Abstract
ABSTRACTPeptidoglycan 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.
Highlights
Peptidoglycan (PGN) encases the bacterial cell, forming a huge, netlike, turgor-resisting and shape-maintaining envelope structure that is composed of glycan strands of two alternating -1,4-linked sugars, N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc), cross-linked by short peptides [1]
SAUSA300_0193–0195 encode proteins with 47%, 37%, and 27% amino acid sequence identities to the respective E. coli proteins, based on analysis of the full-size proteins using the basic local alignment search tool (BLAST)
We show here that three Gram-positive bacteria, S. aureus, B. subtilis, and S. coelicolor, recycle the MurNAc contents of their PGN cell wall during vegetative growth in rich medium, which requires the orthologs of the MurQ enzyme of E. coli
Summary
Peptidoglycan (PGN) encases the bacterial cell, forming a huge, netlike, turgor-resisting and shape-maintaining envelope structure that is composed of glycan strands of two alternating -1,4-linked sugars, N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc), cross-linked by short peptides [1]. Applying continuous radioactive labeling and pulse-chase labeling approaches, the amount of PGN breakdown was measured by determining the radioactivity found in the growth medium relative to that remaining in the insoluble cell wall material These studies came up with rather inconsistent rates of PGN release in Gram-positive bacteria during growth, ranging from no turnover to up to 50% turnover per generation [5, 22,23,24,25]. The first reports revealed PGN turnover in the Gram-positive bacterium Bacillus megaterium, i.e., the release of PGN fragments into the growth medium [4], at a rate of 15 to 20% turnover per generation by measuring the loss of radiolabeled diaminopimelic acid from the prelabeled cell wall. PGN does not affect the growth rates of S. aureus and B. subtilis but is crucial for their survival during stationary phase
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