Bakterioklorofill fluoreszcencia, mint a fotoszintetikus baktériumok fiziológiai állapotának jelzőrendszere

Photosynthesis is a biological process whereby the energy of the Sun is captured and stored by series of events that convert the free energy of light into different forms of free energy needed to feed cellular processes. (Blankenship 2014). The photosynthesis provides the foundation for essentially all life and has altered the Earth itself over geologic time profoundly. It provides all of our foods and most of our energy resources. Since essentially all energy used on Earth can be traced back to the photosynthetic transformation of solar energy into chemical energy, it is not surprising that the study of photosynthesis is at the center of scientific interest (Govindjee et al. 2005; Eaton-Rye et al. 2012; Niederman 2017). In photosynthetic bacteria, the energy conversion processes are considerably simpler than in green plants. While there are two photochemical reactions in green plants, there is only one in the bacteria. In contrast to the linear electron transport chain of green plants, the electron transport in bacteria is cyclic, in which the free energy of the charge pair produced in the reaction center (RC) is utilized by a cyclic pathway of electron building up a proton gradient across the photosynthetic membrane. The reaction center and the cytochrome bc1 complex (via the Q-cycle) constitute a proton-pump mechanism that translocates protons from the cytoplasmic side to the periplasmic side of the membrane. In the modern photosynthesis research, the non-sulfur type of purple bacteria plays a significant role, because the three-dimensional determination of the reaction center at atomic level (Deisenhofer et al. 1984) has made it possible to identify the structure and function of a photosynthetic energy conversion system. Although the details of the transformation of energy may vary in different species, there are structural and functional similarities. The bacterial reaction center has a very high photochemical quantum yield (~ 100%) since nearly all of the absorbed photons create charge pairs (Wraight and Clayton 1974). The highest free-energy loss relates to the reduction of the primary quinone (QA), which also means that physiological conditions make this process irreversible. The photosynthetic bacteria protect and operate their energy conversion system with remarkable efficiency and rate. An important part of this process is the light-dependent production and protection of tripled states of bacteriochlorophylls (BChl) essential for the survival of photosynthetic organisms. The energy of the BChl tripled state can be transmitted easily to triplet molecular oxygen (3O2) that generates harmful singlet excited oxygen (1O2*, strong oxidant). To avoid this reaction, several pathways are operating in all of which carotenoid (Car) pigments play prominent role. In addition to high light intensity, photosynthetic bacteria are exposed to numerous stress effects including heavy metal ions. The organisms can maintain their functions even under harmful conditions. How do they do it and what can be learned from these experiences? What makes the intact photosynthetic bacterium and its reaction center robust and yet flexible enough to function efficiently under different stress conditions? These are the fundamental questions I set in the frontline of the dissertation

Photoprotection in intact cells of photosynthetic bacteria: quenching of bacteriochlorophyll fluorescence by carotenoid triplets.

Upon high light excitation in photosynthetic bacteria, various triplet states of pigments can accumulate leading to harmful effects. Here, the generation and lifetime of flash-induced carotenoid triplets (3Car) have been studied by observation of the quenching of bacteriochlorophyll (BChl) fluorescence in different strains of photosynthetic bacteria including Rvx. gelatinosus (anaerobic and semianaerobic), Rsp. rubrum, Thio. roseopersicina, Rba. sphaeroides 2.4.1 and carotenoid- and cytochrome-deficient mutants Rba. sphaeroides Ga, R-26, and cycA, respectively. The following results were obtained: (1) 3Car quenching is observed during and not exclusively after the photochemical rise of the fluorescence yield of BChl indicating that the charge separation in the reaction center (RC) and the carotenoid triplet formation are not consecutive but parallel processes. (2) The photoprotective function of 3Car is not limited to the RC only and can be described by a model in which the carotenoids are distributed in the lake of the BChl pigments. (3) The observed lifetime of 3Car in intact cells is the weighted average of the lifetimes of the carotenoids with various numbers of conjugated double bonds in the bacterial strain. (4) The lifetime of 3Car measured in the light is significantly shorter (1-2 mus) than that measured in the dark (2-10 mus). The difference reveals the importance of the dynamics of 3Car before relaxation. The results will be discussed not only in terms of energy levels of the 3Car but also in terms of the kinetics of transitions among different sublevels in the excited triplet state of the carotenoid

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria

Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca2+ channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H+ ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 105) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (muM)-1 and 1 (mM)-1, respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures

Characterization of mercury(II)-induced inhibition of photochemistry in the reaction center of photosynthetic bacteria

Mercuric contamination of aqueous cultures results in impairment of viability of photosynthetic bacteria primarily by inhibition of the photochemistry of the reaction center (RC) protein. Isolated reaction centers (RCs) from Rhodobacter sphaeroides were exposed to Hg(2+) ions up to saturation concentration (~ 10(3) [Hg(2+)]/[RC]) and the gradual time- and concentration-dependent loss of the photochemical activity was monitored. The vast majority of Hg(2+) ions (about 500 [Hg(2+)]/[RC]) had low affinity for the RC [binding constant Kb ~ 5 mM(-1)] and only a few (~ 1 [Hg(2+)]/[RC]) exhibited strong binding (Kb ~ 50 muM(-1)). Neither type of binding site had specific and harmful effects on the photochemistry of the RC. The primary charge separation was preserved even at saturation mercury(II) concentration, but essential further steps of stabilization and utilization were blocked already in the 5 < [Hg(2+)]/[RC] < 50 range whose locations were revealed. (1) The proton gate at the cytoplasmic site had the highest affinity for Hg(2+) binding (Kb ~ 0.2 muM(-1)) and blocked the proton uptake. (2) Reduced affinity (Kb ~ 0.05 muM(-1)) was measured for the mercury(II)-binding site close to the secondary quinone that resulted in inhibition of the interquinone electron transfer. (3) A similar affinity was observed close to the bacteriochlorophyll dimer causing slight energetic changes as evidenced by a ~ 30 nm blue shift of the red absorption band, a 47 meV increase in the redox midpoint potential, and a ~ 20 meV drop in free energy gap of the primary charge pair. The primary quinone was not perturbed upon mercury(II) treatment. Although the Hg(2+) ions attack the RC in large number, the exertion of the harmful effect on photochemistry is not through mass action but rather a couple of well-defined targets. Bound to these sites, the Hg(2+) ions can destroy H-bond structures, inhibit protein dynamics, block conformational gating mechanisms, and modify electrostatic profiles essential for electron and proton transfer

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples-without interfering with their 'conventional' fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644-2656, 2013). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall (Ginkgo biloba) stained with Etzold's staining solution

Season dependent changes in the expression of Protein Kinase C isoenzymes in a female patient with Systemic Lupus Erythematosus

Europeanwhitewomen .Peripheral bloodmononuclear cells (PBMC) . Protein kinaseC(PKC) . Season dependence . Systemic lupus erythematosus (SLE