StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway.

• Klíčová slova
• Ser/Thr protein kinase, cell division, cell signalling, phosphorylation, protein-protein interaction,
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• buněčné dělení * MeSH
• fosforylace MeSH
• protein-serin-threoninkinasy * metabolismus   genetika   MeSH
• signální transdukce * MeSH
• Streptococcus pneumoniae * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• protein-serin-threoninkinasy * MeSH

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP-mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.

• Klíčová slova
• GpsB peptidoglycan regulator, KhpA/B RNA binding protein, StkP protein kinase, gene duplication and amplification, peptidoglycan precursor synthesis,
• MeSH
• bakteriální proteiny * genetika   metabolismus   MeSH
• buněčné dělení MeSH
• fosforylace MeSH
• mutace MeSH
• Streptococcus pneumoniae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny * MeSH