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ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. IMPORTANCE The peptidoglycan of the bacterial cell wall is turned over steadily during growth. As peptidoglycan fragments were found in large amounts in spent medium of exponentially growing Gram-positive bacteria, their ability to recycle these fragments has been questioned. We conclusively showed recycling of the peptidoglycan component MurNAc in different Gram-positive model organisms and revealed that a MurNAc-6P etherase (MurQ or MurQ ortholog) enzyme is required in this process. We further demonstrated that recycling occurs predominantly during the transition to stationary phase in S. aureus and B. subtilis, explaining why peptidoglycan fragments are found in the medium during exponential growth. We quantified the intracellular accumulation of recycling products in MurNAc-6P etherase gene mutants, revealing that about 5% and 10% of the MurNAc of the cell wall per generation is recycled in S. aureus and B. subtilis, respectively. Importantly, we showed that MurNAc recycling and salvaging does not sustain growth in these bacteria but is used to enhance survival during late stationary phase.