Ser/Thr protein kinases of eukaryotic type in bacteria
Identification of new substrates of protein kinase StkP
Human pathogen Streptococcus pneumoniae encodes a single Ser/Thr protein kinase, StkP, and a cognate phosphatase, PhpP. StkP plays a role in virulence, stress resistance and the regulation of cell wall synthesis and cell division. We are mainly interested in signal transduction pathways regulated by these two enzymes and the goal is to understand their role in physiology of pneumococcus. Consistent with its role in cell division, StkP was found to phosphorylate several proteins involved in cell wall synthesis and cell division. Using various approaches we identified following StkP substrates: the late cell division protein DivIVA, the early cell division protein LocZ (named also MapZ) and the phosphoglucosamine mutase GlmM. In order to reconstitute the StkP-dependent signaling pathway we continue with identification of other StkP substrates. We collaborate with prof. Dr. Jiří Stulík from the University of Defence (Hradec Králové, Czech Republic) and with Dr. Petr Halada from the Laboratory of Molecular Structure from the Institute of Microbiology AS CR.
Function of protein phosphatase PhpP
In contrast to StkP, the role of its cognate phosphatase, PhpP, is not well defined, in part because it was formerly reported to be essential. However, we successfully prepared phpP knock-out mutant and recently we study its role in physiology and life cycle of S. pneumoniae.
Functions and interactions of StkP substrates
Identification of new substrates of StkP kinase is the first step in understanding its regulatory role in pneumococcus. The second step is to investigate the function of identified substrate proteins and the impact of phosphorylation on their function. Recently, we study the function of early cell division protein LocZ which we found to be responsible for cell division site selection in pneumococcus. We also study the role of phosphorylation of DivIVA and GlmM in pneumococcal cell division and physiology. We closely collaborate with Orietta Massida from the Universita di Cagliari (Italy).
StkP belongs to a distinct group of Ser/Thr protein kinases which regulate cell cycle and cell division in many Gram-positive bacteria. These conserved transmembrane proteins consist of cytoplasmic kinase domain and repeated PASTA (penicillin-binding protein and Ser/Thr kinase-associated) domains in their extracellular region. To understand the mechanism of binding of ligand molecules and activation of protein kinase activity we study StkP molecular structure in collaboration with Juan Hermoso (CSIC, Madrid, Spain). In collaboration with Dr. Evžen Bouřa (ÚOCHaB, Czech Republic) we study crystal structures of several StkP substrates.
The proteomic group of laboratory has a lot of experience with the biology of proteins. We routinely use following methods: separation and analysis of proteins by gel-based methods (FPLC chromatography, one and two dimensional gel electrophoresis, isoelectric focusing), gel-free techniques (the ZOOM® IEF Fractionator), identification of proteins by MALDI TOF, software analysis of protein maps (comparative proteomics; PDQuest 8.0.1.). In our projects we combine gel-free methods and gel-based proteomics.
We are involved in a number of collaborative projects. Recently finished or currently running projects include:
- The identification of proteins in bone marrow plasma of children with acute lymphoblastic leukemia (ALL), that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. Project was performed in collaboration with Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University (Prague, Czech Republic)
- In collaboration with Prof. Diarmaid Hughes group in Department of cell and molecular biology, Biomedical center, Uppsala University (Sweden), we studied proteomes of mutants of the RNA-processing enzyme RNase E harbouring mutant translation factor EF-Tu and presented by the extreme slow-growth phenotype in Salmonella enterica.
- A study on mutual interactions of Pleurotus ostreatus biofilms with bacteria of activated sludge in solid-bed bioreactors is carried out in collaboration with the Laboratory of Environmental Biotechnology, Institute of Microbiology of the CAS.
- Proteomic projects targeting membrane proteomes (separation and 2-D gel analysis of membrane proteins) were performed with model prokaryotic organism Bacillus subtilis (membrane fractions) and a filamentous fungus Pleurotus ostreatus (proteins of microsomal fractions).
- The evolution of antibiotic resistance in Enterococcus faecium strains isolated from hemato-oncological patients is studied in collaboration with Laboratory of Clinical Microbiology and Antibiotic Centre of General University Hospital (CMAC), Prague.
We are opened for new collaborations. If you need help with your proteins, please, do not hesitate to contact Dr. Denisa Petráčková (firstname.lastname@example.org).