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Cytoplasmic membrane of Bacillus subtilis Regulation of the physical parameters
Beranová, Jana ; Konopásek, Ivo (advisor) ; Branny, Pavel (referee) ; Holoubek, Aleš (referee)
Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...
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Regulace vnitřního pH kvasinek - vliv vybraných transportních proteinů
Zalom, Peter ; Chaloupka, Roman (advisor) ; Holoubek, Aleš (referee)
Intracellular pH affects nearly all biochemical processes in yeast, the processes regulating the cytosolic pH includes function of many transport proteins. In this work, the impact of selected sodium transporters on cytosolic pH has been studied in two yeast species: Saccharomyces cerevisiae and Zygosaccharomyces rouxii including wild-type and mutants with affected sodium transport. Measurements of cytosolic pH and buffering capacity have been performed using fluorescent protein probe pHluorin - a pH sensitive derivate of green fluorescence protein. Several procedures for calibration of pHluorin fluorescence response have been compared and the importance of a proper correction of the calibration curve has been demonstrated. It has been shown that cytosolic pH is influenced by the function of Nha1 transport protein in S. cerevisiae as well as in Z. rouxii but not by Sod2-22 transporter in Z. rouxii. It has been demonstrated that the buffering capacity of cytosol decrease in the presence of glucose in all strains studied.
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Study of oligonucleotide penetration through liposomal membrane
Nováková, Jana ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Modified oligonucleotides, short pieces of nucleic acids, have become potential candidates for treatment of viral, malignant or genetic diseases. Efficient application of modified oligonucleotides requires their sufficient penetration inside the cell through the lipid membrane. Diploma thesis employs liposomes, synthetic vesicles formed by the lipid bilayer, as a model system for the study of interaction of lipid and complex of homooligonucleotides dT15 with cationic porphyrin CuTMPyP4 using Raman spectroscopy. Raman spectra were measured in solution on the slide and using special technique of drop coating deposition Raman spectroscopy (DCDR) on integrated Raman confocal microspectrophotometer. Processes of liposome formation and sample preparation were optimized. Experimental data were analysed using factor analysis based on singular value decomposition (SVD) algorithm. It was observed that interaction of oligonucleotide with porphyrin induces fluorescent background increase. Raman spectra of complex of oligonucleotide with porphyrin exhibit the domination of porphyrin vibrational bands. Stretching vibrations of lipid assigned to hydrogens of fatty acids are observed in the spectra of complex with liposomes. The observed spectral changes show the interaction of lipid with complex of oligonucleotide and...
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Study of angular and time characteristics of suspensions by using of elastic scattering
Peikertová, Pavlína ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Elastic light scattering represents relatively simple and noninvasive technique to study angular dependencies of the scattered light intensity that contains important information about scattering objects. In the frame of this thesis the data from adapted computer controlled apparatus Photo-gonio-diffusometre type 42.000 were collected. Experimental set-up was upgraded to use it for small sample volumes. Two model systems were studied: polymer and latex suspensions and real systems: liposomal suspension of various sizes, as well. The size estimation by using of Guinier plot was performed from experimental data. This estimation is in good agreement with the defined sizes in case of model systems. For suspensions of liposomes the results are also in relatively good agreement with expected values. Aggregation process and subsequent sedimentation after addition of porphyrin CuTMPyP4 was observed for silver borohydrid colloid. Acquired time and angular dependencies were processed by factor analysis that is able to make a qualitative and temporal separation of both processes
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Intracellular pH of yeast and its regulation by transport of potassium ions
Hošková, Barbora ; Urbánková, Eva (advisor) ; Holoubek, Aleš (referee)
Cytosolic pH of yeast is regulated and maintained by cooperation of many transport proteins. A fluorescent protein pHluorin was used in this thesis to evaluate cytosolic pH of yeast Saccharomyces cerevisiae. pHluorin is sensitive to pH of its surrounding. The experiments were mostly carried out using spectrofluorometer, however also a method which enables using reader was improved and optimalized for future use. As well a specific arginin-phosphate growth medium was used. An influence of certain proteins on the ability to regulate cytosolic pH was also studied. Some proteins are connected with the regulation directly e. g. Pma1 ATPase, some of them in an indirect way e. g. Ypt6. It was proved, that the value of cytosolic pH depends on yeast strain and cultivation and experimental conditions. None of the gene deletions studied in this thesis changed regulation of pHcyt much, the cells were able to adjust pHcyt even in situation, when the external medium is acidified by sulphuric acid. Buffering capacity was measured with addition of weak base to the cell suspension. The values varied in range from 30 to 60 mM if the cells were cultivated in YNB growth medium. Cells cultivated in arginin-phosphate medium had higher buffering capacity.
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Study of transport proteins of the Nramp family
Surá, Lucie ; Chaloupka, Roman (advisor) ; Holoubek, Aleš (referee)
The Natural Resistance-Associated Macrophage Proteins (Nramp) form functionally conserved family of proton-dependent divalent metal ion transporters. In the present study, we investigated transport properties of a prokaryotic Nramp homolog - MntH transporter from Escherichia coli. H+ transport mediated by MntH was monitored in a bacterial model system using pH-sensitive green fluorescent protein (pHluorin). Our experimental conditions enabled us to observe an uncoupled H+ transport mediated by MntH. Uncoupled H+ flux had been previously described in eukaryotic Nramp proteins, nevertheless this is the first observation of this phenomenon in a prokaryotic homolog. We demonstrated that the uncoupled H+ transport is pH- and temperature- dependent. The uncoupled transport H+ is also affected by specific single-point mutations at functionally important residues Asp34, His211 and Asn401. The second part of the work focused on effect of different ions, which are not MntH substrates, on transport properties of MntH. It was shown that addition of excess calcium or magnesium resulted in increase of H+ transport induced by divalent metal ions, but on the other hand our data suggest that calcium inhibits uncoupled H+ transport.
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