Identification of Multiple Substrates of the StkP Ser/Thr Protein Kinase in Streptococcus pneumoniae

Abstract

Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by twocomponent systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotictype Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mndependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the ␣-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein-and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.Protein phosphorylation is considered the universal language for inter-and intracellular communication in all living organisms. This process, catalyzed by protein kinases, enables translation of extracellular signals into cellular responses and adaptation to a constantly changing environment. Although previous work indicated that histidine kinases of two-component regulatory systems are the most prominent kinases in prokaryotes, recent data provide evidence that eukaryotic-type Ser/Thr protein kinases (ESTPKs) play an important role in prokaryotic cell signaling. The distribution of these enzymes among bacteria is broad but not universal. ESTPKs have been found in nearly two-thirds of the sequenced bacterial strains. Most of these enzymes are encoded by genes in strains belonging to the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, Chloroflexi, Acidobacteria, and Planctomycetes (38). We surveyed sequenced bacterial genomes and found ESTPK-encoding genes also in the bacterial taxa Aquificae,

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

“…4C). When norfloxacin was added, a slightly reduced protein phosphorylation pattern was observed, perhaps caused by an arrested growth in the presence of this antibiotic, consistent with previous reports (33). Strikingly, we observed hyperphosphorylation in the presence of ampicillin, and a previously faintly phosphorylated band (26), representing an as yet unknown protein, became robustly phosphorylated (Fig.…”

“…To test this notion, we examined the in vivo Thr protein phosphorylation profile of cells incubated with vancomycin or ampicillin. A phosphoprotein pattern was observed that included the StkP substrates DivIVA and Spr0334, as previously identified (33). In line with the localization results, protein phosphorylation was reduced significantly by adding vancomycin (Fig.…”

“…Western blot analysis using antibodies raised against DivIVA served as a control of the whole-cell extracts, because DivIVA is the most abundant substrate of StkP (33). In the whole-cell extract of the wild-type strain, phosphorylated DivIVA (p-Div-IVA) was detected readily, whereas in a strain that contained P Zn -gfp-stkP as the only copy of stkP, p-DivIVA was detected only after GFP-StkP was induced ( Fig.…”

“…StkP also plays an essential role for in vivo survival, because stkP mutants were strongly attenuated in virulence in mouse models (29,30). Phenotypic analysis, through both transmission electron microscopy and differential interference contrast microscopy, showed that stkP mutants often are elongated, suggesting a defect in cell division (30,33). Several StkP substrates playing a role in cell-wall metabolism and cell division were identified, including phosphoglucosamine mutase GlmM and the cell-division proteins DivIVA and FtsZ (26,33,34).…”

“…Phenotypic analysis, through both transmission electron microscopy and differential interference contrast microscopy, showed that stkP mutants often are elongated, suggesting a defect in cell division (30,33). Several StkP substrates playing a role in cell-wall metabolism and cell division were identified, including phosphoglucosamine mutase GlmM and the cell-division proteins DivIVA and FtsZ (26,33,34). By using immunofluorescence it was shown that StkP localizes to cell-division sites (34), but the molecular mechanisms underlying the stkP-null phenotype remain unknown.…”