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Actinobacteral communities in agricultural soils at sites with occurence of potato common scab.
Daniel, Ondřej ; Kopecký, Jan (advisor) ; Lichá, Irena (referee)
The diploma thesis is focussed on understanding relationships between soil chemical characteristics, actinobacterial communities of agricultural field soils and occurrence of potato common scab, a disease caused by members of the genus Streptomyces. The aim of monitoring study, on thirty-three sites covering main potato- growing regions in the Czech Republic, was to find relationships suitable for prediction of common scab severity. The second part of the thesis compared actinobacterial communities and incidence of Streptomyces harboring a pathogenic determinant, gene txtA (gene of biosynthetic pathway of phytotoxin thaxtomin A), in soils differing in occurrence of common scab. In the screening study, analysis of terminal restriction fragment length polymorphism (T-RFLP) was employed to compare composition of soil actinobacterial communities. Real-time PCR was used to quantify total actinobacteria and streptomycetes harboring txtA gene in soils differing in scab incidence. The screening study revealed negative correlations between the scab severity and (i) available phosphorus in soil and (ii) diversity of actinobaterial community. The results were used to design a model for scab prediction. A qPCR analysis showed difference in numbers of total actinobacteria and the strains harboring txtA gene in...
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Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus
Tkadlec, Jan ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...
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Streptomycetes surface growth and differentiation on inert microbeads- morphology and proteome study
Tesařová, Eva ; Weiser, Jaroslav (advisor) ; Lichá, Irena (referee)
Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...
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Bacillus subtilis ribosomes: regulation of ribosomal RNA biosynthesis and identification of the new ribosomal protein YbxF
Sojka, Luděk ; Jonák, Jiří (advisor) ; Valášek, Leoš (referee) ; Lichá, Irena (referee)
1 Abstract The biology of the bacterial ribosome of gram positive bacterium Bacillus subtilis is the central point of this thesis that includes studies of both ribosomal components - ribosomal RNA (rRNA) and one of ribosomal proteins. The first part of the thesis focuses on the regulation of rRNA synthesis and the second part focuses on the identification and characterization of a new ribosomal protein, YbxF. rRNA synthesis is mostly regulated at the level of transcription initiation. Initiating nucleoside triphosphates (iNTPs) are important molecule effectors that regulate this process. Varying iNTP concentration in the cell directly affects RNA polymerase (RNAP) at rRNA promoters as these promoters are sensitive to [iNTP] in vivo. Most of the knowledge about this regulation is derived from Escherichia coli, where the rRNA promoter sequence is key for this regulation. Nevertheless, sequence characteristics of [iNTP]-regulated rRNA promoters from gram positive bacterium B. subtilis do not emulate the sequence characteristics derived from [iNTP]-regulated rRNA promoters from gram negative bacterium E. coli. Using a combination of in vitro and in vivo approaches, we determined promoter DNA elements that are responsible for [iNTP] sensitivity of ribosomal and non ribosomal promoters in B. subtilis. The second...
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Study of the impact of physical and chemical stress to development of mutator phenotype in Bacillus subtilis
Šoberová, Tereza ; Lichá, Irena (advisor) ; Schierová, Michaela (referee)
In a bacterium's environment, life conditions are subject to constant changes. One of the proposed mechanisms of adaptation to these changes is the increase in mutation rate. Bacterial mutability is generally kept very low by action of various mechanisms of control and repair, one of the most important ones being the Mismatch Repair, which is the master regulator of genetic stability of organisms. When its function is impaired, larger amounts of mutations occur in cells. In adverse conditions, these might be beneficial for cells' adaptation. The role of these repair mechanisms in adaptive processes in Bacillus subtilis has not yet been definitely resolved. The previous work in our lab focused on establishing an experimental system to measure the extent of mutagenesis in B. subtilis, and the influence of several stresses on mutation rate was assessed. No significant increase in mutability was found to be triggered by nutrient limitation in stationary growth phase, hyperosmotic stress or increased cultivation temperature. Furthermore, a system to monitor the expression of mismatch repair proteins was constructed, which has not revealed significant differences between stressed and nonstressed growth conditions. This thesis follows the results of previous experiments, expanding the range of stresses...
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Function of alternative sigma factors of RNA polymerase in stress response of Corynebacterium glutamicum
Dostálová, Hana ; Nešvera, Jan (advisor) ; Lichá, Irena (referee)
The aim of this thesis was to contribute to understanding of the functions of the alternative sigma factors of RNA polymerase, σH a σM , during stress response of C. glutamicum. The role of σH and σM in the transcription of the gene sigM encoding σM and the genes of the operon dnaK-grpE-dnaJ-hspR which encode proteins involved in heat-shock response of C. glutamicum was studied. The promoters of the tested genes were cloned into the "promoter-probe" vector pET2 and their activity was determined by the measuring of specific activity of the reporter enzyme chloramfenicol acetyltransferase. It was found, that the heat stress has a moderate positive effect on the activity of the promoter of the gene sigM (P-sigM), whereas no effect of the oxidative stress induced by diamide was found. It was proved, that deletion of the gene sigH or sigM itself does not lead to decrease of activity of the promoter P-sigM neither in standard conditions nor after heat-shock. On the other hand, complete abolition of the activity of the promoter P-sigM was observed in the strain C. glutamicum ∆sigH∆sigM. The promoter of the gene sigM is thus recognized in standard conditions and after heat stress is recognized by both sigma factors σH and σM , but not by the sigma factor σA . It was shown, that mutation in the -10 region...
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Structure and evolution of efflux pump in gramnegative bacteria.
Gálová, Diana ; Lichá, Irena (advisor) ; Matyska Lišková, Petra (referee)
Drug resistence in microorganisms is of major concern and there is an increasing number of pathogenic bacteria resistent to clinically used antibiotics. The major mechanism of resistence in Gram-negative is active efflux that prevent the intracellular accumulation of antibiotics to toxic levels. We recognise five families of efflux pumps, mainly using proton motive force to translocate the substrate, less common driven by ATP hydrolysis. The multidrug pumps with broad substrate specifity are of a key role in drug resistance of pathogens, typically represented by RND superfamily. The source of efflux pumps are probably the producers of antibiotics and organisms exposed to toxic compounds in their natural habitat. The genes for resistance can then spread out via horizontal gene transfer to other bacteria. It is presumed that eflux pumps have developed from transporters serving physiological functions like transport of endogenous substrates.
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Regulation of cell cycle in Bacillus subtilis.
Zelenka, Tomáš ; Lichá, Irena (advisor) ; Harant, Karel (referee)
2 Abstract Relations between several events running in bacterial cell during cell cycle were the subject of many studies during last years. More advanced techniques showed, that bacterial cell life has much more variable factors, than we supposed before. Relatively recent researches managed to reveal function and in few events molecular principle of several mechanisms coordinating those events such as progression of replication and its initiation, segregation of newly replicated chromosomes and after all synchronization of complex cell division machinery. Furthermore it showed variability of those events during changing living conditions of the cell. Keywords: Cell cycle, regulation, initiation, replication, segregation of chromosome, cytokinesis, Bacillus subtilis
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Catalytic function of DNA-dependent RNA polymerases
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Lichá, Irena (referee)
DNA-dependent RNA polymerase is a highly organised protein complex that is responsible for gene expression and its regulation. Multisubunit RNA polymerase with its several catalytic activities is responsible for transcription of genes to RNA copies in all cellular organisms. During transcription RNA polymerase undergoes substantial conformational changes depending on the conditions in a particular cell. RNA polymerase in a state designated as an elongation complex passes through repetitive cycles of adding a nucleotide to the growing RNA chain. The active center contains two magnesium ions which coordinate the reactive groups of substrates. Furthermore, the active center contains structural elements that participate in binding of substrate, propper orientation of substrate towards the template strand and translocation of the RNA polymerase. The most important of these mobile structural elements are the bridge helix and the trigger loop whose conformational changes accompanies nucleotide addition cycle. Advances in the structural and biochemical characterization of RNA polymerase open new possibilities in the understanding of the transcription mechanism, its fidelity and control.
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Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters
Koběrská, Markéta ; Janata, Jiří (advisor) ; Lichá, Irena (referee) ; Kormanec, Ján (referee)
Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters Markéta Koběrská PhD thesis 2010 The introductory part of the thesis presents isolation and sequencing of lincomycin gene cluster from type strain Streptomyces lincolnensis ATCC 25466. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin industrial strain Streptomyces lincolnensis 78-11. Analysis of the cluster flanking regions revealed its localization within the genome of S. lincolnensis ATCC 25466. The cluster-bearing cosmid was integrated into the chromosome of lincomycin non-producing strains Streptomyces coelicolor CH 999 and Streptomyces coelicolor M 145. The modified strains heterologously produced lincomycin, but the level dropped to approximately 1-3% of the production in S. lincolnensis ATCC 25466. The exact sequence of lincomycin gene cluster from the type strain allowed isolation and sequence analysis of the gene cluster of structurally related celesticetin. The analysis revealed 24 putative genes, 18 of them homologous with the genes participating in lincomycin biosynthesis. Four celesticetin specific genes are encoding enzymes involved in the salicylate biosynthesis and attachment, one is coding for...
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