An Alternative Route for Recycling of N-Acetylglucosamine from Peptidoglycan Involves the N-Acetylglucosamine Phosphotransferase System in Escherichia coli

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.Peptidoglycan (PG) or murein, the rigid shape-forming layer of the bacterial cell envelope, undergoes extensive degradation and resynthesis during normal bacterial growth. It is estimated that 40 to 50% of the PG is broken down and reused each generation (for a review, see reference 22). PG is a matrix of chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) sugars cross-linked by peptide bridges. Over the last 20 years the pathways for recycling both the peptide and amino sugar portions of the PG have been elucidated, and a number of genes involved in this process have been identified. Most of the genes involved encode dedicated enzymes whose only function seems to be to recover the material produced during PG turnover and to reuse it to synthesize more PG or as a source of energy. However, some of the enzymes shown to be involved have apparently been recruited from another metabolic pathway (e.g., murQ- and nagA-encoded enzymes see below]), while other specialized PG-recycling enzymes have a subsidiary function (e.g., ampG- and ampD-encoded enzymes in β-lactamase induction 20]).The pathway for recycling the amino sugar part of PG in Escherichia coli is shown in Fig. ​Fig.11 (for a review, see reference 22). Periplasmic hydrolases (lytic transglycosylases, Slt) and endopeptidases break the PG backbone, liberating anhydro-muropeptides (principally GlcNAc-anhydro-MurNAc anhMurNAc]-tetrapeptide), which are transported into the cytoplasm by the ampG-encoded transporter (10). The peptide portion is cleaved off either by the membrane-associated amiD-encoded amidase (28) or by the ampD-encoded cytoplasmic amidase (11), liberating the disaccharide. The tetrapeptide is converted to a tripeptide and free d-Ala, both of which are reused to produce UDP-MurNAc-pentapeptide (11). The GlcNAc-anhMurNAc disaccharide is cleaved by the nagZ-encoded β-N-acetylglucosaminidase (2, 32), and then both sugars are converted to their 6-phosphate forms by the specific kinases NagK (29) and AnmK (31). The latter produces MurNAc-6-phosphate (MurNAc6P), which is converted to GlcNAc6P by the murQ-encoded etherase (12, 30). MurNAc6P is also the product of transport of MurNAc by the MurNAc-specific phosphotransferase system (PTS) transporter MurP. The murP and murQ genes form an operon for use of MurNAc as a carbon source (4). Thus, the MurQ protein has both catabolic and recycling functions (12, 30). Similarly, further use of the GlcNAc6P involves an enzyme normally involved in the catabolism of GlcNAc, the nagA-encoded GlcNAc6P deacetylase of the GlcNAc degradation pathway (21). The deacetylase converts GlcNAc6P to glucosamine-6-phosphate (GlcN6P), which can be converted to UDP-GlcNAc, the first dedicated compound for the synthesis of the cell wall components, by the glmM- and glmU-encoded enzymes (16, 17).Open in a separate windowFIG. 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.It has been known for many years that mutations in nagA lead to very high levels of GlcNAc6P (33). Strains carrying nagA mutations are Nag^Sensitive (i.e., they do not grow in medium containing GlcNAc and another carbon source). The toxicity of the accumulated sugar phosphates means that secondary mutations that alleviate this toxicity arise spontaneously in vivo (33). GlcNAc6P is the inducing signal for the NagC repressor of the nag regulon, and the accumulation of GlcNAc6P in the nagA strain results in derepression (endogenous induction) of the nag regulon (25). One class of suppressor mutations result in noninducible versions of NagC that are not sensitive to GlcNAc6P, so that the nag genes stay repressed (23), implying that overexpression of the nag regulon genes is one cause of the toxicity. Amino sugars are essential constituents of the bacterial PG and lipopolysaccharide (LPS) in gram-negative bacteria. In the absence of an exogenous supply of amino sugars, glmS, encoding GlcN6P synthase, is an essential gene (for a review, see reference 7). As GlcNAc6P accumulates in nagA cells growing in medium devoid of amino sugars, it must ultimately be derived from the de novo synthesis of GlcN6P by GlmS, which is destined for synthesis of PG and the LPSs of the outer membrane. As no acetyltransferase for GlcN6P has been characterized, the most likely origin of the GlcNAc6P in nagA strains is recycling of the PG. The LPS of the outer membrane of gram-negative bacteria also contains GlcN, but it is not known to undergo any turnover and the work of Park (21) showed that radioactive GlcN was stably incorporated into the LPS fraction, whereas radioactivity was slowly lost from the PG of isolated sacculi.In this work the effect of mutations in the recycling pathway on the accumulation of GlcNAc6P in vivo was investigated. The results show that mutations in one or more genes of the recycling pathway reduce but do not eliminate GlcNAc6P accumulation in nagA strains. However, when these mutations are present in the same strain with a mutation in the nagE gene encoding the GlcNAc6P-specific transporter of the GlcNAc PTS, GlcNAc6P levels decrease to the background level. This shows that the GlcNAc PTS is another pathway that is involved in recycling the GlcNAc component of PG. The manXYZ-encoded PTS transporter is also capable of GlcNAc uptake, and its effect on the recycling process was also examined.