Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria.

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivax gelatinosus and Rhodobacter sphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.

Correlated clusters of closed reaction centers during induction of intact cells of photosynthetic bacteria.

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 c2 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.

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 Hg2+ ions up to saturation concentration (~ 103 [Hg2+]/[RC]) and the gradual time- and concentration-dependent loss of the photochemical activity was monitored. The vast majority of Hg2+ ions (about 500 [Hg2+]/[RC]) had low affinity for the RC [binding constant Kb ~ 5 mM-1] and only a few (~ 1 [Hg2+]/[RC]) exhibited strong binding (Kb ~ 50 µM-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 < [Hg2+]/[RC] < 50 range whose locations were revealed. (1) The proton gate at the cytoplasmic site had the highest affinity for Hg2+ binding (Kb ~ 0.2 µM-1) and blocked the proton uptake. (2) Reduced affinity (Kb ~ 0.05 µM-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 Hg2+ 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 Hg2+ ions can destroy H-bond structures, inhibit protein dynamics, block conformational gating mechanisms, and modify electrostatic profiles essential for electron and proton transfer.

The biophysics of a critical phenomenon: colonization and sedimentation of the photosynthetic bacteria Rubrivivax gelatinosus.

In response to environmental changes, the photosynthetic bacterium Rubrivivax gelatinosus (Rvx.) can switch from a planktonic lifestyle to a phototrophic biofilm. Like in critical phenomena, the colonization and sedimentation of the cells is abrupt and hard to predict causally, and the underlying biophysics of the mechanisms involved is not known. Herein, we report basic experimental observations and quantitative explanations as keys to understanding microbial turnover of aggregates. (1) The moment of sedimentation can be controlled by the height of the tube of cultivation, by the concentrations of externally added Ficoll (a highly branched polymer) and/or of internally produced polysaccharides (constituents of the biofilm). (2) The observed translational diffusion coefficient of the planktonic bacteria is the sum of diffusion coefficients coming from random Brownian and twitching movements of the bacteria and amounts to 14 (µm)2/s. (3) This value drops hyperbolically with the association number of the cell aggregates and with the concentration of the exopolysaccharides in the biofilm. In the experiments described herein, their effects could be separated. (4) The critical conditions of colonization and sinking of the cells will be achieved if the height of the tube meets the scale height that is proportional to the ratio of the diffusion coefficient and the net mass of the bacterium. The decisive role of the web-like structure of a biofilm, the organization of bacteria from loose cooperativity to solid aggregation, and the possible importance of similar controls in other phototrophic microorganisms are discussed.

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 (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 a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures.

Purple non-sulfur photosynthetic bacteria monitor environmental stresses.

Heavy 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.

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria.

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 µM) of mercury (Hg²âº) 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 Hg²âº 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.