Abstract
The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.
Highlights
Staphylococcus aureus is an opportunistic pathogen that inhabits the human skin and mucosa and causes a large variety of nosocomial and community-acquired infections[1]
In S. aureus the conserved eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp impact bacterial cell signalling, central metabolism[12,13,14], stress response[15,16], antibiotic resistance[16,17,18] and virulence[16,17,19,20,21]
The major components of bacterial cell walls have been known for decades, many details of the complex regulatory network controlling PGN synthesis and the cell division machinery are not well understood and are a topic of current research[7]
Summary
Proper cell wall synthesis is essential to protect bacteria against the extracellular environment and for resisting high intracellular turgor. The detected in vivo phosphorylation site of MraY47 and a decreased tunicamycin sensitivity of the mutant strains indicate Stk-/Stp-dependent regulation All these data support the idea that Stk/Stp regulates cell wall synthesis and cell division at multiple layers. The phosphatase Stp activates cell wall synthesis enzymes like FemX through dephosphorylation of Stk-mediated phosphorylation sites (Fig. 5a,b). This model is supported by the physiological changes observed in the stp mutant. The Stp-deficient cells are not able to dephosphorylate FemX, resulting in permanently phosphorylated FemX In consequence, this less active FemX reduces pentaglycine interpeptide bridge formation of lipid II which results in the incorporation of incomplete muropeptides into the PGN sacculus. Phosphatase inhibitors may be developed as promising antibacterial agents that induce deregulation of bacterial cell wall metabolism and, in combination with cell wall-active antibiotics, could overcome the current shortage in the treatment of S. aureus and other bacteria
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