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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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Parasite-induced apoptosis in mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Parohová, Irena ; Macháček, Tomáš (advisor) ; Kopecký, Jan (referee)
Helminths are capable of induction of apoptosis in host immune cells and thus supress or dampen the host immune response. Nevertheless, apoptosis can affect also other, non-immune cells present in the infected tissues which contributes to the pathology related to the infection. Schistosomes are known to be potent immunomodulators, able to dampen the immune response of the host through induction of apoptosis, which ensures their survival. In the case of mice infected with the avian neuropathogenic T. regenti, the host immune response is responsible for parasite elimination. Therefore it seems that T. regenti can not effectively alter the immune response. On the contrary, parasite induced apoptosis in host cells present in the tissues infected with T. regenti could contribute to pathology. Thus we decided to investigate if T. regenti is capable of induction of apoptosis in infected mice. Firstly, we used TUNEL assay to detect DNA fragmentation in infected tissues. We demonstrated that DNA fragmentation is present in the infected skin 8, 24 a 48 days post infection and spinal cord 14 days post infection. Moreover, the transcriptomic analysis of the infected spinal cords revealed up-regulated expression of pro-apoptotic genes. These observations indicate possible apoptosis of host cells due to the...
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Assembly, successional development and functioning of microbial communities in deadwood
Bernardová, Natálie ; Brabcová, Vendula (advisor) ; Kopecký, Jan (referee)
Dead wood is one of the most important reservoirs associated with forest ecosystems. In natural forests, its volume is counted in hundreds of m3 ha-1 , whereas it reaches only tens of m3 ha-1 in productive commercial forests. In contrast to soil and plant litter, deadwood is unevenly distributed on the forest floor. The specific physicochemical properties such as high content of recalcitrant polymers, low nitrogen level and impermeability negatively affect the rate of decomposition especially in the initial stages of wood deconstruction. The deadwood decomposition is very slow in comparisons with other substrates, it accumulates and thus it represents the important reservoir of nutrients. This thesis is focused on the structure, development and function of microbial (fungal) community in decomposing deadwood in unmanaged forest. Functional screening of fungi isolated from fruit bodies collected from coarse deadwood was set aside. Physico-chemical properties of deadwood including pH, carbon and nitrogen content and microbial biomass were estimated for four wood decomposition stages and three different tree species. New generation sequencing (Illumina MiSeq platform) was applied for fungal community structure analysis based on ITS2 fragment. Fungal functional screening was based on physico-chemical...
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Ergothioneine and mycothiol in the biosynthesis of lincosamides
Seidlová, Bára ; Kameník, Zdeněk (advisor) ; Kopecký, Jan (referee)
Specialized microbial metabolites are described as low-molecular-weight bioactive compounds, which are dispensable for the growth, evolution, or reproduction of its producer. This group of substances includes the lincosamides, which are produced mainly by the bacteria of the Streptomyces genera. Apart from other precursors, two low-molecular-weight thiols, ergothioneine and mycothiol, are essential participants of the lincosamide biosynthesis. Mycothiol (MSH) serves in this pathway as a source of sulphur, on the other hand, ergothioneine (ESH) constitutes a conjugate with the aminosugar moiety of lincosamide structure. The conjugate is condensed with an activated amino acid, which is catalyzed by an unusual enzyme to form a core of the lincosamide molecule. The objective of this diploma thesis is to isolate the conjugate of ESH and aminooctose, which serves as a substrate of the LmbD biosynthetic protein. Another aim is to study the links between the thiol metabolism and the biosynthesis of three lincosamides, lincomycin, celesticetin, and intervencin, which are produced by different bacterial strains. Bacterial strains were cultivated under laboratory conditions and methods of liquid chromatography with UV and MS detection were used for the analysis. The parameters of the methods were developed...
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Soil microbial communities in agroecosystems and natural habitats contributing to resistance and resilience of the soil environment
Sarikhani, Ensyeh ; Kopecký, Jan (advisor) ; Demnerová, Kateřina (referee) ; Větrovský, Tomáš (referee)
Ensyeh Sarikhani Soil microbial communities in agroecosystems and natural habitats contributing to resistance and resilience of the soil environment. Summary The control of common scab of potatoes (CS) includes resistant varieties (cultivars), precise fertilization, increase of soil moisture, and chemical treatments. Yet, these management practices do not have common or reproducible results at differing sites. A monitoring study was done in 32 sites to evaluate the relation between CS and biological/chemical soil parameters. Correlations were observed between scab severity and content of nutrients such as Fe, N, and Ca in soil and periderm, and between disease severity and abundance of actinobacteria and total bacteria, together with the pathogenicity determinant, txtB gene (biosynthetic gene of thaxtomin) in both soil and periderm of potatoes. The findings led to novel conclusions, which can help to understand relationships applicable in scab control. Peat and DTPA chelated iron were supplemented to pots filled with soil conducive for CS in order to determine the effects of soil organic matter, iron and pH on CS development. The results were compared with data obtained for a suppressive soil from a nearby field with naturally low CS severity. Both peat and iron supplements decreased CS and the combination...
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Control circuits of railway lift
Kopecký, Jan ; Lažek, Tomáš (referee) ; Huták, Petr (advisor)
This master thesis deals with the mechanical and electrical design of the drive of the inclined construction lift. Individual components are selected in the design. Furthermore, a theoretical research is carried out regarding wireless modules and logic controllers. The selection of sensors is made and the method of retrieving signals from sensors and converting them into iputs the control circuits is also outlined. A program is written in Arduino IDE that wirelessly controls the motion of a motor via variable frequency drive.
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Development of a new sand fly exposure test to evaluate vector control tools
Willen, Laura Adrienne André ; Volf, Petr (advisor) ; Kopecký, Jan (referee) ; Valenzuela, Jesus G. (referee)
In the Mediterranean basin, human visceral leishmaniasis caused by the protozoan parasite Leishmania infantum is a zoonotic disease that gives rise to 1,200 to 2,000 new cases annually. The domestic dog constitutes its main reservoir, of which some may suffer from a severe chronic disease, canine leishmaniasis (CanL). The sand fly Phlebotomus perniciosus is considered to be the principle vector. Saliva of bloodfeeding vectors of diseases has been used in the past to assess host exposure to vector bites and to evaluate vector control tools. This Ph.D. focused on saliva of P. perniciosus to identify exposure markers that could be used in the preparation of a new vector exposure tool. The first part of this Ph.D. aimed at validating the use of a recombinant salivary protein of P. perniciosus - rSP03B - in endemic settings of CanL. During a cross-sectional study, no significant differences between the antibody (Ab) response against whole saliva or the rSP03B were observed between different regions across the Mediterranean basin. Furthermore, the rSP03B was shown to resemble the native protein. During a subsequent study this protein was used to assess the seasonal dynamics of the canine Ab response to P. perniciosus in an endemic area of L. infantum. This study elucidated that also in a heterogeneous...
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Gene pool of the secondary metabolism in soil bacterial communities
Patrmanová, Tereza ; Kopecký, Jan (advisor) ; Brabcová, Vendula (referee)
The need for new antibiotics and other biologically active compounds is the reason for an increased interest in secondary metabolites of soil bacteria. The phylum Actinobacteria has the dominant position in the soil environment thanks to the potential of producing a broad spectrum of antibiotics and the presence of a number of defense mechanisms preventing the effects of antibiotics. The aim of this thesis was to determine the number of copies of selected secondary metabolic genes in the soils of two sites using designed primers and primers from literature. The design of effective new primers for the detection of selected genes in the soil environment was not achieved in this work, and therefore only primers from literature that had been verified for their specificity were used. In samples taken from soil profiles of two sites, abundances of bacteria, actinobacteria, type II polyketide synthase genes and Erm methyltransferase genes mediating resistance to MLSB antibiotics (macrolides, lincosamides and streptogramins B) were determined by digital PCR. The comparison of the determined copy numbers gave an information about the structure of the bacterial community and the relative abundance of bacteria carrying selected secondary metabolic genes depending on the soil condition changes due to the...
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