|
|
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...
|
|
|
The phenomenon of persistence in bacteria - the role of toxin-antitoxin systems.
Váchal, Martin ; Lichá, Irena (advisor) ; Seydlová, Gabriela (referee)
Most bacterial species currently studied are able to generate a small fraction of heterogeneous persister cells which are tolerant to antibiotics or other antimicrobials and still genetically identical to the susceptible parental population. Bacterial persisters emerge as a result of the stochastic regulation of cellular processes. Persistence can be triggered by stressful environmental stimuli or emerge spontaneously under favourable growth conditions. According to their origin, persistent subpopulations were divided into type I and type II persisters. Many recent studies indicate that toxin-antitoxin (TA) systems increase persistence. TA systems are ubiquitous genetic elements in prokaryotes and consist of a stable toxin, inhibiting essential cellular functions in persister cells, and an unstable antitoxin, which counteracts the activity of its toxin. Overexpression of toxin parts in excess of their corresponding antitoxin leads to multidrug tolerance (MDT). This work summarizes causes of persister formation and their hypothetical survival strategies and deals primarily with TA systems, controlling bacterial persistence of model organism Escherichia coli. The emphasis is put on the description of type I TA system TisB/IstR-1, type II TA systems HipBA, RelBE, MazEF, DinJ-YafQ, MqsRA, type V TA...
|
|
|
Characterization of Ms1, a newly identified small RNA from Mycobacterium smegmatis
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...
|
|
|
The effect of vanZTei and vanZg expression on resistance to glycopeptide antibiotics in Staphylococcus aureus
Zieglerová, Leona ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...
|
|
|
Microbial consortia and metagenome of industrially polluted soil: occurrence of genes encoding AEH
Pitkina, Anastasiya ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
Soils contain highly diverse consortia of bacteria making them very attractive starting points for both culture-dependent and metagenomic discovery efforts. The present diploma thesis analyses the composition of the microbial community from pharmaceutically polluted soil, with the employment of next-generation Illumina sequencing of 16S rDNA region. This analysis revealed high complexity of the soil microbial environment and confirmed that anthropogenic activity (represented by production of beta- lactam antibiotics) influences the variability and abundance of the species, yet without reducing the microbial diversity. In the second part of the thesis, isolation and heterologous expression of a novel gene encoding alpha-amino acid ester hydrolase (AEH) from a cultivable soil microorganism B. cereus is described. AEHs possess industrial potential for biocatalytic synthesis of semi-synthetic beta-lactam antibiotics, which are presently of great clinical importance. Powered by TCPDF (www.tcpdf.org)
|
|
|
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...
|
|
|
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)....
|
|
|
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...
|
|
|
SCCmec and other mobile genetic elements associated with methicillin resistance in staphylococci.
Kubištová, Lucie ; Lichá, Irena (advisor) ; Plocek, Vítězslav (referee)
Staphylococci are common part of human flora but also they are a dangerous pathogen. Among staphylococci strains, methicillin resistance is widespread. The mecA gene, organized in mec complex, is responsible for methicillin resistance. The mec complex is part of mobile genetic element - staphylococcal chromosome cassette SCCmec. SCCmec is large variable mobile genetic element and it is always composed of three parts - mec complex, ccr complex and J regions. Complex mec consists of mecA gene and its regulatory genes mecR1 and mecI. Complex ccr encodes recombinase genes, they are responsible for excision and insertion of SCCmec. J regions are remaining parts of SCCmec, which include other mobile genetic elements that directly influence methicillin resistance genes expression or carry genes for resistance to other antimicrobial agents. SCCmec or its parts can be transferred by horizontal gene transfer between staphylococci both intraspecific and interspecific, although mechanism of its transfer is still unknown. Eleven types of SCCmec have been described so far. In this thesis, I summarized the findings about molecular composition of SCCmec, horizontal gene transfer of the genes encoding methicillin resistance and molecular evolution of SCCmec. Mobile genetic elements play a key role in evolution and...
|
|
|
Characterization of the HelD protein from Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
BACKGROUND: Bacterial RNA polymerase (RNAP) is an extensively studied enzyme required for gene expression. In our Laboratory we found a new protein named HelD. HelD copurifies with B. subtilis RNAP. HelD is a ~90 kDa protein from the UvrD/Rep helicase family, which contains protein with the 3'-5' DNA unwinding activity. The molecular role(s) HelD in cell are still unknown and its potential role in transcription has not been studied so far. OBJECTIVE: The main aim of this Diploma project was to describe HelD. APPROACHES: The characterization was carried out on three levels: (i) bioinformatics analysis in silico was used to identify HelD homologs in other bacteria; (ii) growth tests in vivo were used to determine the phenotype(s) of the HelD-null mutant strain compared to wt; and (iii) biochemical experiments in vitro were utilized to describe the effects of HelD on transcription, and to test whether HelD has DNA binding and DNA unwinding activities. RESULTS: The in silico analysis revealed that HelD is present in Firmicutes, an industrially and medicinally important group of G+ bacteria. The phenotypic experiments showed that HelD is required for rapid adaptations to nutritional changes in the environment. The biochemical experiments showed that HelD stimulates transcription despite the fact that it...
|
guest ::
