Molecular Characterization of the β-N-Acetylglucosaminidase ofEscherichia coliand Its Role in Cell Wall Recycling

Abstract

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 ...

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…aeruginosa harbours two AmpG homologues, PA4393 (AmpG) and PA4218 (AmpP) ( Table 2) [150,151]. Both AmpG and AmpP are inner membrane permeases with 14 and 10 transmembrane helices, respectively [151].…”

The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide

“…Turnover is carried out by periplasmic autolysins that excise GlcNAc-␤(134)-1,6-anhydroMurNAc-peptide fragments from the PG sacculus (9), and the fragments are transported to the cytosol by the AmpG permease (10) for recycling. In the cytosol, the nonreducing GlcNAc residue is removed from the fragments by the glycosidase NagZ (11)(12)(13) to yield a series of 1,6-anhydroMurNAc-tripeptides, -tetrapeptides, and -pentapeptides. The amidase AmpD cleaves the amide bond linking the remaining sugar, 1,6-anhydroMurNAc, to the peptide stems (14,15), and the resulting pool of monosaccharide and peptide catabolites are subsequently used to build UDP-MurNAc-pentapeptide, an essential anabolite of cell wall synthesis (5).…”

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…6 The enzyme releases GlcNAc from the cytosolic peptidoglycan recycling GlcNAc-1,6-anhydroMurNAcpeptide intermediates to create 1,6-anhydroMurNAcpeptides. 7 These catabolites serve as a signal to induce transcription of chromosomal AmpC b-lactamase 8 and thereby promote resistance of Gram-negative bacteria to an extended-spectrum of b-lactam antibiotics including cephalosporins, cephamycins, and monobactams. 9 This inducible resistance mechanism is found in a number of Gram-negative bacteria, including, for example, the opportunistic human pathogens Pseudomonas aeruginosa and Enterobacter cloacae.…”