Abstract
The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.
Highlights
In gram-positive bacteria a thick multilayered peptidoglycan (PG) constitutes the major component of the cell wall and is essential for survival, maintenance of cell shape and counterbalance of turgor pressure [1]
Biosynthesis starts in the cytoplasm, where the MurA-F ligases catalyze the formation of the ultimate soluble cell wall precursor UDP-MurNAc-pentapeptide [2,3,4,5]
The peptidoglycan of many Gram positive pathogens such as Staphylococcus aureus is almost fully amidated which appears to reduce the susceptibility towards innate host defenses
Summary
In gram-positive bacteria a thick multilayered peptidoglycan (PG) constitutes the major component of the cell wall and is essential for survival, maintenance of cell shape and counterbalance of turgor pressure [1]. The biosynthesis of PG is a multistep process which requires numerous enzymatic reactions, occurring in three compartments of a bacterial cell; the cytoplasm (synthesis of nucleotide-bound precursors), the inner face of the membrane (synthesis of the cell wall building block lipid II and lipid II modifications) and the outer face of the membrane (polymerisation of lipid II into the growing PG network). Biosynthesis starts in the cytoplasm, where the MurA-F ligases catalyze the formation of the ultimate soluble cell wall precursor UDP-MurNAc-pentapeptide [2,3,4,5]. Following synthesis and assembly the S. aureus PG undergoes further modifications including O-acetylation of N-acetyl-muramic acid [17,18,19] and the addition of structures that are covalently linked, such as wall teichoic acids [20], proteins and capsules [21,22]
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