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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.

Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...