Abstract
The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-β-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-β-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.
Highlights
Staphylococcus aureus is a small, spherical bacterium (∼1 μm diameter) belonging to the phylum firmicutes that can cause life-threatening infections due to the emergence of multi-drug resistance (Hiramatsu et al, 2014)
The gene SAUSA300_0192, named mupG, encodes an uncharacterized, hypothetical protein that is classified as a member of a protein family of unknown function (DUF871)
We recently showed that the PTS transporter MurP and the MurNAc 6-phosphate etherase MurQ are required for the recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) in S. aureus and other Gram-positive bacteria (Borisova et al, 2016)
Summary
Staphylococcus aureus is a small, spherical bacterium (∼1 μm diameter) belonging to the phylum firmicutes that can cause life-threatening infections due to the emergence of multi-drug resistance (Hiramatsu et al, 2014). As a Grampositive bacterium, S. aureus is encased in a thick layer of peptidoglycan (PGN), which maintains cell shape and protects the cells from rupture due to high turgor pressure (Cabeen and Jacobs-Wagner, 2005). The general structure of the PGN is conserved in all eubacteria, consisting of a glycan backbone of two alternating β-1,4-glycosidically linked sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) and polypeptides, connected to the D-lactyl moiety of MurNAc, that are partially crosslinked (Vollmer et al, 2008). The PGN polymer is synthesized by glycosyltransferases and transpeptidases [involving shape, elongation, division and sporulation (SEDS) family peptidoglycan synthases and penicillin binding proteins (PBPs)] through polymerization of GlcNAc-β-1,4-MurNAc-peptide building blocks, provided by lipid II precursors (Cho et al, 2016; Meeske et al, 2016). About 80% of the glycan chains of the mature cell wall have a predominant length of 3– 10 disaccharides, the average degree of polymerization is 6 disaccharides, and only about 15% of the glycan chains have a degree of polymerization more than 25 (Boneca et al, 2000)
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