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Regulation of transcription in Streptococcus pneumoniae
Zajíčková, Adéla ; Doubravová, Linda (advisor) ; Hnilicová, Jarmila (referee)
Regulation of transcription in Streptococcus pneumoniae Streptococcus pneumoniae, an opportunistic human pathogen, encodes a single eukaryotic-like Ser/Thr kinase, StkP, which plays a crucial role in cell growth and division. In a phosphoproteomic study, StkP-dependent phosphorylation of SigAunder antibiotic- induced stress conditions was demonstrated in vivo. SigA, a housekeeping factor, in complex with RNA polymerase, is responsible for the transcription of most genes in S. pneumoniae. Therefore, phosphorylation of this important regulator might play an important role in transcriptional regulation. In this work, we confirmed that StkP phosphorylates SigA at residues T291 and T306 in vitro. However, we could not detect SigA phosphorylation in vivo, and the role of phosphorylation remains to be elucidated. We detected all RNAP subunits, including SigA, in the RNAP complex of S. pneumoniae by mass spectrometry analysis. The protein Spr0726 was identified as part of the RNAP complex. The reciprocal interaction between Spr0726 and RNAP was confirmed by immunoprecipitation experiments. In functional studies, we found that deletion of the spr0726 gene leads to a decrease in the amount of the subunits β and α in the RNAP complex. Our data suggest that the Spr0726 protein may affect the stability of the RNAP...
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Preparation of genetically manipulated producers of hybrid manumycins
Krýslová, Dita ; Petříčková, Kateřina (advisor) ; Doubravová, Linda (referee)
Streptomyces are one of the most prolific producers of various secondary metabolites. Manumycin antibiotics represent an important class of these compounds. They belong to a big class of polyketide metabolites. While their antibiotic effect is not very significant, their other biological properties have a big potential in the treatment of inflammations, tumors, etc. They are characterized by two short polyketide chains, which are attached to a central subunit. At the end of the lower polyketide chain, a C5N cyclic unit is frequently attached. This thesis originates from the colabomycin E, an antibiotic, which was discovered by our team. This antibiotic is a new member of manumycin-type metabolites and is produced by Streptomyces aureus SOK1/5-04 strain. Previous studies on the function of individual genes in the biosynthetic gene cluster of colabomycin E inspired us to consider editing of the current biological activity of colabomycin E by replacement of the C5N unit with another, structurally similar bioactive subunit. Due to high structural similarity, we have selected 4,7- dihydroxycoumarin unit of novobiocin, an aminocoumarin-type metabolite produced also by Streptomyces. The 4,7-dihydroxycoumarin unit is pharmacophore with a cancerostatic activity. We expected that the cancerostatic activity...
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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Doubravová, Linda (advisor) ; Svobodová, Jaroslava (referee)
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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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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Regulation of penicillin-binding protein Pbp2a in Streptococcus penumoniae
Kubeša, Bohumil ; Doubravová, Linda (advisor) ; Pospíšil, Jiří (referee)
Regulation of penicillin-binding protein Pbp2a in Streptococcus pneumoniae Streptococcus pneumoniae is an extracellular human pathogen that encodes a unique eukaryotic-type Ser/Thr protein kinase StkP in its genome. This enzyme is involved in other cellular processes, such as cell division and cell wall synthesis, through phosphorylation with its substrates. A transmembrane protein MacP has been identified as a substrate of StkP. It is an activator of penicillin-binding protein PBP2a, which is involved in the synthesis of peptidoglycan with its transpeptidase and transglycosylase activities. We found that MacP is phosphorylated by the protein kinase StkP at positions T32 and T56. We confirmed that proteins MacP and PBP2a interact with each other and that phosphoablative and phosphomimetic mutations of the major phosphorylated residues of the MacP protein do not affect the interaction with PBP2a and do not fundamentally affect the function of MacP in vivo. Furthermore, we showed that the ∆macP mutation is synthethically lethal with the ∆pbp1a mutation, confirming that MacP is an activator of the PBP2a protein. MacP is located in the cell septum and interacts with a number of S. pneumoniae cell division proteins. Keywords: Streptococcus pneumoniae, cell division, MacP, PBP2a, phosphorylation
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Ser/Thr protein kinases in mycobacteria
Borovcová, Taťána ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
The Mycobacterium tuberculosis genome encodes 11 Ser/Thr protein kinases. These protein kinases are structurally related to eukaryotic protein kinases. The phosphoproteome contains hundreds of proteins phosphorylated on Ser/Thr residues that influence all aspects of cell biology, which supports the critical role of phosphorylation in the regulation of physiology. Particularly important role in regulation belongs to protein kinases PknA, PknB, PknG and PknL, these protein kinases occur in all species of mycobacteria. Although only PknA and PknB are essencial for the M. tuberculosis, they regulate cell shape through the regulation of cell wall synthesis and cell division. Another important protein kinase is PknG, although not essential for growth it is necessary for virulence, because it promotes the survival of pathogen inside macrophages of the host. As a result, Ser/Thr protein kinases represent an interesting target for inhibitor development that could be used as drugs against tuberculosis.
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela ; Doubravová, Linda (advisor) ; Lichá, Irena (referee) ; Petříčková, Kateřina (referee)
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,...
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