffer with 0.5 mM of each primer and 0.1 g of chromosomal DNA as a template. After initial denaturation for 3 min at 92°C, touchdown PCR (29) was performed with 0.5 min of annealing, 1 min of extension at 72°C, and 0.5 min of denaturation at 92°C. During the PCR, the annealing temperatures were lowered to 61, 59, 57, 55, and 53°C for three cycles each and finally to 51°C for another 15 cycles.

Cloning of nagZ and Construction of an Overproducing System
The coding sequence of ycfO was amplified by using PCR. Both the wt allele (from E. coli AB1157) and the copy from the glucosaminidase deficient mutant strain E593 were amplified (specific primers 5Ј-AAA-GAATTCGTGGGTCCAGTAATGTTG-3Ј and 5Ј-AAAGTCGACCGCTT-CCTCACATAAGCC-3Ј) and cloned into the EcoRI and SalI sites of the vector pBC SK ϩ (Stratagene, La Jolla, CA). The constructs were termed pBC-nagZ and pBC-nagZ mut , respectively. The addition of an N-terminal His 6 tag to the protein was obtained after cloning of the PCR product (template chromosomal DNA from E. coli AB1157; primers: 5Ј-AAAGGATCCATGGGTCCAGTAATGTTGG-3Ј and 5Ј-AAAGTCGA-CCGCTTCCTCACATAAGCC-3Ј) into pQE30 (Qiagen) cut with BamHI and SalI. The resulting plasmid was named pHN5. All constructs were sequenced; in the case of the mutant allele several independently obtained clones were subject to sequencing.

Purification of NagZ
To overproduce the His 6 -tagged NagZ 800 ml of culture of E. coli XL-1 blue harboring pHN5 were grown in LB medium with moderate shaking at 37°C. Expression of the protein was induced at an A 578 of 0.2 by addition of 0.5 mM isopropyl-␤-D-thiogalactopyranoside. After 4 h the cells were harvested by centrifugation, washed with 50 ml ice-cold buffer A (100 mM sodium phosphate, 100 mM NaCl, pH 7.0), and resuspended in 5 ml buffer A containing 10 mM imidazol (buffer B). After disrupting the cells by three passages through a precooled French pressure cell at 15,000 p.s.i. and centrifugation (20 min, 90,000 ϫ g), the supernatant was used for purification of the enzyme by metal chelation chromatography on Superflow nickel-nitrilotriacetate (Qiagen). After loading of the column (bed volume, 3 ml; equilibrated with buffer B), the column was washed with 60 ml of buffer B at a flow-rate of 4 ml/h. The protein was eluted with buffer A containing 250 mM imidazol. Glucosaminidase activity in the eluted fractions was determined by meas-uring hydrolysis of PNP-␤-GlcNAc, and fractions showing high activity were pooled. After extensive dialysis against buffer A, the protein was stored at Ϫ20°C.

Purification of Muropeptides
Murein sacculi were isolated as described before and treated with ␣-amylase and pronase (30). Murein from 20 liters of culture was completely digested into noncross-linked disaccharide units by incubation with purified MepA (3 g) and purified Slt70 (2 g) in a volume of 4 ml of buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.0). Anhydro-disaccharide-tripeptide and anhydro-disaccharide-tetrapeptide were isolated by rpHPLC (30). To obtain unsubstituted anhydrodisaccharides isolated sacculi were degraded with purified Slt70 (2 g) and isolated AmiC (4 g). The anhydro-disaccharides were isolated by rpHPLC on 5-m Hypersil-ODS (4.6 mm x 125 mm; Bischoff, Leonberg, Germany). Chromatography was performed at room temperature at a flow rate of 1 ml/min using 0.1% trifluoroacetic acid. After 10 min a gradient (0% to 30% acetonitril) that was built up in 10 min was applied before equilibrating to 0.1% trifluoroacetic acid for 10 min. Fractions containing muropeptides were collected and dried.
Assays for ␤-1,4-N-acetylglucosaminidase Activity Spectrophotometric Assay-Cell extracts or protein fractions were incubated at room temperature for 20 min in a volume of 200 l containing 0.66 mM PNP-␤-GlcNAc, 50 mM Hepes, pH 7.1, and 500 mM NaCl. The reaction was quenched with 800 l of 300 mM sodium carbonate, and absorbance measured at 405 nm. ␤-1,4-N-acetylglucosaminidase activity in whole cells was measured as described before (24).
HPLC-based Assay-To determine the activity on different muropeptides, a HPLC-based detection system was established. Routinely 50 ng of purified enzyme was incubated for 20 min at room temperature in 100 l of buffer (50 mM Hepes, 500 mM NaCl, pH 7.1) with varying amounts of substrate. After addition of 5 l of 2% phosphoric acid, the mixture was boiled and centrifuged. 90 l of the reaction were used for separating the amino-sugars by rpHPLC on 5-m Hypersil-ODS (column size, 4.6 ϫ 125 mm; Bischoff). Chromatography was performed at room temperature at a flow rate of 1 ml/min using 50 mM sodium phosphate, pH 4.0 using a methanol gradient (4 to 15%) that was built up in 20 min. Alternatively, the chromatography conditions as described for the isolation of muropeptides were employed. Substrate and product peaks were quantified using calibrated standards (kindly provided by Astrid Ursinus).
␤-Lactamase Assay-␤-Lactamase activity was determined by measuring hydrolysis of nitrocefin, a chromogenic ␤-lactam (31). In contrast to published assay systems, activity was determined in whole cells, which were permeabilized by treatment with SDS and chloroform (32). Activity was calculated from ⌬A 492 /min⅐amount of cells (A 578 ). Briefly, 0.1-8 ml of a bacterial culture (A 578 ϭ 0.2-1.2) were centrifuged, and the cells were resuspended in 750 l of 50 mM sodium phosphate buffer, pH 7.0. Cells were permeabilized by the addition of 1 drop of chloroform and one drop of 0.05% SDS solution and vigorous shaking on a vortex mixer for 20 s. 100 l of different dilutions of permeabilized cells were mixed with an equal volume of a Nitrocefin solution (25 g/ml nitrocefin in 50 mM sodium phosphate buffer, pH 7.0), and hydrolysis was monitored over 20 min in a microtiter plate reader at 492 nm. The rate of hydrolysis was expressed as nM nitrocefin hydrolyzed per 1 A 578 of cells/min.

Identification and Molecular Cloning of the nagZ Gene-
Mapping data from Hrebenda (33) and Park (15) suggest a chromosomal location for the gene of a ␤-1,4-N-acetylglucosaminidase active on muropeptides at around 26 min of the E. coli genome. To identify this gene, the possible reading frames from this region (25 min to 27 min) were subject to data base comparisons using the BLAST algorithm (34) on the Swiss-Prot data base. One out of the approximately 100 checked reading frames showed a significant similarity to known glycosyl hydrolases. To show that such an enzyme is encoded by ycfO (located at 25.1 min, 1163 kilobase pairs of the physical map of the E. coli genome), the gene was amplified by PCR and subcloned into the cloning vector pBC SK ϩ . Transformation of E. coli E593, a mutant deficient in ␤-1,4-N-acetylglucosaminidase activity, with this plasmid restored enzyme ac-tivity as detected with a whole cell ␤-N-acetylglucosaminidase assay. Transformation of the same strain with a plasmid harboring a plasmid with the ycfO allele from E593 showed no measurable increase in glucosaminidase activity (data not shown). Both alleles were sequenced, and it could be shown that the wt copy is identical to the published sequence of ycfO (35). For the mutant allele a single base pair change was detected (changing Gly 101 to Asp) and therefore is responsible for the total loss of activity. The identified open reading frame was renamed to nagZ as proposed by Park (15).
Sequence Analysis-The reading frame from the ycfO gene encodes a protein of a predicted size of 37 kDa. Using the TopredII algorithm (36), no leader sequence was detected; therefore, a cytosolic localization is expected. These results are in agreement with data from Yem and Wu (23), who characterized the cytosolic ␤-N-acetylglucosaminidase from E. coli AB1157 mapping at 25 min.
Further similarity searches (using the PSI BLAST algorithm; Ref. 37) performed with the coding sequence of NagZ put the protein to family III of glycosyl hydrolases, a group of enzymes mainly containing ␤-glucosidases. Recently, the active site aspartic acid of ExoII, a highly similar protein from Vibrio furnissi was identified (38,39), and, indeed, this amino acid is conserved in all members of the family including the newly characterized enzyme. The mutation found in the nagZ allele from E593 maps close to another conserved region in the protein (data not shown). Highly similar orthologs were found within most Enterobacteriaceae sequenced to date, and searching the unfinished microbial genomes data base (last update, May  Characterization of a nagZ Mutant-Initially, no obvious phenotype for E593, the mutant isolated by Yem and Wu (23,24) was observed. Neither growth rate or cell shape was changed in LB medium (data not shown). To find out whether NagZ is indeed an enzyme that is involved in muropeptide recycling, an indirect approach was chosen. Muropeptides are known effectors of the transcriptional activator AmpR that controls the expression of AmpC type ␤-lactamases. This activator promotes ␤-lactamase transcription and is needed to establish ␤-lactam resistance. Activation of AmpR only occurs upon binding of certain muropeptides that are formed during the cytosolic degradation steps of turnover material from the cell wall. During normal growth conditions, the intracellular concentration of these murein metabolites stays low because they are efficiently recycled. In bacteria treated with ␤-lactams (e.g. cefoxitin), a high amount of muropeptides gets released and accumulates in the cytoplasm. Consequently, AmpR activation is found, and AmpC production is induced (18). The muropeptides activating AmpR are anhydro-monosaccharides (anhydro-N-acetyl-muramoyl-peptides) that are products of an N-acetyl-glucosaminidase acting on the released turnover material. Therefore, a strain carrying a nagZ mutation was tested for its capability to induce expression of AmpC type ␤-lactamases by determination of the sensitivity against ␤-lactam antibiotics. E. coli E593 was transformed with pJP1 (9), a plasmid that carries the ampR-ampC operon from E. cloacae, and the influence of the nagZ mutation on the induction process was examined. In an agar diffusion assay the sensitivity to cefoxitin, a cephalosporin that is a potent inducer of AmpC expression, was determined. In the corresponding control strain (AB1157), the transformation with pJP1 leads to high resistance against ␤-lactams. In contrast, the mutant E593 harboring the plasmid stayed as sensitive against cefoxitin as a strain not possessing the resistance plasmid (Table I). Introducing a second plasmid containing the cloned wild type allele of nagZ can complement this defect; the control transformation with the mutated nagZ from E593 on a plasmid did not lead to increased resistance to the cephalosporin. Therefore, it is concluded that the identified glucosaminidase is involved in muropeptide recycling and is needed for the establishment of expression of the inducible ␤-lactamases of the AmpC type.
To get further insight into the induction process, ␤-lactamase induction was measured in cultures of AB1157 and E593 carrying pJP1 (ampR ampC) after treatment with 2.5 g/ml cefoxitin. Activity was determined in culture aliquots (taken every 15 min) in a newly established whole cell assay (see "Experimental Procedures"). The hydrolysis of nitrocefin, a chromogenic substrate for ␤-lactamases was monitored, and induction factors could be calculated for the strains carrying the resistance plasmid (Table II). For the control a 17-fold induction (as compared with the untreated control) was found after 45 min, in the mutant the calculated induction factor was 4.5. Still a substantial amount of ␤-lactamase (37.2 nano mol/ min ϫ A 578 ) was measured in the induced nagZ mutant. Al-TABLE II ␤-Lactamase induction in a nagZ mutant carrying ampC/ampR from E. cloacae ␤-Lactamase production was induced by treating growing cells (A 578 ϭ 0.35) with 2.5 g/ml Cefoxitin. ␤-Lactamase activity was determined in permeabilized cells by monitoring the rate of hydrolysis of the chromogenic ␤-lactam nitrocefin at 492 nm as described under "Experimental Procedures." ␤-Lactamase activity is expressed as nano mol/min ϫ A 578 . The induction factor (in brackets) is the quotient from induced and uninduced cells.   38 1 a A nagZ mutant and its corresponding parent were transformed with a plasmid carrying the ampR ampC operon from E. cloacae and with different derivatives of the cloning vector pBC SK ϩ containing wt nagZ or mutated nagZ mut .
b Sensitivity was determined in an agar diffusion assay. Filter disks (5 mm in diameter) loaded with 200 g of cefoxitin were placed onto an agar plate inoculated with the indicated strain. After incubation overnight the diameter of the zone of inhibition was measured.
c The minimal inhibitory concentration (MIC) was determined on agar plates containing the indicated concentration of the antibiotic. though this level of ␤-lactamase production does not lead to any increase in ␤-lactam resistance, it opens the question how induction can occur in the nagZ mutant. A possible explanation might be the presence of a second enzyme able to hydrolyze anhydro-muropeptides. This enzyme could be identical to BglX, a periplasmic ␤-D-glucosidase also belonging to the family III of the glycosyl hydrolases (40). This enzyme, together with an transporter specific for the released anhydro-monosaccharides would constitute an unknown way to reutilize turnover material.
Purification of His 6 -NagZ-To allow a simple purification of the identified enzyme, the coding region of nagZ was subcloned into the gene fusion vector pQE30 (Qiagen), and an N-terminal His 6 extension was created. The fusion protein was active in the whole cell glucosaminidase assay (Ref. 23 and data not shown). A high degree of overexpression of the soluble fusion protein could be obtained upon addition of 0.5 mM isopropyl-␤-D-thiogalactopyranoside (Fig. 1). Metal chelation chromatography was used for purification on a nickel-nitrilotriacetate-Sepharose column, and a simple one-step purification procedure was established. Essentially pure protein as judged by SDS-polyacrylamide gel electrophoresis and Coomassie staining could be obtained (Fig. 1). The apparent molecular mass (37 kDa) agrees with the calculated value from the predicted amino acid sequence. The protein concentration was found to be 0.3 mg/ml, and the specific activity on PNP-␤-GlcNAc was 8.8 mol/min mg protein in the standard assay.
Characterization of NagZ-An initial characterization of the enzyme was performed using PNP-␤-GlcNAc, a chromogenic substrate for ␤-1,4-glucosaminidases that had been shown to be accepted by NagZ (23). In a buffer system containing 50 mM Hepes, the pH optimum of the enzyme was detected at 7.1, and the temperature optimum was found at 38°C. The addition of 1 M NaCl stimulated the activity 5-fold (Fig. 2). All experiments described below were therefore performed in 50 mM Hepes buffer, pH 7.1, containing 0.5 M NaCl. The apparent kinetic constants with PNP-␤-GlcNAc as substrate (calculated from a Lineweaver-Burk diagram) were: K m 310 M, V max 13.3 mol/ min mg protein at 25°C.
Activity of NagZ on Muropeptides-The role of NagZ in muropeptide recycling suggests three possible natural substrates. The major muropeptide released during cell wall turnover by E. coli is the anhydro-disaccharide-tetrapeptide (41). This disaccharide can be converted to anhydro-disaccharide and to anhydro-disaccharide-tripeptide by the action of the intracellular amidase AmpD (19) and the LD-carboxypeptidase LdcA (21), respectively. To test activity of NagZ on these muropeptides, they were isolated in a larger scale. Isolated murein sacculi were degraded to anhydro-disaccharides by incubation with isolated Slt70 (42); cross-linked muropeptides were converted to anhydro-disaccharide-tetrapeptide and anhydro-disaccharide-tripeptide by enzymatic cleavage of the cross-bridge by addition of purified MepA (11). Disaccharides lacking the peptide side chain were obtained from isolated sacculi treated with purified AmiC 2 and isolated Slt70. The muropeptides were isolated by rpHPLC and quantified using authentic standards (kindly provided by A. Ursinus). An initial characterization showed that the isolated NagZ accepts all three muropeptides as substrates (data not shown).
Kinetic Characterization and Inhibition of NagZ-To find 2 M. Templin, manuscript in preparation. out about a preference for any of these substrates, a kinetic characterization of the action of NagZ on the three muropeptides was performed. The substrate concentration was varied from 1 to 300 M, and the initial reaction velocity was estimated using the rpHPLC based assay (Fig. 3A). The apparent kinetic constants for the different muropeptides were calculated with the EKI data analysis program that uses a nonlinear regression of the Michaelis-Menten equation (43). The K m values determined were 32 M for anhydro-disaccharide-tetrapeptide, 29 M for anhydro-disaccharide-tripeptide, and 35 M for anhydro-disaccharide. These values are nearly identical; it is concluded that all muropeptides formed during the turnover and recycling process are substrates for NagZ.
In the assay employed, strong inhibition of enzyme activity was observed at high substrate concentrations. The graphical presentation of the kinetic data in a Lineweaver-Burk diagram visualizes this effect (Fig. 3B). The apparent inhibition constant for the anhydro-disaccharide-tetrapeptide was estimated from a Dixon plot and found to be approximately 100 M. Although this value is higher then the amount of muropeptides found during normal growth conditions, it is in the range of the intracellular muropeptide concentration found during ␤-lactamase induction.
The observation that NagZ activity can be inhibited was further studied. The influence of different amino-sugars on enzyme activity was determined. Strong inhibition was found with N-acetylglucosamine, a product of the glucosaminidase and with the chromogenic substrate of the enzyme the PNP-␤-GlcNAc. MurNAc, GlcNAc-6-phoshate, and chitobiose had no effect on the enzyme. Interestingly, bulgecin, a glycopeptide that has been shown to inhibit the soluble lytic transglycosylase Slt70, did inhibit NagZ activity (Table III). In vivo, inhibitors of NagZ need to be present in the cytosol, and uptake systems internalizing such compounds are needed. Because the identified inhibitors are all different amino-sugars, it seems possible that known sugar transporters will faciliate the uptake of such compounds. DISCUSSION By reusing the turnover material, which is released during growth of the murein sacculus, E. coli saves a substantial amount of energy. Indeed, the recycling process has to be seen as a major energy saving pathway by which precursors for cell wall synthesis are formed. During exponential growth about 50% of the cell wall gets released per generation, and about a third of the peptides found in newly formed precursor molecules originate from recycled material (44). With the identification of the uptake systems (MppA/Opp and AmpG), the tripeptide releasing enzymes (AmpD and LdcA) and the muropeptide ligase Mpl all enzymes needed for the reuse of the peptide are known (15). In contrast, it is not clear what happens with the disaccharide moiety taken up by the cell. Although the presence of an intracellular ␤-1,4-N-acetylglucosaminidase active on muropeptides has been described (23), its physiological function has not been clarified. This work could show that this enzyme leads to the formation of cytosolic monosaccharides. For one of the reaction products of NagZ, N-acetylglucosamine, a reutilization by feeding it into the described metabolic pathways using this amino-sugar seems likely. Still, a cytosolic sugar kinase converting this aminosugar into N-acetylglucosamine-phosphate has to be identified. The other product, 1,6-anhydro-muramic acid substituted with a Inhibition of NagZ was determined in the rpHPLC based assay with 50 ng of purified His 6 -NagZ and 50 M anhydro-disaccharide-tetrapeptide as substrate under standard conditions. FIG. 3. Substrate specificity of His 6 -NagZ. Different amounts of isolated muropeptides were incubated with 50 ng of purified NagZ under standard conditions. Ⅺ, anhydro-disaccharide; E, anhydro-disaccharide-tripeptide; q, anhydro-disaccharide-tetrapeptide. The initial reaction velocity was calculated from the amount of product formed as quantified by rpHPLC (A). The Lineweaver-Burk diagram was chosen to visualize the apparent kinetic constants (B). a side peptide at the lactyl moiety is subject to further enzymatic degradation. The action of the cytosolic N-acetylmuramyl-L-alanine amidase AmpD leads to the release of the anhydro-muramic acid. This is an unusual sugar containing an intramolecular ring from C-1 to C-6 formed during the enzymatic cleavage of the ␤-1,4-glycosidic bond between the Nacetylmuramic acid and the N-acetylglucosamine by the action of the lytic transglycosylases. These muramidases catalyze the cleavage of the glycosidic bond found in the glycan strands of the polymer during the turnover of the murein sacculus. The cleavage occurs not by hydrolysis but by an intramolecular transglycosylation reaction (10,45). Part of the energy released during splitting of the bond gets used to create the intramolecular 1,6-ring, and anhydro-muropeptides are formed. It has been argued that this is a way of saving energy that can be used during the metabolism of the anhydro-sugars (46). Therefore the reuse of 1,6-anhydro-muramic acid might be accomplished by a way different from known sugar utilizing systems. A phosphorylation mechanism taking advantage of the energy stored in the intramolecular ring might be involved in metabolizing these sugars. Even a direct conversion into an UDP activated muramic acid, an early precursor for cell wall synthesis, might be envisioned.
The muropeptide recycling pathway is not only important for saving energy, it is used for signaling and sensing processes in Gram-negative bacteria (47,48). During growth, turnover material is released in a controlled manner and gets recycled. Disturbance of this process, either by mutations in the recycling pathway or by interfering with cell wall metabolism, leads to changes in the pools of intracellular molecules formed during their reuse (48,49). Gram-negative bacteria are able to detect deviations from normal conditions and thereby sense the state of the cell wall. Work on the inducible ␤-lactamases of the AmpC type has shown how pools of intracellular metabolites from degradation and synthesis of the murein are used for signaling. Changes in the amount of the anhydro-N-acetylmuramyl-peptides (tri-, tetra-, or pentapeptides) lead to differences in the expression pattern of the AmpC ␤-lactamases (41). The sensing device coupling the muropeptide recycling pathway to precursor synthesis is the transcriptional activator AmpR. It has the ability to complex UDP-MurNAc-pentapeptide and stays inactive upon binding. In the presence of high amounts of the anhydro-N-acetylmuramyl-peptides, this murein precursor gets displaced, and AmpR gets functional as transcriptional activator (48). Therefore, by measuring the relative amounts of precursors and degradation products, information about the state of the cell wall can be obtained. In the case of the inducible ␤-lactamase, the increased degradation of the murein caused by the presence of ␤-lactams is detected and leads to expression induction of AmpC. Actually most of the proteins needed for recycling have been identified during studies of the expression control of ␤-lactamases, and only later could their primary function be elucidated (15). Here an opposing approach was chosen. By identifying and characterizing an enzyme needed for muropeptide recycling, information about its role in the establishment of ␤-lactam resistance was obtained. The need of a ␤-1,4-N-acetylglucosaminidase for the formation of the positive effectors (anhydro-N-acetylmuramylpeptides) of AmpR is obvious because they are formed as reaction products of this enzymatic activity. Consequently, a mutant lacking NagZ is not able to produce the activating monosaccharides. This work could show that the enzyme is indeed needed for establishing the resistant state. The sensitivity against ␤-lactams that is found for a nagZ mutant transformed with a plasmid carrying the ampRC operon is compa-rable with a situation where no ampC gene is present. Therefore, the inactivation of this glucosaminidase can be seen as a possibility to inhibit expression of inducible ␤-lactamases and lock bacteria capable of producing AmpC in a sensitive state.
The biochemical characterization of the identified ␤-1,4-Nacetylglucosaminidase NagZ demonstrated some properties of the enzyme that are of interest in this context. The possibility to inhibit enzyme activity by addition of various amino-sugars might allow the design of specific inhibitors of NagZ. Such compounds would act as indirect effectors on AmpC expression and prevent expression induction of AmpC type ␤-lactamases. These enzymes are important virulence factors in a variety of pathogenic Gram-negative microorganisms, and novel approaches to treat infections caused by these bacteria are needed. By using inhibitors of NagZ, production of ␤-lactamases could be prevented, and strains resistant against ␤-lactam antibiotics could be rendered sensitive again.