We identified a pathway in Bacillus subtilis that is used for recovery of N-acetylglucosamine (GlcNAc)-Nacetylmuramic acid (MurNAc) peptides (muropeptides) derived from the peptidoglycan of the cell wall. This pathway is encoded by a cluster of six genes, the first three of which are orthologs of Escherichia coli genes involved in N-acetylmuramic acid dissimilation and encode a MurNAc-6-phosphate etherase (MurQ), a MurNAc-6-phosphate-specific transcriptional regulator (MurR), and a MurNAc-specific phosphotransferase system (MurP). Here we characterized two other genes of this cluster. The first gene was shown to encode a cell wall-associated -N-acetylglucosaminidase (NagZ, formerly YbbD) that cleaves the terminal nonreducing N-acetylglucosamine of muropeptides and also accepts chromogenic or fluorogenic -N-acetylglucosaminides. The second gene was shown to encode an amidase (AmiE, formerly YbbE) that hydrolyzes the N-acetylmuramyl-L-Ala bond of MurNAc peptides but not this bond of muropeptides. Hence, AmiE requires NagZ, and in conjunction these enzymes liberate MurNAc by sequential hydrolysis of muropeptides. NagZ expression was induced at late exponential phase, and it was 6-fold higher in stationary phase. NagZ is noncovalently associated with lysozyme-degradable particulate material and can be released from it with salt. A nagZ mutant accumulates muropeptides in the spent medium and displays a lytic phenotype in late stationary phase. The evidence for a muropeptide catabolic pathway presented here is the first evidence for cell wall recovery in a Gram-positive organism, and this pathway is distinct from the cell wall recycling pathway of E. coli and other Gram-negative bacteria.Bacteria are covered by an exoskeleton-like cell wall that protects the fragile membrane-enclosed cell, the protoplast, and withstands the high internal pressure of the cell, the turgor pressure (27). Despite its stabilizing function, the cell wall is not rigid and static but is highly flexible. It undergoes continuous resynthesis, remodeling, and degradation (turnover) in which a substantial amount of the murein (peptidoglycan), the stabilizing component of the bacterial cell wall, is released during logarithmic growth (5, 47). The Gram-positive model organism Bacillus subtilis was shown to release about 50% of its murein into the medium in one generation during growth (9, 42, 43). Gram-negative bacteria have an outer membrane that keeps most of the cell wall turnover products in the periplasmic space, but Gram-positive bacteria lack such a membrane barrier and therefore cannot retain their turnover products. So far, more than 30 peptidoglycan hydrolases of B. subtilis have been characterized or identified as candidate autolysins on the basis of amino acid sequence identity (50), but the nature of the turnover products remains ambiguous. Moreover, a cell wall recycling-recovery pathway has not been identified in this organism or in any other Gram-positive bacterium so far.In contrast, cell wall turnover and recycling in the Gramneg...
