The ␤-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissii ExoII. NagZ hydrolyzes the ␤-1,4 glycosidic bond between N-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel ␤-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.␤-N-Acetylglucosaminidase in Escherichia coli K-12 was first described by Yem and Wu in 1976 (18, 19). It was shown to be a cytoplasmic enzyme active against both p-nitrophenyl-␤-Nacetyl-D-glucosaminide and a muropeptide released by lysozyme from E. coli cell wall murein (peptidoglycan). However, based on indirect evidence, it was clear that the enzyme would also be active against anhydro-muropeptides (6). These muropeptides contain N-acetylglucosamine (GlcNAc) linked ␤-1,4 to anhydro-N-acetylmuramyl peptides (aMurNAc-peptides) (15). aMurNAc, which possesses a 1,6 anhydro bond, is formed by the lytic transglycosylases of E. coli that digest murein during normal growth as the initial step of the murein tripeptide recycling pathway. The murein tripeptide recycling pathway is a major metabolic pathway of E. coli (3) in which, during each generation of growth, about 40% of the cell wall murein is broken down into anhydro-muropeptides (3, 6). The anhydro-muropeptides are then transported into the cytoplasm via AmpG permease (6) and are rapidly degraded by the combined actions of ␤-N-acetylglucosaminidase (NagZ), anhydro-N-acetylmuramyl-L-alanine amidase (AmpD) (5, 7), and an LD-carboxypeptidase (LdcA) (17) to release GlcNAc, aMurNAc, D-alanine, and the murein tripeptide (L-alanyl-␥-D-glutamylmeso-diaminopimelic acid). The tripeptide is then linked to UDP-MurNAc by the murein peptide ligase, Mpl (10), and efficiently recycled to form murein de novo.Previous results imply the participation of a ␤-N-acetylglucosaminidase in recycling (6), and the present results identify NagZ as the enzyme involved. In this work, we also identify the gene encoding ␤-N-acetylglucosaminidase (nagZ), characterize a null mutation and a mutation in the structural gene, and report initial observations on the specificity of NagZ. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The E. coli K-12 strains and plasmids used in this work are listed in Table 1. Bacteria were grown aerobically at 37°C in L broth, which is LB broth (12) modified to contain only 5 g of NaCl per liter. Ampicillin (100 g/ml), kanamycin (25 g/ml), and chloramphenicol (10 g/ml) were used as required.Isolation of a ␤-N-acetylglucosaminidase-deficient mutant. E. coli TP71 cells were treated with nitrosoguanidine (10, 20, or 40 g/ml in 0.1 M citrate buffer ...

AmpG was originally identified as a gene required for induction of ␤-lactamase. Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides. We have now studied the specificity of the AmpG permease. The principal requirement is for the presence of the disaccharide, N-acetylglucosaminyl-␤-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc). These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth. AmpG permease is sensitive to carbonylcyanide mchlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force.AmpG is a cytoplasmic membrane protein required for recycling of murein tripeptide as well as induction of Citrobacter freundii ␤-lactamase (4, 6, 7). AmpG has been presumed to be the permease for N-acetylglucosaminyl-␤-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc)-peptides, since anhydro-N-acetylmuramyl-L-alanyl-␥-D-glutamyl-meso-diaminopimelic acid (anhMurNAc-tripeptide) accumulates in the cytoplasm of ampD cells but not in ampG, ampD cells (4) and since the only ␤-N-acetylglucosaminidase in Escherichia coli is cytoplasmic (12, 13). GlcNAcanhMurNAc-peptides are the products of breakdown of the murein sacculus of E. coli by multiple lytic transglycosylases (11). During active growth, well over half of the side wall of the sacculus is broken down each generation (1). To determine the specificity of the AmpG permease, a number of radioactive ligands were prepared from E. coli cells labeled with D-[6-3 H(N)]glucosamine (except for three ligands labeled with 3 Hdiaminopimelic acid [ 3 H-Dap] as noted). These ligands were used to compare uptake by freeze-thawed cells of E. coli ampG ϩ with those of ampG freeze-thawed cells. MATERIALS AND METHODSBacterial strain, plasmids, and growth conditions. The E. coli K-12 strains and plasmids used in this study are listed in Table 1. Cells were grown aerobically at 37°C in L broth, which is Luria-Bertani broth (10) modified to contain only 5 g of NaCl per liter. Ampicillin (100 g/ml) and chloramphenicol (10 g/ml) were used as required.Preparation of freeze-thawed cells. Cells from 40 ml of overnight culture were harvested, washed with 0.1 M phosphate buffer (pH 7.0) in the cold, and resuspended in 1.3 ml of the same buffer to give a suspension usually containing 3 to 5 mg of protein/ml. Stationary-phase cells were used because they were found to be more active than mid-log-phase cells. The cell suspension was adjusted to contain 1 mg of protein/ml. Two-hundred-microliter aliquots were rapidly frozen in a dry ice alcohol bath and were then thawed in a water bath and held at room temperature for 15 to 30 min. After this period, radioactive substrate was added and the incubation was continued for 50 min. An incubation time of 50 min at room temperature was chosen since significant uptake continued for that length of time. Therea...