An alternative route for recycling of N-acetylglucosamine from peptidoglycan involves the N-acetylglucosamine phosphotransferase system in Escherichia coli

Abstract

A set of enzymes dedicated to recycling of the amino sugar components of peptidoglycan has previously been identified in Escherichia coli. The complete pathway includes the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase, of the catabolic pathway for use of N-acetylglucosamine (GlcNAc). Mutations in nagA result in accumulation of millimolar concentrations of GlcNAc6P, presumably by preventing peptidoglycan recycling. Mutations in the genes encoding the key enzymes upstream of nagA in the dedicated recycling pathway (ampG, nagZ, nagK, murQ, and anmK), which were expected to interrupt the recycling process, reduced but did not eliminate accumulation of GlcNAc6P. A mutation in the nagE gene of the GlcNAc phosphotransferase system (PTS) was found to reduce by 50% the amount of GlcNAc6P which accumulated in a nagA strain and, together with mutations in the dedicated recycling pathway, eliminated all the GlcNAc6P accumulation. This shows that the nagE-encoded PTS transporter makes an important contribution to the recycling of peptidoglycan. The manXYZ-encoded PTS transporter makes a minor contribution to the formation of cytoplasmic GlcNAc6P but appears to have a more important role in secretion of GlcNAc and/or GlcNAc6P from the cytoplasm.

FIG. 1.

Scheme for recycling of PG in E. coli. The enzymes and substrates are described in the text. Slt is the major soluble lytic transglycosylase. OM, outer membrane; PP, periplasm; IM, inner membrane. The enzymes involved in converting UDP-GlcNAc into the components of the PG and outer membrane are not shown. Arrows with a question mark indicate the pathways postulated to exist based on the results described in this work.

FIG. 2.

Effect of recycling and nagE mutations on levels of GlcNAc plus GlcNAc6P and nagB-lacZ expression. (A) Soluble extracts of strains belonging to the different mutant groups (see panel B) were tested to determine the levels of GlcNAc plus GlcNAc6P by the modified Morgan-Elson method. Values for different strains belonging to the same mutant group are combined, and the results are the means and standard deviations for between 2 to 10 independent cultures. (B) Genotypes of strains in the different mutant groups. (C) β-Galactosidase activities of the nagB-lacZ fusion in strains belonging to mutant groups 1 to 8 and the isogenic manXYZ groups 1m to 9m. The data are the means and standard deviations for 2 to 10 independent cultures. (D) Total GlcNAc reacting material was measured by the Morgan-Elson method in soluble extracts of mutant group 1 to 8 strains and the isogenic manXYZ strains (mutant groups 1m to 8m) before and after treatment of the extracts with NagA (GlcNAc6P deacetylase). The first bar in each set of four bars indicates the total amount of GlcNAc plus GlcNAc6P in the manXYZ⁺ strain; the second bar indicates the amount of GlcNAc6P in the manXYZ⁺ strain; the third bar indicates the total amount of GlcNAc plus GlcNAc6P in the manXYZ strain; and the fourth bar indicates the amount of GlcNAc6P in the manXYZ strain. A subset of the extracts tested to obtain the data in panel A were reanalyzed in this test. The data are the means and standard deviations for two to six independent cultures. 5OD, 5 OD₆₅₀ units.