|
|
Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
|
|
|
Identification of residues of acylated domain of RTX toxins involved in acyltransferase binding
Grobarčíková, Michaela ; Mašín, Jiří (advisor) ; Černý, Ondřej (referee)
Both adenylate cyclase toxin (CyaA) and α-hemolysin (HlyA) are members of Repeats in ToXins (RTX) cytolysins that play key roles in the virulence of Bordetella pertussis and Escherichia coli, respectively. Bacterial RTX toxins represent a growing group of proteins produced by gram- negative bacteria. These pore-forming RTX toxins share several notable common features: (1) they require post-translational activation by attachment of fatty acid chains to two lysine residues; (2) they contain a hydrophobic domain that forms cation-selective pores in target cell membranes; (3) they are secreted by a type I secretion system; (4) after secretion, they become biologically active by binding of Ca2+ to the nonapeptide glycine- and aspartate-rich repeats. CyaA translocates a unique AC enzyme to the cytosol of phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. CyaA and HlyA also permeabilize the cell membrane of eukaryotic cells through cation-selective pores. Both toxins preferentially bind to cells expressing β2 integrins but can also interact with a variety of cells that do not express integrins or with naked lipid membranes. Both toxins are activated from protoxin form by post- translational acylation mediated by a specific acyltransferase. CyaA is activated by...
|
|
|
Preparation of mutant variants of Kingella kingae RtxA cytotoxin for membrane topology research
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Malý, Petr (referee)
Kingella kingae is a facultative anaerobic, β-hemolytic, gram-negative bacterium. It has been shown, that K. kingae is an important cause of invasive infections in young children, especially between 6 to 36 months of age. The most common diseases caused by K. kingae are septic arthritis, osteomyelitis, bacteremia and infective endocarditis. The key virulence factor of K. kingae is the secreted RtxA toxin, which belongs to the RTX toxins family (Repeats in ToXin). These are divided into two categories, hemolysins and leukotoxins, based on the cellular specificity of their action. The broad specificity of the RtxA toxin indicates that RtxA can be classified as a cytolytic RTX hemolysin. RtxA molecules are inserted into the host cell membrane and form cation-selective membrane pores that trigger cation flux. This disrupts normal cell physiology and eventually leads to cell lysis. The aim of this bachelor thesis was to prepare mutant variants of the K. kingae RtxA cytotoxin with lysine substitutions in the pore-forming domain for future study of the membrane topology of the toxin using biotin binding to the lysine residues. In order to observe the topology of the RtxA toxin in the host cell membrane, the toxin must be able to insert to the cell membrane. Therefore, another objective was to determine...
|
|
| |
|
|
Engineering of microbial glycosidases for modifying synthetic potential
Hovorková, Michaela ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
Glycosidases (EC 3.2.1.) alias glycoside hydrolases are enzymes that catalyze the cleavage of a glycosidic bond between two carbohydrates or between a carbohydrate and an aglycone. Under suitable conditions (especially reduction of water activity in the reaction mixture), these enzymes are also able to synthesize a glycosidic bond. By targeted mutagenesis of the catalytic centre of the enzymes, it is possible to suppress or completely abolish their hydrolytic activity. Enzyme synthesis using glycosidases makes it possible to prepare bioactive galactosides, for example galectin ligands. The present work deals mainly with β-galactosidase from Bacillus circulans, its recombinant expression and mutagenesis. In the first part of the work, the commercially prepared plasmid of -galactosidase from B. circulans isoform A that I designed was used for recombinant expression in E. coli. It was necessary to optimize the conditions of the enzyme production. As it is a large protein (189 kDa), the expression vector pCOLD II and cold production at 15 ř C were used. The enzyme is specific for the formation of the β-1,4 glycosidic bond and has been used to synthesize complex tri- and tetrasaccharide ligands that cannot be prepared with a crude commercial preparation containing undesirable enzyme activities....
|
|
| |
|
|
Mechanism of auxin transport across plasma membrane through PIN auxin efflux carriers
Lefnar, Radek ; Petrášek, Jan (advisor) ; Nodzynski, Tomasz (referee)
Phytohormone auxin and its directional distribution plays an essential role in the regulation of numerous processes during vegetative and reproductive plant development. Regulation of the expression, localization and activity of the PIN-FORMED (PIN) proteins is important for proper polar auxin transport in plant tissues. PIN proteins have been described as the major auxin efflux carriers regulating auxin's directional flow to build up gradients that provide information for the coordination of plant development. PIN protein structure topology prediction through bioinformatic analysis is still insufficient to understand their transport mechanism. Experimental analysis of PIN protein domains can provide valuable insight into understanding their role in mediating auxin transport. In this study, the C-terminal part of PINs have been modified by gradual trimming to determine the existence of relevant functional domains, which could be important for auxin transport. Seven modified PIN proteins from Arabidopsis thaliana and Nicotiana tabacum were prepared. Transiently transformed tobacco cell line Bright Yellow-2 (BY-2) was used to monitor differences in PIN transport activity. This approach allowed indirect monitoring of intracellular auxin levels using the DR5 reporter system. Transiently expressed...
|
|
|
Study of function and molecular architecture of fungal nitrilases applicable in biocatalysis
Veselá, Alicja Barbara ; Martínková, Ludmila (advisor) ; Macek, Tomáš (referee) ; Teisinger, Jan (referee)
Nitrilases are enzymes which catalyze the hydrolysis of a nitrile into the corresponding carboxylic acid and ammonia. These enzymes are potentially applicable in biocatalysis and bioremediation because of their advantages over the conventional (chemical) methods of nitrile hydrolysis (lower demand for energy, safety, simplicity, high yields, selectivity). In this work, genome mining was used to search for the sequences of hypothetical nitrilases from filamentous fungi. The amino acid sequences of previously characterized fungal nitrilases were used as the templates. Then the new synthetic genes together with other genes from our nitrilase library were expressed in E. coli and the substrate specificities of the enzymes thus produced were compared. Significant attention was focused on the relationships between the sequence of the enzyme and its substrate specificity. The arylacetonitrilases from Arthroderma benhamiae (NitAb) and Nectria haematococca (NitNh) were purified and characterized. Their substrate specificities, kinetic parameters, pH and temperature profiles and subunit and holoenzyme size were assessed. NitAb and NitNh together with other recombinant fungal nitrilases were employed in the hydrolysis of high concentrations of (R,S)-mandelonitrile in a batch or fed-batch mode. Nitrilase from...
|
|
|
Processing peptidases of M16B family and their evolutionary relationships
Hanušová, Iva ; Stiborová, Marie (advisor) ; Šulc, Miroslav (referee)
The theoretical part of this bachelor thesis deals with the group of processing peptidase of the M16B family. The main focus is on the structure and the evolution of mitochondrial and hydrogenosomal processing peptidase and also the hypothetical peptidase from the bacterium Rickettsia prowazekii. In the practical part of this work, the constructs coding the α-subunit of hydrogenosomal processing peptidase (α-HPP) with the substituted tryptophan residue in the position 236 for phenylalanine and tyrosine were prepared using the site-directed mutagenesis. Subsequently, the new reporter tryptophan residue was introduced in α-HPP in positions 256, 260, 267 or 271 in the so-called glycine-rich loop.
|
|
|
Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
|
guest ::
