The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn 2؉ as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.The phosphorylation of proteins is a key regulatory mechanism in the signal transduction pathways of both prokaryotes and eukaryotes. Typically, extracellular signals are translated into cellular responses. The phosphorylation of proteins is carried out by specific protein kinases and is coupled to dephosphorylation reactions catalyzed by protein phosphatases. In prokaryotes sensing of extracellular signals and transduction of information are usually mediated by two-component signal transduction systems consisting of histidine kinase sensors and their associated response regulators (42). In contrast, signal transduction in eukaryotes occurs via phosphorylation of serine, threonine, and tyrosine residues. Serine/threonine and tyrosine kinases and phosphatases control reversible phosphorylation of target proteins in eukaryotes and are essential for cell cycle control and differentiation (17,19).It has recently been shown in a number of studies that eukaryotic-type serine/threonine protein kinases (STPKs) and phosphatases are also expressed in many prokaryotes (2). Prokaryotic STPKs regulate various cellular functions, such as stress responses, biofilm formation, sporulation, and metabolic and developmental processes (20,23,30,34,37,39,46). STPKs also play a role in the virulence of many bacterial pathogens, such as streptococci, Mycobacterium tuberculosis, Yersinia pseudotuberculosis, and Pseudomonas aeruginosa (11,16,21,36,47). Although the functional roles of protein kinases have been described in previous studies, only a small number of target substrates have been identified so far. Moreover, the impact of phosphorylation and dephosphorylation of target protein functions has been investigated in only some cases (33,38).A single STPK has been found to be conserved in all sequenced strains of Staphylococcus aureus. Origi...

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Little is known about intracellular metabolite pools in pathogens such as Staphylococcus aureus. We have studied a particular metabolome by means of the presented LC-MS method. By investigating the central carbon metabolism which includes most of the energy transfer molecules like nucleotides, sugar mono- and biphosphates, and cofactors, a conclusion about phenotypes and stress answers in microorganisms is possible. Quantitative metabolite levels of S. aureus grown in complex lysogeny broth and in minimal medium were compared in the wild-type S. aureus strain 8325 and the isogenic eukaryotic-like protein serine/threonine kinase (DeltapknB) and phosphatase (Deltastp) deletion mutants. Detection of several remarkable differences, e.g., in nucleotide metabolism and especially cell wall precursor metabolites, indicates a previously unreported importance of serine/threonine kinase/phosphatase on peptidoglycan and wall teichoic acid biosynthesis. These findings may lead to new insights into the regulation of staphylococcal cell wall metabolism.

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.