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Y1 and Y2 transposases, mechanisms of transposition, biological function.
Zahradník, Jiří ; Lichá, Irena (advisor) ; Schierová, Michaela (referee)
Transposases are enzymes that catalyse cleavage, transmission and re-inserting of mobile genetic element into the DNA. Tyrosine transposase take between these enzymes completely independend status. Their uniqueness is determined by their structure and different mechanism of the transposition reaction, in which the covalent phosphotyrosine intermediate plays major role. Mandatory presence of the catalytic tyrosine gives name to these enzymes and it enables their further classification into a group that carries only a single catalytic tyrosine - Y1 transposases and a group carrying two tyrosines - Y2 transposases. This thesis summarizes the current knowledge about tyrosine transposases. It covers their occurrence, structure, reaction mechanism and biological function. The reaction mechanism of the most studied Y1 transposase, associated with IS608 element, is described in detail. The work also focuses on other members of the tyrosin transposases family which carry the characteristic HUH motive. These include transposases associated with the insertion sequence of IS200/IS605 family (Y1), transposases associated with REP elements (so called RAYT proteins), transposases associated with IS91 family (Y2), transposases of ISCRs family (Y1) and unusual eukaryotic transposases of the Helitron family (Y2)....
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Bacteriophages - current knowledge and possibilities for their therapeutic use
Glendová, Kristýna ; Lichá, Irena (advisor) ; Čáp, Michal (referee)
Bacteriophages, as viruses of bacteria, are the most abundand entity, populate every biotope where also bacteria live. One of the alternatives to combat infections caused by resistant strains of bacteria currently appear bacteriophage therapy, consists in the application of lytic bacteriophage, or only bacteriophage enzymes to inhibit bacterial growth. Thesis mentions the history of phage therapy, a crucial part of the thesis deals with a summary of current trends in bacteriophage therapy, beginning to develop in recent years. Many studies are dedicated to the possibilities of treatment of bacterial infections by phage lysates, including genetically modified bacteriophages and also application of bacteriophage enzymes themselves - endolysins, or a combination of the phage lysates and endolysins with antibiotics. The main interests in studies are the efficiency, specificity and safety of therapy. The effectiveness of bacteriophage therapy was already proved by many studies, both in vitro and in vivo. The safety of therapy for clinical usage needs to be prove by in vivo experiments. Key words: bacteriophage, bacteriophage therapy, endolysins, enzybiotics, multiresistence
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Antibiotic rezistance genes in soil actinobacteria
Patrmanová, Tereza ; Kopecký, Jan (advisor) ; Lichá, Irena (referee)
Actinobacteria are important members of the soil ecosystems, where they are involved in organic matter decomposition. It is worth mentioning that their secondary metabolism allows them to produce a variety of different compounds. These compounds include antibiotics, among them aminoglycosides have a place in clinical practice. These antibiotics are significant due to a broad spectrum of activities against both gram-negative and gram-positive bacteria. However, their use currently carries a risk, mainly their toxicity and development of antibiotic resistance in bacteria. Resistance is the cause of low effectiveness of some of those antibiotics. Actinobacteria as aminoglycoside producers must protect themselves from these compounds, so a variety of resistance types was developed, out of which enzymatic inactivation is the most studied one. Actinobacteria have evolved several mechanisms, which contribute to a resistance to the agents with antimicrobial effects. Genes encoding antibiotic resistance are abundant in soil environment. Their variability is influenced by many factors, especially the selection of bacteria in soil contaminated with antibiotics and also with strains originating from human and animal waste. Significant role has a horizontal gene transfer, which allows distribution of resistence...
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Bacterial REP elements: origins, variability and application.
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Pačes, Jan (referee) ; Melter, Oto (referee)
4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...
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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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The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav ; Nešvera, Jan (advisor) ; Branny, Pavel (referee) ; Lichá, Irena (referee)
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
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Role of small effector molecules in bacterial signalling.
Kolář, Petr ; Lichá, Irena (advisor) ; Hilská, Markéta (referee)
Small effector molecules play an important role in bacterial physiology. There are many types of them in the bacterial cell. One group are small signalling molecules which participate in quorum sensing, enabling bacterial cell-cell communication as part of a extracellular signalling. These molecules mediate information about the cell density and different qualities of the extracellular matrix. Next group of small effector molecules are modified nucleotides. They participate in intracellular signalling pathways which regulate the switch of bacterial lifestyle according to changing environment. Best studied are signalling pathways using the molecules - c-di-GMP and (p)ppGpp. Detail studies were done in case of gram positive (Bacillus subtilis) and gram negative (Escherichia coli, Vibrio cholerae) bacteria, where was proved the connection between c-di-GMP signalling pathways and quorum sensing (Vibrio cholerae, Xanthomonas campestris). Important discovery in field of small effector molecules is the c-di-AMP signalling pathway in Bacillus subtilis. New regulatory mechanisms were determined in well-known small effector molecule cAMP, regarding the signalling pathways connections (Vibrio cholerae). Recent studies considering the cooperation of the extracellular and intracellular signalization pathways...
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Oxidative stress in bacteria - with an emphasis on the model organism of Escherichia coli
Moravcová, Andrea ; Lichá, Irena (advisor) ; Čáp, Michal (referee)
Most bacterial species encounter aerobic conditions during their life, which can be toxic. This leads to a state of oxidative stress. Toxicity of aerobic environment results from the chemical properties of molecular oxygen and its reactive species (ROS). Bacteria had to adapt to these conditions in the past to ensure preservation and prosperity. This thesis is preferably focused on oxidative stress adaptations in the most elaborated bacterial model - Escherichia coli. Regulation of adaptations at the regulation of transcription, translation and metabolism level are described with emphasis on molecular mechanisms. The main adaptation mechanism against oxidative stress is the deactivation of ROS, as well as the repair of damaged cell structures (macromolecules). These enzyme activities are regulated by several transcriptional regulators. The transcriptional regulators OxyR and SoxRS have been well studied in E. coli. Even though these regulators are conserved across the bacterial spectrum, they may not have the same function in all organisms. For this reason, also other, more or less studied bacterial species - Bacillus subtilis, Streptomyces coelicolor, Pseudomonas putida, Pseudomonas aeruginosa - were included in this thesis. The various strategies of how these bacteria use not only OxyR and SoxRS...
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A heterologous expression of alpha-amino acid ester hydrolase from the strain Achromobacter sp. CCM 4824 in Escherichia coli BL21(DE3)
Schneiderová, Michaela ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
On the chromosomal DNA of the microorganism Achromobacter sp. CCM 4824, which was gained in the Laboratory of enzyme technology MBU AVCR v.v.i., there was identified a gene coding an enzyme capable of hydrolyzation of semi-synthetic β-lactam antibiotics ampicillin and cephalosporin with a D-phenylglycine as a side chain. This enzyme belongs to a group of α-amino acid esterases, which are interesting because of a potential use in kinetically controled synthesis of β-lactam antibiotics. In several aspects α-amino acid esterases might be better than actually used penicillin acylases and that is why these enzymes are subjects of a big interest. The gene for α-amino acid esterase coded by chromosomal DNA was cloned, characterized and heterologously expressed in constructed highly-producing bacterial system Escherichia coli BL21(DE3)JM5. Produced enzyme was purified and its properties important for possible use in the production of semi-synthetic β-lactam antibiotics were determined. Key words: alpha-amino acid ester hydrolase, Achromobacter sp., recombinant expression system, β-lactam antibiotics
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