|
|
Mode of action and nature of different susceptibility of bacteria to antibacterial compounds lipophosphonoxins
Havlová, Noemi ; Mikušová, Gabriela (advisor) ; Krásný, Libor (referee)
Lipophosphonoxins (LPPO) are small synthetic antibacterial compounds targeting the cytoplasmic membrane. 1st generation of LPPO (LPPO I) displays an antimicrobial activity against Gram positive bacteria, however they do not show any activity against Gram negatives. After the modification of the iminosugar module (bearing the positive charge) the 2nd generation of LPPO (LPPO II) were synthetized. LPPO II exhibit broadened activity against Gram positive bacteria and also kill Gram negatives, including multiresistant strains. This work focuses on the mode of action of LPPO - the pore-forming activity of these substances is investigated on model membranes as well as in vivo. It also deals with the nature of different activity against Gram positive and Gram negative bacteria using model bacteria Bacillus subtilis and Escherichia coli. The results show that the insensitivity of Gram negative bacteria against LPPO I is probably caused by the different cell wall structure and the presence of the outer membrane that LPPO are almost unable to overcome. Also, the composition of phospholipids in the target membrane influences the antimicrobial activity of LPPO. Higher proportion of phospholipids with neutral charge reduces the LPPO pore-forming activity but is also responsible for low cytotoxicity in...
|
|
|
SigN from Bacillus subtilis: Functional characterization.
Kambová, Milada ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Bacillus subtilis strain 3610 is an ancestral undomesticated strain. It diers from the laboratory strain 168 in many aspects. One dierence in strain 3610 is the presence of plasmid pBS32 encoding the sigma factor N (σN). This σ factor is activated when DNA damage occurs and induces the bacteria's cell death. The aim of the Thesis was a systematic characterisation of σN-dependent transcription. First, I showed that plasmid-borne but not chromosome-borne predicted σN-dependent promoters were ac- tive in transcription in vitro. Next, the anities of RNAP with σN for DNA, initiating NTP (iNTP) were determined for both relaxed and supercoiled DNA templates. Sur- prisingly, the activity of RNAP on relaxed σN-dependent promoters was higher than on their supercoiled versions, an opposite trend than displayed by RNAP associated with other σ factors. This property of σN-dependent promoters was not encoded by the core promoter sequence. In summary, this Thesis contributed to our understanding of the bacterial transcription apparatus. 1
|
|
|
Regulation of transcription in Gram-positive bacteria
Rabatinová, Alžběta ; Krásný, Libor (advisor) ; Bobek, Jan (referee) ; Valášek, Leoš (referee)
Bacteria are the most abundant organisms on the planet. They live almost in all environments, including those that are most extreme. All land and water ecosystems depend heavily upon their activity. Bacteria play essential roles in cycling of nutrients such as carbon, nitrogen, and sulphur. Due to their short cell cycle, they must be able to swiftly adapt to the conditions of their habitat to survive. Microbial growth itself is an autocatalytic process. There are three distinct phases of the growth curve: lag, exponential (log), and stationary. Bacterial cells must change their gene expression between these phases in order to adapt to the new conditions. The first stage of gene expression is transcription. The key enzyme of this stage is RNA polymerase (RNAP) that transcribes DNA into RNA. RNAP is regulated by a number of accessory proteins and also small molecule effectors. Understanding how RNAP functions is essential for understanding how bacteria cope with changing environments. This Thesis presents studies of selected aspects of bacterial gene expression regulation at the level of transcription, using Bacillus subtilis as the model organism. The first part of this Thesis focuses on protein determinants of the ability of RNAP to be regulated by the concentration of the initiating nucleoside...
|
|
|
Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
|
|
|
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....
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
guest ::
RSS feed.