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Differences in physiology between r and K bacterial strategists.
Moserová, Andrea ; Konopásek, Ivo (advisor) ; Krištůfek, Václav (referee)
Differences in physiology between r and K-bacterial strategists. The definition of bacterial r/K-strategists is currently based on the time interval they need to form a colony on agar plate. Also, their growth rate which is often used to identify r/K-position within a pair of bacterial strains. To date it was evidenced that also other physiological characteristics relate to bacterial r/K-status, for example their different ability to 1) adapt for changing conditions 2) utilize complex or very diluted substrates, 3) use secondary metabolites to cope with other strains and possibly also others. The intersection of macro- and microbiological r/K-conceptions lies in time distribution of r/K-strategists during succession. The aim of this study was to verify the basic r/K-characteristics on nine chosen strains and to correlate them with their physiological differences that are implicitly regarded as characteristic for r/K-groups. The study deals with growth rates measurements on both liquid and solid media, identification of fatty acid composition and membrane fluidity of strains cultivated at near-optimum and cold temperature in order to track the differences in cold adaptation. The study also deals with the description of possible new K-strategist characteristic: the lack of correlation between the colony...
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Cold adaptation in stationary phase in Bacillus subtilis
Beranová, Anna ; Konopásek, Ivo (advisor) ; Svobodová, Jaroslava (referee)
Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...
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Z-scan Fluorescence Correlation Spectroscopy Applied in Studies of Model Phospholipid Membranes
Kulakowska, Anna ; Hof, Martin (advisor) ; Konopásek, Ivo (referee) ; Dejneka, Alexander (referee)
6. Summary First of all, the issue whether and to what extent lipid mobility in the bilayer is altered when the bilayer is being deposited on the solid support was addressed. We designed a method that allows a direct comparison of the diffusion coefficients of labelled-lipids in the free-standing lipid bilayer of GUVs and that of the bilayer interacting with the mica surface (SPBs) in the glucose solution. The lateral diffusion analysis strongly indicate that the observed phenomenon is an unrestricted 2D diffusion. The results clearly show that the diffusion is slowed by more than 2 times for the interaction with the support. Moreover, we believe that the quantitative comparison of lipid diffusion in two frequently used model membranes can be helpful when comparing data reported in the literature. Additionally, the obtained data clearly indicate the existence of an inter-leaflet coupling. The lateral diffusion of investigated membrane probes and phospholipid analogues depend on the character and structure of the used fluorescent probe. The obtained lateral diffusion of the flavone probe, F2N12S is significantly faster than that of the fluorescent lipid analogues under study. Moreover, flavone probe exhibits approximately an order of magnitude faster flip-flop in comparison to the Atto633-DOPE lipid analogue...
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Adenylate cyclase toxin of bacteria Bordetella pertussis: mechanism of potassium efflux from macrophages
Pospíšilová, Eva ; Konopásek, Ivo (referee) ; Mašín, Jiří (advisor)
The adenylate cyclase toxin-hemolysin (ACT or CyaA) is a key virulence factor of Bordetella pertussis, the agent of the human respiratory disease whooping cough. CyaA penetrates phagocytes expressing the CD11b/CD18 integrin and exhibits two different activities. One toxin conformer oligomerizes in cell membrane and permeabilizes it by forming small cation-selective pores. Another toxin conformer appears to act as monomer. It forms a calcium influx path in the membrane, concomitantly with translocating into cells the adenylate cyclase enzyme domain that binds calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. We show here that CyaA causes efflux of K+ from CD11b+ cells by a mechanism that requires binding of CyaA to integrin CD11b/CD18 and permeabilization of the cellular membrane by pore-forming conformer of CyaA. Intact CyaA and the enzymatically inactive CyaA-AC- toxoid unable to generate cAMP produced the same kinetics of K+ efflux showing that elevation or signaling of cAMP had no role in this activity. The truncated CyaA variant (CyaA-AC) devoid of its invasive AC domain failed to promote K+ efflux despite a normal pore forming activity on erythrocyte and artificial membranes. However, binding of the MAb 3D1, which recognizes the distal segment of the AC domain...
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Effect of environmental stresses on mutability of Bacillus subtilis - role of mismatch-repair system
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Konopásek, Ivo (referee)
The everchanging nature of bacterial environment requires adaptation to emerging novel conditions. One proposed way of adaptation involves increased generation of genetic variability in response to harmful conditions - a phenomenon called adaptive mutagenesis. However, the details of mechanisms of adaptive mutagenesis, and even its very existence, are far from clear. Our goal was to subject the Gram-positive model bacterium Bacillus subtilis to variety of environmental stresses, examine the rate of mutagenesis occuring and compare it to unstressed conditions. Next we wondered if there was a role for mismatch-repair system (MMR), the major pathway for mutation avoidance, in these processes. To accomplish this, we constructed systems to monitor the expression of MMR components both on transcription and translation level. We also developed a mathematical model for precise mutation rate determination in order to quantify the intensity of mutagenic processes. The monitoring of MMR proteins translation failed due to high background endogenous fluorescence present in B. subtilis cells. However, we found out, using transcription reporter system, that the expression of MMR is not influenced by imposition of harsh hyperosmotic shock upon cells. The expression of MMR was also barely influenced by nutrient limitation...
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