The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (~100 µM) of mercury (Hg 2+ ) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg 2+ sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c2 and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc1 complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.
The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0-4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10(4) s(-1)) and yield (F v/F max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10(4) s(-1)). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.
a b s t r a c tHeavy metal ion pollution and oxygen deficiency are major environmental risks for microorganisms in aqueous habitat. The potential of purple non-sulfur photosynthetic bacteria for biomonitoring and bioremediation was assessed by investigating the photosynthetic capacity in heavy metal contaminated environments. Cultures of bacterial strains Rhodobacter sphaeroides, Rhodospirillum rubrum and Rubrivivax gelatinosus were treated with heavy metal ions in micromolar (Hg 2+ ), submillimolar (Cr 6+ ) and millimolar (Pb 2+ ) concentration ranges. Functional assays (flash-induced absorption changes and bacteriochlorophyll fluorescence induction) and electron micrographs were taken to specify the harmful effects of pollution and to correlate to morphological changes of the membrane. The bacterial strains and functional tests showed differentiated responses to environmental stresses, revealing that diverse mechanisms of tolerance and/or resistance are involved. The microorganisms were vulnerable to the prompt effect of Pb 2+ , showed weak tolerance to Hg 2+ and proved to be tolerant to Cr 6+ . The reaction center controlled electron transfer in Rvx. gelatinosus demonstrated the highest degree of resistance against heavy metal exposure.
Antenna systems serve to absorb light and to transmit excitation energy to the reaction center (Rc) in photosynthetic organisms. As the emitted (bacterio)chlorophyll fluorescence competes with the photochemical utilization of the excitation, the measured fluorescence yield is informed by the migration of the excitation in the antenna. In this work, the fluorescence yield concomitant with the oxidized dimer (p +) of the RC were measured during light excitation (induction) and relaxation (in the dark) for whole cells of photosynthetic bacterium Rhodobacter sphaeroides lacking cytochrome c 2 as natural electron donor to p + (mutant cycA). The relationship between the fluorescence yield and p + (fraction of closed RC) showed deviations from the standard Joliot-Lavergne-Trissl model: (1) the hyperbola is not symmetric and (2) exhibits hysteresis. These phenomena originate from the difference between the delays of fluorescence relative to P + kinetics during induction and relaxation, and in structural terms from the non-random distribution of the closed Rcs during induction. the experimental findings are supported by Monte Carlo simulations and by results from statistical physics based on random walk approximations of the excitation in the antenna. The applied mathematical treatment demonstrates the generalization of the standard theory and sets the stage for a more adequate description of the long-debated kinetics of fluorescence and of the delicate control and balance between efficient light harvest and photoprotection in photosynthetic organisms. Photosynthesis is responsible for the genesis, development and regulation of vast majority forms of life on the Earth by using the ultimate free energy source of the sun. The conversion of (sun)light to chemical energy is initiated by the absorption of the photons in the closely packed network of protein-pigment complexes (antenna) followed by funneling of the excitation energy (exciton) to a specially organized (B)Chl dimer (P) in the reaction centers (RC) 1. Here an electron is stripped from P (P→P +) converting the energy of the exciton into chemical (redox) energy of P/P +. The electron is transferred via the primary quinone acceptor Q A to the secondary quinone acceptor Q B producing a series of transient charge separated states (P + Q-). While Q A can accept one electron only, Q B performs two-electron chemistry: by binding two protons and forming reduced quinone QH 2 , it is exchanged for an oxidized quinone from the quinone pool in the membrane 2-4. To describe the functional cooperation of the antenna pigments in light collection, the loose concept of the photosynthetic unit (PSU) was introduced 5. According to the present knowledge, the structure of the PSU of photosynthetic bacteria can be identified as the core complex including the photochemical RC and the closely attached light-harvesting (core) antenna (LH1, B870 in Rhodobacter (Rba.) sphaeroides) together (if exists) with the peripheral antenna (LH2, B800-850 complex in Rba. sphaeroides) loosely arranged in ...
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 the 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 Ca 2+ 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 10 5 ) greater than their own masses by well defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM) -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 sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury contaminated aqueous cultures.2
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