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Metabolic control of bacterial division.
Valtová, Aneta ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Metabolic control of cell cycle has been study for a long time, but it is not completely known. Mechanisms of metabolic control described for a several decade has been explained on molecular level with using a modern methods. Regulation of cell cycle in consideration of metabolism and nutritional status is going on at the several level of cell replication. The most known is about assembly of bacterial cell divisiome. Changes in nutrient availability induce stress response that use low-molecular substances in signaling pathways leading to changes in the cell cycle. One of the most studied is (p)ppGpp that participates in stringent response and affect sigma factors, directly inhibits the initiation of replication by binding to the DnaG primase and indirectly inhibits the elongation of replication. Current researches has revealed that some enzymes with already known enzymatic function in the major metabolic pathways (glycolysis or TCA) also has a function as sensors that transmit the nutritional change signal directly into the cell dividing process. These signals most often inhibits FtsZ protein or affect its helper proteins and subsequent ring formation. Analogues of these enzymes were found in gram-positive (Bacillus subtilis) and gram-negative bacteria (Escherichia coli, Caulobacter crescentus)....
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General stress response in Staphylococcus aureus and its role in adaptation to hyperosmotic stress
Kotková, Hana ; Lichá, Irena (advisor) ; Seydlová, Gabriela (referee)
Hyperosmotic stress induces a stress reponse in a halotolerant bacteria Staphylococcus aureus that helps it to survive even the osmotic conditions that are inhibiting for most of the bacteria. Stress responses of S. aureus include a general stress response that activates rapidly on a wide range of stresses and is ensured by expression of genes induced by an alternative sigma B subunit of RNA polymerase (SigB), and more specific responses to hyperosmotic stress associated with transporters of potassium and transporters of compatible solutes. Also, some virulence factors (such as the adhesive Ebh protein) allow S. aureus to survive hyperosmotic stress within the host, thereby helping to colonize it. It is currently known that the general stress response directly and indirectly participates in the specific stress adaptation through the Kdp transport system for potassium (Kdp transporter) and transporters of compatible solutes (PutP and OpuD). Expression of the Kdp transporter and the large S. aureus Ebh protein is SigB dependent, via Agr or ArlRS virulence regulator systems. The formation of a biofilm, in which SigB is significantly involved, may also enable to colonize host niches with low water activity. Hyperosmotic stress by triggering SigB initiates a complex response that is associated not only...
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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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Regulation of expression of Ms1, a sRNA from Mycobacterium smegmatis
Páleníková, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...
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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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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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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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Morphine alkaloid metabolism in bacteria
Zahradník, Jiří ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
Morphine alkaloids and their derivatives are pharmaceutically important substances. Huge production and consumption of these compounds predetermines them to be significant pollutants in the environment. Some of them have been detected in surface waters. The aim of this study was to characterize effects of morphine alkaloids on the physiology of three model organisms: Agrobacterium sp. R89-1, Escherichia coli XL-1 (Blue), and Raoultella sp. kDF8, and elucidation of the mechanisms leading to toxicity. The biotransformation potential and utilization ability were characterized for model organisms. It was demonstrated that the microorganism Agrobacterium sp. R89-1 is capable of rapid biotransformation of codeine to its 14-OH derivatives. The manifestation of morphine compounds toxic effects for the strain R89-1 is the highest. In contrast, microorganism Raoultella sp. KDF8 is able to utilize codeine as a carbon and energy source. The accumulation of 14-OH-derivatives was not observed. Escherichia coli XL-1 (Blue) is not able to biotransform or utilize codeine. Α, β-unsaturated ketones (morphinone, codeinone, 14-OH-morphinone and 14-OH-codeinone) were found as a most toxic intermediates of codeine metabolism. Bacterial cell growth (strains R89-1 and KDF8) in the presence of codeine is characteristic with...
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Molecules involved in immune evasion encoded within the US2-US11 genomic region of human cytomegalovirus
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Lichá, Irena (referee)
Human cytomegalovirus (HCMV) from the family Herpesviridae causes lifelong latent infections in immunocompetent patients. HCMV encodes multiple molecules which interfere with host immune responses, thus helping the virus to escape from them and persist in the host. Inhibition of antigen presentation by MHC class i glycoproteins is an important immune evasion mechanism, which enables the virus to protect infected cells from recognition by CD8+ T lymphocytes. Most of the molecules inhibiting MHC class i presentation is encoded withing the US2 to US11 region of HCMV genome, including five glycoproteins (US2, US3, US6, US10 a US11) and one miRNA (US4-1). gpUS2 and gpUS3 are also able to block MHC class II antigen presentation and supress the response of CD4+ T lymphocytes. This thesis summarises current knowledge about the immune evasion molecules encoded within the US2-US11 genomic region of HCMV. Keywords: human cytomegalovirus (HCMV), immune evasion, MHC I, MHC II, US2, US3, US4-1, US6, US10, US11
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Endogenous retroviral elements and their functions in the human genome
Famfulíková, Mirka ; Pačes, Jan (advisor) ; Lichá, Irena (referee)
In addition to the coding sequences, the human genome contains a so noncoding DNA, among which we count transposable elements capable of transposition in the genome. The remnants of the past retrovirus infections - endogenous retroviruses (human endogenous retroviruses - HERVs) belong to the transposable elements, which contain the LTR sequences. Human endogenous retroviruses make up to 8% of the size of the human genome. The retroviruses are not only passive relicts, but they have gained some key functions - too. They increase the plasticity of the human genome and some HERV LTRs can serve as binding sites for transcription factors like. Env protein from the families HERV-W and HERV- FRD were coopted by the human genome and are nowadays expressed as proteins Syncitin-1 and Syncitin-2, which are necessary by the forming of human placenta. Unfortunately, the HERV elements can have a negative health impacts. In the last decades they are subject of a debate in connection with various diseases, such as multiple sclerosis, schizophrenia, HIV proliferation and some types of tumorigenesis. The role of HERVs in the human genome is not completely known yet and it is important to continue with their research. Powered by TCPDF (www.tcpdf.org)
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