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.

• Keywords
• Ser/Thr protein kinase, cell division, cell signalling, phosphorylation, protein-protein interaction,
• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Cell Division * MeSH
• Phosphorylation MeSH
• Protein Serine-Threonine Kinases * metabolism   genetics   MeSH
• Signal Transduction * MeSH
• Streptococcus pneumoniae * metabolism   genetics   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Protein Serine-Threonine Kinases * 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.

• Keywords
• GpsB peptidoglycan regulator, KhpA/B RNA binding protein, StkP protein kinase, gene duplication and amplification, peptidoglycan precursor synthesis,
• MeSH
• Bacterial Proteins * genetics   metabolism   MeSH
• Cell Division MeSH
• Phosphorylation MeSH
• Mutation MeSH
• Streptococcus pneumoniae * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Bacterial Proteins * MeSH

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.

• MeSH
• Aminoacyltransferases genetics   metabolism   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Wall metabolism   MeSH
• Cell Division genetics   physiology   MeSH
• Virulence Factors genetics   metabolism   MeSH
• Phosphorylation MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mutation genetics   MeSH
• Peptidoglycan biosynthesis   MeSH
• Penicillin-Binding Proteins genetics   metabolism   MeSH
• Streptococcus pneumoniae genetics   metabolism   MeSH
• Base Composition genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Aminoacyltransferases MeSH
• Bacterial Proteins MeSH
• Virulence Factors MeSH
• GpsB protein, Streptococcus pneumoniae MeSH Browser
• Membrane Proteins MeSH
• penicillin-binding protein 2b, Streptococcus MeSH Browser
• Peptidoglycan MeSH
• Penicillin-Binding Proteins MeSH

Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.

• Keywords
• FtsA, Gram-positive cocci, Streptococcus pneumoniae, cell division,
• Publication type
• Journal Article MeSH

BACKGROUND: Reversible protein phosphorylation catalyzed by protein kinases and phosphatases is the primary mechanism for signal transduction in all living organisms. Streptococcus pneumoniae encodes a single Ser/Thr protein kinase, StkP, which plays a role in virulence, stress resistance and the regulation of cell wall synthesis and cell division. However, the role of its cognate phosphatase, PhpP, is not well defined. RESULTS: Here, we report the successful construction of a ΔphpP mutant in the unencapsulated S. pneumoniae Rx1 strain and the characterization of its phenotype. We demonstrate that PhpP negatively controls the level of protein phosphorylation in S. pneumoniae both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase, StkP. Catalytic inactivation or absence of PhpP resulted in the hyperphosphorylation of StkP substrates and specific phenotypic changes, including sensitivity to environmental stresses and competence deficiency. The morphology of the ΔphpP cells resembled the StkP overexpression phenotype and conversely, overexpression of PhpP resulted in cell elongation mimicking the stkP null phenotype. Proteomic analysis of the phpP knock-out strain permitted identification of a novel StkP/PhpP substrate, Spr1851, a putative RNA-binding protein homologous to Jag. Here, we show that pneumococcal Jag is phosphorylated on Thr89. Inactivation of jag confers a phenotype similar to the phpP mutant strain. CONCLUSIONS: Our results suggest that PhpP and StkP cooperatively regulate cell division of S. pneumoniae and phosphorylate putative RNA binding protein Jag.

• Keywords
• Cell division, Jag, Phosphorylation, Protein kinase, Protein phosphatase, Signal transduction, Streptococcus,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Wall metabolism   MeSH
• Cell Division physiology   MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Gene Knockout Techniques MeSH
• Mutant Proteins genetics   metabolism   MeSH
• Oxidative Stress physiology   MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Phosphoprotein Phosphatases genetics   metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Sequence Deletion MeSH
• Signal Transduction MeSH
• Streptococcus pneumoniae cytology   enzymology   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Mutant Proteins MeSH
• Protein Serine-Threonine Kinases MeSH
• Phosphoprotein Phosphatases MeSH
• RNA-Binding Proteins MeSH
• Recombinant Fusion Proteins MeSH