|
|
Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis
Vaňousová, Kateřina ; Mikušová, Gabriela (referee)
After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry. We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular...
|
|
|
Characterization of the ABC-F protein Sco0636 in Streptomyces coelicolor
Pinďáková, Nikola ; Balíková Novotná, Gabriela (advisor) ; Mikušová, Gabriela (referee)
The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...
|
|
|
Mode of daptomycin action and mechanism of resistance
Helusová, Michaela ; Mikušová, Gabriela (advisor) ; Lichá, Irena (referee)
Daptomycin is one of antimicrobial peptides. These molecules are part of immune system of all organisms. Daptomycin consists of a cyclic peptide core and a lipophilic tail. Daptomycin is produced by Streptomyces roseosporus and is used to treat serious gram-positive infections. Daptomycin is active also against methicillin-resistant Staphylococcus aureus. Its antimicrobial effect depends on the presence of calcium ions and phosphatidylglycerol. Daptomycin targets bacterial cytoplasmic membrane, where it forms oligomers. Mode of action of daptomycin probably includes pore-forming effect leading to membrane damage. This disturbance causes ion efflux from cytoplasm which leads to membrane potential disruption, which results in inhibition of macromolecular synthesis and cell death. Daptomycin also causes changes in cell morphology. Despite its unusual mode of action, several mechanisms of resistance have emerged in some pathogenic strains. These are for example decrease in the amount of phosphatidylglycerol in the membrane, increase in the amount of lysylphosphatidylglycerol, release of membrane phospholipids or mutations in genes which control peptidoglycan synthesis.
|
|
|
Microbial degradation of high-molecular polycyclic aromatic hydrocarbons
Bachmannová, Christina ; Cajthaml, Tomáš (advisor) ; Mikušová, Gabriela (referee)
Polycyclic aromatic hydrocarbons are anthropogenic pollutants of the environment that reprsent danger to human health. Thus, there is a great need for knowledge of their degradation mechnaims that could be utilized for bioremediation of the contaminated environment. Polycyclic aromatic hydrocarbons with higher molecular weight are rather insoluble substances with very low bioavailability and one of them i.e. benzo[a]pyrene was found to cause carcinogenic effects and other polycyclic aromatic hydrocarbons are potential carcinogens. In this thesis, I describe information of microbial degradation pathways of suspected carcinogens such as benzo[a]pyrene, benz[a]nthracene, dibenz[a]anthracene, benzo[k] fluoroanthene, benzo[b]fluoroanthene, chrysene and indeno[1,2,3-c,d]pyrene. I also discuss enzymes of degradation pathways, namely ring-hydroxylating dioxygenases, dihydrodiol dehydrogenases, ring-cleaving dioxygenases and their adaptations to catabolism of heavy polycyclic aromatic hydrocarbons. Key words: polycyclic aromatic hydrocarbons, Pseudomonas, biodegradation, benzo[a]pyrene
|
|
|
Expression of genes for the conversion of nitriles and amides in Rhodococcus erythropolis
Kracík, Martin ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The strain Rhodococcus erythropolis A4 is a source of enzymes nitrilhydratase and amidase, that catalyse conversion of nitriles and amides. These enzymes are used in industrial biotransformation and bioremediation. Since it was difficult to carry out genetic manipulations aimed at increasing the production of these enzymes in the strain A4, the corresponding genes (ami and nha1 + nha2) of a related strain R. erythropolis CCM2595, in which both plasmid and chromosome manipulations can be routinely performed, were identified and analyzed in this diploma theses. The ami and nha1 + nha2 genes from the strain R. erythropolis CCM2595 were isolated and sequenced together with the flanking regions (5.5 kb in total). The organization of these genes and the expected regulatory genes was described in the strain CCM2595 and mechanisms of regulation of expression of these genes were studied. For the analysis of transcription of amidase and nitrilhydratase genes from both strains of R. erythropolis, the promoter-probe vector pEPR1 replicating in Escherichia coli and R. erythropolis was used. Transcriptional fusion of Pami promoters of the strains A4 and CCM2595 and the reporter gfp gene were constructed. The activity of the Pami promoter was measured by means of fluorescence of gfp gene product (green fluorescent...
|
|
|
Interaction of influenza virus with cell defence mechanisms
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Mikušová, Gabriela (referee)
Influenza virus infection is one of the most current problems nowadays. Its unique ability to strongly inhibit cell immune response on many different levels and its pandemic potential make it a subject of interest of many research groups. The Influenza virus uses mainly NS1 protein to inhibit the immune response. NS1 protein is able, on one hand, to bind RNA and mask it against recognition by cellular sensors and other proteins. On the other hand, NS1 protein possesses a catalytic domain. Using this domain it interacts with many cellular proteins, interferes with signal transduction and guarantees successful infection. NS1 protein is one of principal pathogenicity instruments of the Influenza virus and it deserves appropriate attention.
|
|
|
Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina ; Svobodová, Kateřina (advisor) ; Mikušová, Gabriela (referee)
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
|
guest ::
