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Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP.
Mašková, Kateřina ; Ulrych, Aleš (advisor) ; Fišer, Radovan (referee)
Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP The extracellular human pathogen Streptococcus pneumoniae encodes in its genome only a eukaryotic-type serine/threonine protein kinase (StkP) and the corresponding PP2C phosphatase (PhpP) in its genome. This makes it a unique model organism for the studying of signaling pathways in bacteria. To date, several substrates of this signaling protein have been identified and characterized, including DivIVA, KhpB, FtsA, FtsZ, MacP, GlmM, GpsB, ComE and others. These proteins are involved in various cellular processes, including cell division and peptidoglycan biosynthesis. In a global phosphoproteome study based on the LC- MS approach, one of the potential new substrates was the hypothetical protein Spr1962, whose phosphorylation was detected only in the hyperphosphorylated strain with deleted PhpP phosphatase. The aim of this work was to characterize this new substrate Spr1962 and to clarify its possible function. Based on the significant structural similarity to Spr1962 the FloT flotillin protein of Bacillus subtilis, it was suggested that it might be a protein with an analogous function in pneumococci. Deletion of this gene in different genetic backrounds was found to cause an...
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6S RNA as a tool for increased production of clinically relevant antibiotics
Kostílek, Štěpán ; Bobek, Jan (advisor) ; Ulrych, Aleš (referee)
Streptomyces belong to the group of soil microorganisms, and they are significant producers of biologically active substances used in a wide range of industries, whether it is medicine, agriculture or biotechnology. It is for this reason that the research of these microorganisms is of interest to a number of research institutions. One of the most important groups of these biologically active substances are antibiotics. A significant part of antibiotics produced in nature is produced precisely by streptomyces, and therefore it is in our interest to understand the mechanisms that influence this production. A wide variety of different regulatory mechanisms controlling antibiotic production have been found so far in streptomyces. One of these regulatory mechanisms is the utilization of scr3559 RNA. Scr3559 RNA is a small, non-coding RNA molecule that regulates antibiotic production at the level of DNA transcription. These small RNA molecules mimic the structure of the target promoter sequence and are able to interact with RNA polymerase. The presence of scr3559 RNA has not been confirmed in streptomyces as of yet, but new scr3559 RNA molecules are still being sought. To find new molecules, in silico methods are used, where the similarity of the scr3559 RNA sequence of S. coelicolor with homologous...
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Study of persistence mechanisms to antibiotics in Staphylococcus aureus
Balgová, Tamara ; Lichá, Irena (advisor) ; Ulrych, Aleš (referee)
In clinical practice, infections caused by persistent bacteria have become a worldwide problem. We are talking about a subpopulation of cells that are able to withstand lethal doses of antibiotics and after their elimination are capable of resuscitation and re-induction of the disease. The opportunistic pathogen Staphylococcus aureus is one of this bacteria and causes various serious chronic infections. During the long-term persistence in patients, persistent bacteria acquire adaptive mutations. The aim of this diploma thesis was to monitor the degree of persistence in selected clinical isolates, and at the same time to demonstrate the effect of adaptive mutations on the degree of persistence as well as to molecularly characterize the persistent state by gene expression. I had chronological isolates of S. aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of one year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment. I found that this...
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Study of the unique signaling pathway of Ser/Thr protein kinase StkP and phosphatase PhpP in Streptococcus pneumoniae
Keil, Jan ; Ulrych, Aleš (advisor) ; Bobková, Šárka (referee)
The major human pathogen Streptococcus pneumoniae is a unique model for the study of eukaryotic-type serine/threonine protein kinases and its cognate phosphatases in bacteria, since it encodes only a single signaling pair composed of the StkP protein kinase and PhpP phosphatase. This signaling pair plays a role in several cellular processes, mainly in cell wall biosynthesis and cell division. StkP and PhpP proteins with a pleiotropic effect appear to regulate a complex signaling cascade by phosphorylation of many substrates. However, only a few have been characterized so far. Using MS analysis, we have identified about 90 phosphopeptides that are potential substrates for the StkP kinase and PhpP phosphatase. This diploma thesis is focused on the characterization of the new substrate Spr0929 and its role in pneumococcal physiology. One of the objectives was to investigate cell morphology of strains carrying deletion of the spr0929 gene in different genetic backgrounds. It turned out that the role of Spr0929 in cell morphology is strain specific. The growth curves of strains with this deletion were compared to that of the wild type in various physiological conditions as well. As Spr0929 contains a nucleoid-associated domain called NdpA, determination of its cell localization was an important...
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The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor
Ulrych, Aleš
The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor WD-40 domains, also known as beta-transducin repeats, are highly conserved repeating amino acid units, which are found in a wide variety of eukaryotic proteins that have a range of different functions. In the late 1990s, the first WD-40 containing proteins were identified in prokaryotes, however the knowledge about their function is scarce. Streptomyces coelicolor is a gram-positive bacterium with complicated morphological and physiological differentiation in the course of its life cycle. The genome of Streptomyces coelicolor encodes 6 potential genes encoding proteins with WD-repeat motifs. To determine the function of two of these WD-40 genes (wdpB and wdpC), the deletion replacement mutants in both genes were prepared. Both mutants exhibited medium-dependent phenotypes, which are markedly evident on modified R3 plates. Phenotypic studies revealed that deletion of wdpB gene resulted in substantial reduction of aerial hyphae formation and reduced production of undecylprodigiosin. In addition, the hyphae of ΔwdpB mutant were unusually branched and showed the signs of precocious lysis. Delayed spore-containing hyphae were irregularly septated....
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The effect of antibiotics on human gut microbiome and the influence of probiotics on its restoration
Hloucalová, Nikola ; Lichá, Irena (advisor) ; Ulrych, Aleš (referee)
Antibiotics are used for treatment of bacterial infections. They negatively affect not only the pathogens, but also other microorganisms in the gut, including the beneficial bacteria. Antibiotic treatment changes the proportion of good versus bad bacteria in the gut, causes a decrease in the number of commensal bacteria and leads to the overgrowth of opportunistic pathogens. We should consume probiotics during and after the antibiotic treatment, otherwise it results in an unhealthy stool and moreover it affects the immune system which then leads to physical and mental illnesses. This thesis summarizes the influence of probiotics on human gut during dysbiosis caused mainly by antibiotics.
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The cell wall biosynthesis in gram-positive bacteria and inhibitory effect of antibiotics.
Mašková, Kateřina ; Ulrych, Aleš (advisor) ; Lichá, Irena (referee)
The cell wall of Gram-positive bacteria includes, in addition to the core peptidoglycan molecule, unique polysaccharides such as teichoic acids, capsular polysaccharides and exopolysaccharides, and covalently bound surface proteins. Together, they create a strong and durable layer that provides protection but also communication with the external environment. Peptidoglycan biosynthesis in Gram-positive bacteria can be divided into three phases: cytoplasmic, membrane and extracytoplasmic phase. The individual phases consist of specific reactions that are catalyzed by often conserved bacterial enzymes, which are potential targets for antibiotic molecules. Most known antibiotics effective against Gram-positive bacteria are aimed at inhibiting the process of cell wall synthesis. The mechanisms of action of individual antibiotics are described with varying degrees of detail. Some are known and widely used in medicine or veterinary practice, and some have so far only shown the potential to become drugs. Another use of antibiotics is in the basic research itself, especially in the study of cell wall biosynthesis and bacterial division. In this work, I have compiled a summary of knowledge about cell wall biosynthesis of Gram-positive bacteria and a list of antibiotics and a description of the mechanisms of...
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Identification of new substrates of Ser/Thr protein kinase StkP
Kleinová, Simona ; Ulrych, Aleš (advisor) ; Konopásek, Ivo (referee)
Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...
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Functional analysis of phosphoglucosamine mutase GlmM in Streptococcus pneumoniae
Mühldorfová, Tereza ; Ulrych, Aleš (advisor) ; Lišková, Petra (referee)
Phosphoglucosamine mutase (GlmM), an enzyme taking part in biosynthesis of cell wall, has been recently proven to be essential for Streptococcus pneumoniae. The main goal of this thesis was to prove in vivo that GlmM serine residues S99 and S101 phosphorylation is essential while the necessity of it was already proven indirectly based on transformation efficiency. For this purpose we have prepared a strain with two copies of the glmM gene - the first one with amino acid changes on monitored serine residues located at native locus; and the second ectopic copy of the wild allele of glmM gene under control of inducible zinc promoter. We have observed morphology, growth, and GlmM expression with and without the presence of an inductor. All the observed parameters show that the cells are not viable without ectopic glmM expression, thus the essential protein GlmM is functional only when phosphorylated on S99 and S101 residues. Further, we have attempted to localize the enzyme in the S. pneumoniae cell. We have fused GlmM with fluorescent marker GFP and by using the florescent microscopy we have proved that GlmM is cytoplasmic protein. Another goal of this thesis was to find an unknown third phosforylation site of the GlmM protein which is dependent on the protein kinase StkP. From in vitro kinase assay and...
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The mechanism of action of phage tail-like bacteriocins on target cells and artificial membrane systems.
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Ulrych, Aleš (referee)
Fonticins are phage tail-like bacteriocins produced by gram-negative bacterium Pragia fontium from the family Enterobacteriaceae. Phage tail-like bacteriocins can be divided into two different families: flexible ones (F-type) and contractile particles (R-type). Pragia fontium produces R-type particles that adsorb on the surface of sensitive bacterial cell and form pores probably during the contraction using mechanism similar to Type VI Secretion System. The pore-forming activity of fonticins was tested in vivo using bacterial cells. It was also characterized in vitro on artificial lipid membranes. On Black Lipid Membranes fonticins create large channels into the membranes; single channel conductance (G) is about two times higher than single channel conductance of well known α-hemolysine produced by Staphylococcus aureus. Further, we tested the voltage-dependent blocking of fonticin pores by native and unfolded proteins, dsDNA, ssDNA, polyethylene glycol and diamond nanoparticles. The rigid structure of fonticin nanotube in combination with constant conductivity makes it a promising device for analysing the size and shape of nanoparticles and large macromolecules. Key words: fonticin, bacteriocine, nanopore, Pragia fontium, blocking, pore-forming activity, black lipid membranes.
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