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The role of protein kinase StkP in regulation of the cell division in Streptococcus pneumoniae
Malíková, Eliška ; Doubravová, Linda (advisor) ; Kuthan, Martin (referee)
Protein phosphorylation by protein kinases is a key mechanizm that enables both eukaryotic and prokaryotic organizm sense and read environmental signals and convert these signals into changes in gene expression and thus proper biological response. One of the main phosphorylation systems in bacteria consists of eukaryotic-like Ser/ Thr protein kinases. The genome of human pathogen Streptococcus pneumoniae contains single Ser/ Thr protein kinase StkP. StkP regulates virulence, competence, stress resistance, gene expression and plays an important role in the regulation of cell division cycle. Analysis of phosphoproteome maps of both wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the cell division protein DivIVA (NOVÁKOVÁ et al., 2010). DivIVA in S. pneumoniae is localized at midcell and at the cell poles. It was proposed to be primarily involved in the formation and maturation of the cell poles (FADDA et al., 2007). The aim of this thesis was to investigate phosphorylation of the cell division protein DivIVA in S. pneumoniae. Gene divIVA was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of DivIVA by StkP was examined in a kinase assay. We confirmed that DivIVA is a direct...
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Svobodová, Jaroslava (referee) ; Doubravová, Linda (advisor)
The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...
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