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The function of ABCF proteins in bacteria
Mičke, Bianka ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Translation belongs to the most basic processes which happens in the living cells. It is the last step of proteosynthesis when genetic information encoded by the mRNA is transformed into the protein on a ribosome. Organisms have developed a wide range of mechanisms that can regulate it's needs. I focused on one of them - ABCF proteins. This protein group is a member of the ABC transporters superfamily but they haven't a transmembrane domain and their purpose is protect the ribosomes from antibiotics that bind 50S ribosomal subunit or interact with the ribosomes and influence ribosomal functions. Today, we can divide ABCF proteins into the two functional groups: antibiotic resistence proteins (ARE) and proteins with the regulatory functions. The translational regulatory function has been confirmed There is 45 ABCF protein subfamilies spread through the bacteries and eukaryotes but many essential informations like the structure and exact function of them are still missing. My bachelor thesis is analysis and summary of facts that are known about the bacterial ABCF proteins. Key words: ABCF proteins, antibiotic resistence, ARE, translational regulation, ribosome, translation, translational factors
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The function of ClpX chaperone in bacteria
Kýr, Jan ; Balíková Novotná, Gabriela (advisor) ; Šiková, Michaela (referee)
Intracellular proteolysis is an essential regulatory process that affects cellular physiology. Since proteolysis destroys proteins irreversibly, this process must be strictly controlled. The AAA+ proteins are the key factors in regulated proteolysis in bacteria. These proteins consist of two functional domains, the AAA+ chaperone domain and the protease domain. One particular group of these AAA+ protein is the Clp protein family. Functional domains of the Clp family are formed by seperate proteins. The hexameric unfoldase ClpX is a member of this protein family. This unfoldase can interact with the highly conserved ClpP protease to form a ClpXP proteolytic complex. This proteolytic complex utilizes the energy of ATP binding and hydrolysis to unfold and translocate the specifically tagged substrate into the ClpP degradation chamber. Substrate recognition is mediated by the binding of ClpX to short unstructured sequences called degradation tags. ClpX recognizes several degradation tags, but the most important one is recognition of the ssrA degradation tag, which is the output of the tmRNA ribosome rescue system. Although ClpX interacts with ClpP, it affects a variety of cellular processes such as the expression of virulence factors or the adaptation to stress factors, ClpX can work independently of...
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Control of the gene expression by cis-acting non-coding RNAs and their importance in regulating genes conferring resistance to antibiotics
Novotná, Michaela ; Balíková Novotná, Gabriela (advisor) ; Pospíšil, Jiří (referee)
This work is focused on the regulation of gene expression mediated by intrinsic regulatory RNA elements that are part of the 5' end of mRNA untranslated region. These so-called r5'UTR elements are able to bind a wide spectrum of different types of molecules, from ions and small metabolites through the transfer RNA to large protein complexes. Based on this interaction, they modulate the expression of downstream gene, which therefore becomes inducible. This type of riboregulation is widely spread in bacteria and is employed even in the control of many antibiotic resistance genes. Modulation of such genes is considerably advantageous for the cell, as it provides reduction of the negative impact on the fitness of bacteria, which is often connected to the expression of resistance genes. The aim of this work is at the molecular level to characterize all types of intrinsic regulatory elements and outline how the knowledge of these systems could be used in clinical practise for the treatment of infections caused by bacteria resistant to antibiotics. Key words Regulation of gene expression, ncRNA, cis-acting regulatory RNA, r5'UTR element, attenuation, antibiotic resistance
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Characterization of the ABC-F protein Sco0636 in Streptomyces coelicolor
Pinďáková, Nikola ; Balíková Novotná, Gabriela (advisor) ; Mikušová, Gabriela (referee)
The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...
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Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
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Development and genetic basis of glycopeptide resistance in coagulase-negative staphylococci
Prášilová, Jana ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...
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Resistance to macrolides, lincosamides and streptogramins in coagulase-negative staphylococci in ČR
Novotná, Gabriela ; Janata, Jiří (advisor) ; Petráš, Petr (referee) ; Sigler, Karel (referee)
VÝSLEDKY Analýza okolí genu msr(A) 45 součástí kompletního transpozonu Tn552 a s rekombinačním místem resbinR je identická až k místu rekombinace (resbinR site I). Druhá, v pozici JCSC1435 261884-2618311, je součástí sekvence transpozonu Tn5404. Kompletní transpozon Tn5404 je na vnějších okrajích ohraničen 7 bp dlouhou přímou repeticí (AGTAACT), jež vznikla zdvojením místa inzerce transpozonu (Obr. 5.3.), stejné inzerční místo pro Tn552 bylo pozorováno v chromozómu Entereococcus faecalis CH116 (146, 155). Shrnutí výsledků: Gen msr(A) byl u 41 izolátů lokalizován na chromozómu ve společném místě, pouze u dvou izolátů byl gen lokalizován na plazmidu. Oblast chromozomálně kódovaného genu msr(A) je u všech patnácti podrobněji studovaných izolátů (Tab. 5.1.) vysoce homologní s plazmidem πSh1, integrovaným v genomu S. haemolyticus JCSC1435 izolovaného v Japonsku (179). Uspořádání genů na tomto plazmidu je konzervovaným souborem jednotlivých genových motivů, jehož menší fragmenty byly odděleně popsány v několika dřívějších studiích. Rozdíly v msr(A) hybridizačních profilech jsou dány výhradně variabilitou v úseku kódujícím resolvasu Bin a rekombinasu Sin. Nalezli jsme pět různých typů uspořádání msr(A) oblastí, které se liší od JCSC1435 tímto: i) vložením části transpozonu Tn552 (typ KM50; 7,2kb msr(A)...
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Reeducation of mechanical knowledge in mathematics during private supplementary tutoring
Novotná, Gabriela
The diploma thesis is aimed at reeducation of mechanical knowledge in the area of fractions. Since every student has different needs and pace in his/her process of gaining knowledge, the reeducation is set into their private supplementary tutoring lessons. In the theoretical part, the basic concepts (how is the private supplementary tutoring understood in this thesis, which students are taken into consideration, and what is mechanical knowledge) are explained based on Czech and foreign literary sources, subsequently, the constructivist way of teaching, which is suitable for the reeducation of mechanical knowledge, is described. The second part of this thesis is constituted by the practical implementation of the theoretical findings into three case studies. Three students were diagnosed with mechanical knowledge in the area of fractions. Subsequently, their knowledge was being reeducated during their private supplementary tutoring lessons. The process of reeducation of each student is described in detail, moreover, a kind of manual describing what should not be avoided when dealing with this topic is included.
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Pronounciation and perceptual acceptability of the sound r in Czech speeches by foreign speakers whose L1 is German
Novotná, Gabriela ; Veroňková, Jitka (advisor) ; Martínek, František (referee)
The topic of this diploma thesis belongs to the field of language acquisition and language learning. It aims to explore Czech as a second and foreign language of speakers whose mother tongue is German, namely concerning the pronunciation of the r-sound and its perceptual acceptability for Czech native speakers. The theoretical part summarizes the area of pronunciation learning and acquisition in a second and foreign language and outlines various pronunciation difficulties German speakers tend to have in Czech. It deals with the rhotics in general and subsequently focuses on the r- sound, both in Czech and in German, as well as in mutual comparison. In the last chapter, the combinatorial qualities of the r-sound which are used as a basis for the practical part are discussed. The practical part describes the conducted research. Five recordings of German students were collected. The students were staying in Prague for one semester with the Erasmus programme exchange and were learning Czech for the first time. For the recordings a written text was constructed which the students read individually. The text emphasized words including the r-sound. From the recordings, various key words were cut off and a perceptual test was compiled and given to Czech native speakers to evaluate the pronunciation of the...
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Influence of expression of lmr(C) on the biosynthesis of lincomycin in Streptomyces lincolnensis: Resistance or production?
Veselá, Ludmila ; Balíková Novotná, Gabriela (advisor) ; Beranová, Jana (referee)
The genus Streptomyces produces more than a half of the known bioactive substances, ranking it among the most important bacterial taxons. Streptomyces lincolnensis ATCC 25466 encodes a biosynthetic gene cluster for lincomycin biosynthesis in its genome. Apart from the biosynthetic and regulatory genes, the cluster also contains three resistance genes, lmr(A), lmr(B) a lmr(C), which could protect of the host from the toxicity of a synthesized antibiotic. The Lmr(C) protein belongs to ARE proteins which generaly confer resistance to clinically important classes of antibiotics: macrolides, streptogramins, lincosamides and pleuromutilins. In addition to antibiotic producers, ARE proteins are also present in pathogenic microorganisms. However, the resistance mechanism conferred by these protins which belong to ABC transporters, even though they lack the transmembrane domain, have not been characterized yet. This makes the ARE proteins an interesting subject of the research. Using deletion mutants in resistance genes lmr(A), lmr(B) a lmr(C) we studied their effect on the lincomycin production and resistance to lincosamides, lincomycin and clindamycin with special focus on the function of the lmr(C). We have found that deletion of lmr(C) does not significantly influence lincomycin production and...
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