Emese Asztalos

Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg(2+)) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg(2+) is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Export or recombination of charges in reaction centers in intact cells of photosynthetic bacteria.

The kinetics and thermodynamics of forward and reverse electron transfer around the reaction center of purple bacterium Rhodobacter sphaeroides were studied in vivo by flash-excited delayed fluorescence, prompt fluorescence (induction) and kinetic difference absorption. By protection of the photomultiplier from intense bacteriochlorophyll prompt fluorescence evoked by laser excitation, the time resolution of the fluorometer was reduced typically 10 micros. Two precursor states of the delayed fluorescence were identified: P(+)Q(A)(-) and cyt c(2)(3+)Q(A)(-) whose enthalpy levels were 340 meV and 1020 meV below A, respectively. The free energy of the P(+)Q(A)(-) state relative to A* was -870 meV in whole cells. Similar values were obtained earlier for isolated reaction center and chromatophore. The free energies of cyt c(2)(3+)Q(A)(-) and P(+)Q(A)(-) states showed no or very weak (-6 meV/pH unit) pH-dependence, respectively, supporting the concept of pH-independent redox midpoint potential of Q(A)/Q(A)(-) in intact cells. In accordance with the multiphasic kinetics of delayed fluorescence, the kinetics of re-opening of the closed reaction center is also complex (it extends up to 1 s) as a consequence of acceptor and donor-side reactions. The control of charge export from the reaction center by light regime, redox agents and inhibitors is investigated. The complex kinetics may arise from the distribution of quinones in different redox states on the acceptor side (Q(B) binding site and pool) and from organization of electron transfer components in supercomplexes.

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.

Mutational control of bioenergetics of bacterial reaction center probed by delayed fluorescence

The free energy gap between the metastable charge separated state P(+)QA(-) and the excited bacteriochlorophyll dimer P* was measured by delayed fluorescence of the dimer in mutant reaction center proteins of the photosynthetic bacterium Rhodobacter sphaeroides. The mutations were engineered both at the donor (L131L, M160L, M197F and M202H) and acceptor (M265I and M234E) sides. While the donor side mutations changed systematically the number of H-bonds to P, the acceptor side mutations modified the energetics of QA by altering the van-der-Waals and electronic interactions (M265IT) and H-bond network to the acidic cluster around QB (M234EH, M234EL, M234EA and M234ER). All mutants decreased the free energy gap of the wild type RC (~890meV), i.e. destabilized the P(+)QA(-) charge pair by 60-110meV at pH8. Multiple modifications in the hydrogen bonding pattern to P resulted in systematic changes of the free energy gap. The destabilization showed no pH-dependence (M234 mutants) or slight increase (WT, donor-side mutants and M265IT above pH8) with average slope of 10-15meV/pH unit over the 6-10.5pH range. In wild type and donor-side mutants, the free energy change of the charge separation consisted of mainly enthalpic term but the acceptor side mutants showed increased entropic (even above that of enthalpic) contributions. This could include softening the structure of the iron ligand (M234EH) and the QA binding pocket (M265IT) and/or increase of the multiplicity of the electron transfer of charge separation in the acceptor side upon mutation.

Calculation of Connectivity of Photosynthetic Units in Intact Cells of Rhodobacter Sphaeroides

The kinetics of the bacteriochlorophyll fluorescence of intact cells of photosynthetic bacterium Rhodobacter sphaeroides was measured under rectangular shape of intense excitation in the microsecond time range. The interest was focused to the initial (sigmoidal) rise of the fluorescence to characterize the connectivity of the photosynthetic units. As the rate of the primary photochemistry (charge separation) was set to be much larger than that of the re-reduction of the oxidized dimer of the reaction center, there was reciprocity between light intensity and photochemical rise time, and therefore a simple model of single fluorescence (photochemical) quencher could be applied. By linearization of the fluorescence induction kinetics, the connectivity parameter p could be directly obtained (p = 0.47 ± 0.01) and was independent on the intensity of the light excitation. The mean value of the number of steps (visits) in the antenna was calculated before an exciton is either trapped by an open reaction center (it is utilized by photochemistry) or dissipated in form of fluorescence emission. According to these calculations, the mean number of steps is less than 1 if p < 0.5, even if all of the reaction centers are closed. The observed small p value includes highly restricted mobility of excitions among the photosynthetic units.

Oxygen-Dependent Production and Arrangements of the Photosynthetic Pigments in Intact Cells of Rhodobacter Sphaeroides

The development and re-arrangement of photosynthetic membrane of Rhodobacter sphaeroides were studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence when the cells grown under semiaerobic conditions were inoculated to aerobic medium and, after accommodation to aerobiosis, transferred to anaerobic conditions during constant illumination. After sudden transition from semiaerobic to aerobic cultures, the photochemical yield characterized by the relative variable fluorescence Fv/Fmax decreased from 0.70 gradually to 0.30, the photochemical rate constant dropped from 5·104 s−1 to 1·104 s−1 and the contribution of the peripheral antenna (LH2) to the RC-LH1 core complex became less significant. The aerobic growth is characterized by bleaching of the pigment system. The transfer to anaerobiosis resulted in photosynthetic growth with opposite tendency of the parameters and the initial values were restored after a couple of cell cycling periods of doubling time of ∼4 h. The LH2 to LH1 ratio increased by a factor of 5. It was demonstrated that the fast fluorescence induction is a sensitive and non-invasing method to monitor the physiological state of the photosynthetic apparatus upon variation of the oxygen concentration of the culture.