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Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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Clostridium difficile: Molecular typing of clinically significant isolates
Krůtová, Marcela ; Nyč, Otakar (advisor) ; Čermák, Pavel (referee) ; Krásný, Libor (referee)
Currently, Clostridium difficile is a leading nosocomial pathogen due to the spread of epidemic strains. Molecular typing of clinical isolates is an important part of C. difficile occurrence and spread control in hospitals as well as in the community. A total of 2201 clinical C. difficile isolates from 32 hospitals cultured between 2013-2015 were characterized by PCR ribotyping and toxin gene multiplex PCR. A total of 166 different ribotyping profiles were identified, of which 53 ribotyping profiles were represented by at least two isolates for each profile. The most frequently found ribotypes were 176 (n=588, 26.7%) and 001 (n=456, 20.7%) followed by 014 (n=176, 8%), 012 (n=127, 5.8%), 017 (n=85, 3.9%) and 020 (n=68, 3.1%). Out of 2201 isolates, 2024 (92%) isolates were toxigenic and carried genes for toxin A and B, and of these, 677 (33.5%) also carried genes for binary toxin. The remaining 177 (8%) isolates were non-toxigenic. Subtyping of C. difficile isolates using a multilocus variable-number tandem repeats analysis (MLVA), that compared the sum of tandem repeats differences, was performed in C. difficile isolates of ribotype 176 (n=225, 17 hospitals) and in C. difficile isolates of ribotype 001 (n=184, 14 hospitals) cultured in 2014. The clonal relatedness in C. difficile isolates belonging...
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Analysis and mapping of binding sites of gene expression regulators in the genus of Streptomyces.
Šmídová, Klára ; Bobek, Jan (advisor) ; Krásný, Libor (referee) ; Kopecký, Jan (referee)
Streptomyces are medically important soil-living bacteria that undergo morphological changes from spores to aerial hyphae and are important producers of bioactive compounds including antibiotics. Their gene expression is tightly regulated at the early level of transcription and translation. In the transcriptional control, sigma factors play a central role; the model organism Streptomyces coelicolor possesses astonishing 65 sigma factors. The expression of sigma factors themselves is controlled on the post-transcriptional level through the action of sRNAs that modify their mRNA level. However, only several sigma factors in Streptomyces have known regulons and also their sRNAs-mediated regulation has not been studied so far. According to previously measured gene expression data, we selected several highly expressed sigma factors. Using mutant strains with HA-tagged sigma factors, regulons of two important sigma factors, SigQ and HrdB, were analyzed by ChIP-seq procedure. Other sigma factors were further studied to see if they possess asRNAs, using 5' and 3' RACE method and northern blotting. Our data confirm the essentiality of HrdB sigma factor during the vegetative phase of growth. The other sigma factor, SigQ, has been revealed to be an important regulator of nitrogen metabolism and osmotic...
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The role of elF3 a Rps3 in stop codon readthrough
Poncová, Kristýna ; Valášek, Leoš (advisor) ; Vopálenský, Václav (referee) ; Krásný, Libor (referee)
Translation represents a highly regulated, interconnected process of protein synthesis in the cell. It could be divided into 4 phases: initiation, elongation, termination, and ribosomal recycling. Our laboratory is involved in in-depth studies of a complex eukaryotic initiation factor 3 protein (eIF3). We are interested not only in revealing its molecular roles in the translational cycle in general but also in specific mechanisms that allow translational regulation according to specific cellular needs. In the budding yeast, the eIF3 is composed of five essential subunits (a/Tif32, b/Prt1, c/Nip1, g/Tif35 and i/Tif34). In mammals, the protein is even more complex, comprising of 12 subunits (a-i, k-m). eIF3 is a key player not only in translation initiation but also in ribosomal recycling and, surprisingly, in translation termination and stop codon readthrough as well. The latter process harbors important clinical potential, as approximately 1/3 of genetically inherited diseases is caused by the presence of a premature termination codon in the protein-coding region. Therefore, understanding the molecular mechanism underlying this phenomenon provides important tools for the targeted and less toxic drug development approaches needed for patient therapy. In this Ph.D. Thesis, I uncovered the role of...
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Regulation of mycobacterial transcription
Kafka, Vojtěch ; Krásný, Libor (advisor) ; Dostálová, Hana (referee)
RNA polymerase (RNAP) is the enzyme that catalyzes synthesis of RNA. Mycobacterial RNAP significantly differs from RNAPs from other bacterial species. It requires special transcription factors such as RbpA or CarD. Another difference is the presence of a small RNA (sRNA), Ms1, that binds to mycobacterial RNAP. Ms1 regulates the amount of RNAP in the cell. In our laboratory we recently discovered MoaB2, a new binding partner of mycobacterial A (encoded by sigA), an RNAP subunit, which is essential for recognition of the initial promoter sequence and initiation of transcription. The function of MoaB2 in the regulation of transcription and gene expression is still unknown. The first aim of this Thesis is contribute to elucidation of the mechanism by which Ms1 affects the amount of RNAP. The experiments revealed that this regulation occurs at the level of transcription; Ms1 affects the activity of promoter(s) that drive the transcription of rpoB- rpoC that encode the two catalytic subunits of RNAP. The second aim of this Thesis is to characterize the interactions of MoaB2 with protein of the transcription apparatus. The results confirmed the interaction of MoaB2 with A and showed that neither RNAP nor transcription factors RbpA and CarD are required for this interaction. Finally, a role of the...
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The effect of selected endogenous and exogenous factors on bacterial growth
Šiková, Michaela ; Krásný, Libor (advisor) ; Valášek, Leoš (referee) ; Vopálenský, Václav (referee)
The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...
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The effect of the environment on bacterial DNA topology and gene expression.
Mikesková, Klára ; Krásný, Libor (advisor) ; Večerek, Branislav (referee)
Biological processes in the cell are affected by DNA topology, i. e. by DNA structure and shape. An important topological parameter is the level of supercoiling - additional twisting of DNA is relieved by positive (twisting in the same direction as the helix turns) or negative (twisting in the opposite direction) supercoils. In this Thesis I review the role of supercoiling in gene expression regulation. I describe how supercoiling is involved in homeostatic mechanisms that control the transcriptional output from some genes. Environmental changes such as shifts in temperature, oxidative stress, extreme pH and antibiotics and other inhibitors affect the level of DNA supercoiling. DNA supercoiling then affects the expression of enzymes, which influence DNA topology, as well as some other genes/proteins. In summary, this Thesis describes how changes in the environment influence bacterial DNA topology and gene expression with a brief mention of this type of regulation in eukaryotes.
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Riboswitches as tools for gene expression regulation
Jureček, Matěj ; Folk, Petr (advisor) ; Krásný, Libor (referee)
Riboswitches are segments of pre-mRNAs and mRNAs, present in their UTRs or introns, which are able to bindsmall molecules (usually a metabolite, ion or nucleotide) and in response "switch" between two conformations, which can affect the downstream gene expression process. In most cases, at least in bacteria, the mRNA which the riboswitch regulates encodes a component of the metabolism of the riboswitch-binding ligand. Contrary to other mechanisms of gene expression regulation, riboswitches do not require the participation of a protein. They may represent a way for the hypothetical early forms of life to regulate their genes. Riboswitches are abundant in bacteria and there is only a handful in eukaryotes. The scope of the study of riboswitches is ever more increasing and today it could very well be an independent branch of molecular biology.
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Rhomboid family intramembrane proteases in prokaryotes: mechanism, substrate repertoires and biological functions in the Gram-positive bacterium Bacillus subtilis.
Began, Jakub ; Stříšovský, Kvido (advisor) ; Bařinka, Cyril (referee) ; Krásný, Libor (referee)
Rhomboid proteases are a class of serine intramembrane proteases, a large family of enzymes that catalyze the proteolytic cleavage of membrane proteins within their transmembrane regions, in the hydrophobic environment of cellular lipid membranes. Rhomboid proteases were discovered in 2001 in Drosophila. In their pioneering study, Lee et al. identified the essential role of Rhomboid-1 protein (Rhom-1), which proteolytically activates the epidermal growth factor (EGF) receptor signaling pathway, in the early stages of fly eye development. Members of the rhomboid superfamily - active proteases (rhomboids) as well as their catalytically-dead counterparts (rhomboid-like proteins, including iRhoms and Derlins) - are widely conserved, implying their biological significance. Rhomboids are present in all kingdoms of life from archea to humans, while proteolytically inactive rhomboid-like proteins are present in eukaryotes only. Rhomboid superfamily proteins play roles in a wide range of processes, as diverse as signaling in metazoan development, mitochondrial biogenesis in yeast, host- cell invasion by protozoan parasites, protein quality control in the endoplasmic reticulum (ER) or bacterial quorum sensing. Rhomboids are the best understood intramembrane proteases from a structural and mechanistic points...
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Molecular details of translation reinitiation in budding yeast
Mohammad, Mahabub Pasha ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee) ; Krásný, Libor (referee)
Eukaryotic translation initiation is a complex multi-step process. It begins with the formation of 43S pre-initiation complex (PIC), which binds to the 5' cap of the mRNA, scans downstream searching for an appropriate AUG start site. Upon AUG recognition by 48S PIC, most of the initiation factors dissociate. However, some initiation factors, such as eIF3 and may be eIF4G, remain transiently associated with the 48S PIC. Here, we have developed a yeast in vivo RNA-Protein Ni2+ -Pull down (Rap-Nip) assay for in vivo detection of eIF3 bound mRNA fragments. In Saccharomyces cerevisiae, we demonstrated showing that, after initiation, eIF3 persistently associated with the elongating ribosomes for few elongation cycles. Further, it stabilizes the post- termination 40S complex on the stop codon of both uORF1 and 2 by establishing a contact between the N-terminal domain of a/TIF32 subunit and the Reinitiation Promoting Elements (RPEs) of corresponding uORFs on GCN4. Furthermore, employing the β-galactosidase reporter assays we revealed that AU-rich motif (AU1-2A/ UUAU2 motif) that occurs immediately following the stop codon of reinitiation (REI)- permissive uORFs promotes REI in position-specific autonomous fashion. We also have shown that the exact length and the last coding triplet of the REI-permissive...
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