| Signal transduction in prokaryotes is normally attributed to two component regulatory systems in which a membrane-bound sensor kinase facilitates phosphotransfer to a cytosolic response regulator, which can then appropriately modulate transcription. However, one-component regulatory systems have emerged as critical signaling molecules in prokaryotes as well. Members of this group include eukaryotic-like serine/threonine kinases (ESTKs) and phosphatases (ESTPs), named due to similarities to their eukaryotic counterparts. These enzymes are implicated in nearly all steps of bacterial pathogenesis, however their role in Staphylococcus aureus (S. aureus ) unknown. This resilient organism is one of the most common causes of hospital-acquired and community-associated infections, affecting immunocompromised and immunocompetent hosts alike. We characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of the bacterial cell wall. Both enzymes are functional in vitro. Using gene knockout strategies, we created S. aureus N315 mutant strains lacking stp and/or stk. The strain lacking both stp and stk displayed notable cell division defects including multiple and incomplete septa and bulging, as observed by transmission electron microscopy. Mutant strains lacking stp alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, as compared to wild-type. Despite these observations, the core structure in peptidoglycan isolated from each strain was found to be the same, as determined by LC/MS/MS. Penicillin-binding proteins (PBPs), the enzymes responsible for peptidoglycan assembly in bacteria, were differentially expressed in mutant strains. Transcripts of all pbps (A, 2, 2x, 3, and 4) were up-regulated in the absence of stp, while pbps A and 2 were down-regulated in the absence of stk or stp and stk. Additionally, mutant strains lacking stk or both stk and stp displayed increased sensitivity (up to 50-fold in the case of N315DeltaSTP/STK and ertapenem) to cell wall-acting beta-lactam antibiotics. Finally, both STK and STP reversibly phosphorylate the response regulator WalR in vitro. This protein is a key regulator of cell division and cell wall gene transcription. Together, these results indicate that S. aureus STK- and STP-mediated reversible phosphorylation plays a critical role in peptidoglycan synthesis by modulating pbp expression. |
