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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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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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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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Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a homologue of Escherichia coli protein YjjG
Vacková, Zuzana ; Branny, Pavel (advisor) ; Lichá, Irena (referee)
ANGLICKÝ ABSTRAKT Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a likely homolog of Escherichia coli protein YjjG. Bacterial cells are constantly exposed to innumerable toxic substances, either in their external environment or by by-products of their own metabolism. For these reasons, the bacterial cells evolved several mechanisms to cope with this challenge. These mechanisms are represented by: blocking the uptake, export by specific transporters as well as specific inactivation of these substance by enzymes. A particular group of these toxic substances are noncanonica nucleotides, which can directly inhibit bacterial cell DNA replication or can result in increased mutation rate. Enzymes recognizing these modified derivatives are known as "house-cleaning" nucleotide phsphateses, which can inactivate the potentially mutagenic nucleotides and prevent their incorporation into DNA and RNA. Some of the "house- cleaning" enzymes belong to a group of haloacid dehalogenase enzymes (haloacid dehalogenase-like hydrolase superfamily), which are found in many bacterial species. This thesis is focused on the function of hypothetical protein Spr1057 of Streptococcus pneumoniae with an unknown function. Sequence comparison revealed that Spr1057 has a significant...
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Role of the yxkO gene of Bacillus subtilis in responce to environmental stress.
Petrovová, Miroslava ; Lichá, Irena (advisor) ; Nešvera, Jan (referee)
ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.
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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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