M. Picaud

A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts.

A detailed study of the photo-induced decline in chlorophyll a fluorescence intensity (Kautsky phenomenon) in coupled isolated chloroplasts from a high level (P) to a low stationary level (S) is presented. 1. A linear relationship between P leads to S quenching and intrathylakoid H+ concentration was found. When the light-induced proton gradient was abolished by uncoupling, the fluorescence emission at room temperature was lowered proportionally to increased H+ concentration in the medium. 2. Fluorescence spectra at -196 degrees C of samples frozen at the P and S states showed no significant differences in the Photosystem I/Photosystem II ratio of fluorescence emission. Furthermore, freezing to -196 degrees C reversed the P leads to S quenching. This indicates that the P leads to S quenching is not related to an increase of spillover of excitation energy from Photosystem II to Photosystem I. 3. When Mg2+ was added to thylakoids suspended in a medium free of divalent cations, the inhibition of spillover required lower Mg2+ concentrations (half saturation at 0.6 mM). Increased proton concentration in the medium also inhibited spillover. 4. The results are interpreted in terms of two sites of Mg2+ and H+ effects on excitation deactivation in Photosystem II. One site is located on the outer face of the thylakoid membrane; action of both Mg2+ and H+ at this side diminishes spillover. The second site is located on the inner face of the membrane; as Mg2+ is displaced there by protons, a non-photochemical quenching of Photosystem II fluorescence is induced, which is manifested by the P leads to S decline.

Rubrivivax gelatinosus acsF (Previously orf358) Codes for a Conserved, Putative Binuclear-Iron-Cluster-Containing Protein Involved in Aerobic Oxidative Cyclization of Mg-Protoporphyrin IX Monomethylester

This study describes the characterization of orf358, an open reading frame of previously unidentified function, in the purple bacterium Rubrivivax gelatinosus. A strain in which orf358 was disrupted exhibited a phenotype similar to the wild type under photosynthesis or low-aeration respiratory growth conditions. In contrast, under highly aerated respiratory growth conditions, the wild type still produced bacteriochlorophyll a (Bchl a), while the disrupted strain accumulated a compound that had the same absorption and fluorescence emission spectra as Mg-protoporphyrin but was less polar, suggesting that it was Mg-protoporphyrin monomethylester (MgPMe). These data indicated a blockage in Bchl a synthesis at the oxidative cyclization stage and implied the coexistence of two different mechanisms for MgPMe cyclization in R. gelatinosus, an anaerobic mechanism active under photosynthesis or low oxygenation and an aerobic mechanism active under high-oxygenation growth conditions. Based on these results as well as on sequence analysis indicating the presence of conserved putative binuclear-iron-cluster binding motifs, the designation of orf358 as acsF (for aerobic cyclization system Fe-containing subunit) is proposed. Several homologs of AcsF were found in a wide range of photosynthetic organisms, including Chlamydonomas reinhardtii Crd1 and Pharbitis nil PNZIP, suggesting that this aerobic oxidative cyclization mechanism is conserved from bacteria to plants.

Chlorophyll fluorescence as a probe for the determination of the photo-induced proton gradient in isolated chloroplasts.

Gramicidin D-treated chloroplasts show an acid-induced quenching of the chlorophyll fluorescence, which is composed of a reversible and irreversible part. The reversible quenching is analogous to the photo-induced quenching in coupled chloroplasts and can be taken to determine the light induced delta pH.

Photooxidative stress stimulates illegitimate recombination and mutability in carotenoid‐less mutants of Rubrivivax gelatinosus

Carotenoids are essential to protection against photooxidative damage in photosynthetic and non‐photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid‐less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild‐type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid‐less strains (crtB−) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic‐deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non‐functional chimeric genes. The R.gelatinosus crtB− strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions.

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24–48 h according to various steps. For O2 evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.

Molecular analysis of psb A mutations responsible for various herbicide resistance phenotypes in Synechocystis 6714

Mutations conferring herbicide resistance in 3 mutant strains of the cyanobacterium Synechocystis 6714 have been characterized by gene cloning and sequencing. The mutants display very different phenotypes: DCMU-IIA is DCMU-resistant and atrazine-resistant, DCMU-IIB is DCMU-resistant and atrazine-sensitive, and Az-V is DCMU-sensitive, atrazine-resistant and presents particular photoinhibition properties. These mutants were originally obtained either by one-step selection (DCMU-IIA) or by two-step selection (DCMU-IIB and Az-V). psbA copies carrying herbicide resistance have been identified by transformation experiments as psbAI in all cases. Sequences of the psbAI copy of each mutant have been compared to the wild-type sequence. In the single mutant DCMU-IIA, a point mutation at codon 264 (Ser→Ala) results in resistance to both DCMU and atrazine. In the double mutants DCMU-IIB and Az-V, two point mutations were found. DCMU-IIB was derived from DCMU-IIA and had acquired a second mutation at codon 255 (Phe→Leu) resulting in a slight increase in DCMU resistance and complete abolition of atrazine resistance. Az-V contains two changes at codons 211 (Phe→Ser) and 251 (Ala→Val) resulting in high atrazine resistance but only slight DCMU resistance.

Relationship between O2 evolution capacity and cytochrome b-559 high-potential form in Photosystem II particles

Abstract As previously shown for inside-out vesicles by Larsson et al. (Larsson, C., Jansson, C., Ljungberg, U.L., Akerlund, H.E. and Anderson, B. (1984) in Advances in Photosynthesis Research, Vol. I, pp. 363–366 (Sybesma C., ed.), Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, The Netherlands), we observed that NaCl 1 M washing of Photosystem II particles prepared by Triton X-100 treatment of spinach thylakoids induces both an inactivation of oxygen evolution and transformation of cytochrome b -559 from its high-potential to its low-potential form. A partial reactivation of water oxidation by 24 kDa polypeptide refixation is accompanied by a partial restoration of the cytochrome b -559 high-potential (HP) form. In contrast, reconstitution of water splitting by Ca 2+ addition is not associated to a reestablishment of the cytochrome (HP) form. We conclude that cytochrome b -559 HP plays no role in water oxidation.

Mutations responsible for high light sensitivity in an atrazine-resistant mutant of Synechcystis 6714

The primary target of photoinhibition is the photosystem II reaction center. The process involves a reversible damage, followed by an irreversible inhibition of photosystem II activity. During cell exposition to high light intensity, the D1 protein is specially degraded. An atrazine-resistant mutant of Synechocystis 6714, AzV, reaches the irreversible step of photoinhibition faster than wild-type cells. Two point mutations present in the psbA gene of AzV (coding for D1) lead to the modification of Phe 211 to Ser and Ala 251 to Val in D1. Transformation of wild-type cells with the AzV psbA gene shows that these two mutations are sufficient to induce a faster photodamage of PSII. Other DCMU-and/or atrazine-resistant mutants do not differ from the wild type when photoinhibited. We conclude that the QB pocket is involved in PSII photodamage and we propose that the mutation of Ala 251 might be related to a lower rate of proteolysis of the D1 protein than in the wild type.

Global Regulation of Photosynthesis and Respiration by FnrL THE FIRST TWO TARGETS IN THE TETRAPYRROLE PATHWAY

Fnr is a regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. To assess the role of Fnr in photosynthesis in Rubrivivax gelatinosus, a strain carrying a null mutation in fnrL was constructed. It was unable to grow anaerobically in the light, but, intriguingly, it was able to produce photosynthetic complexes under high oxygenation conditions. The mutant lacked all c-type cytochromes normally detectable in microaerobically-grown wild type cells and accumulated coproporphyrin III. These data suggested that the pleiotropic phenotype observed in FNR is primarily due to the control at the level of the HemN oxygen-independent coproporphyrinogen III dehydrogenase. hemN expression in trans partially suppressed the FNR phenotype, as it rescued heme and cytochrome syntheses. Nevertheless, these cells were photosynthetically deficient, and pigment analyses showed that they were blocked at the level of Mg2+-protoporphyrin monomethyl ester. Expression of both hemN and bchE in the FNR mutant restored synthesis of Mg2+-protochlorophyllide. We, therefore, conclude that FnrL controls respiration by regulating hemN expression and controls photosynthesis by regulating both hemN and bchE expression. A comprehensive picture of the control points of microaerobic respiration and photosynthesis by FnrL is provided, and the prominent role of this factor in activating alternative gene programs after reduction of oxygen tension in facultative aerobes is discussed.

Development of gene transfer methods forRubrivivax gelatinosus S1: construction, characterization and complementation of apuf operon deletion strain

Gene transfer systems were developed inRubrivivax (Rx.) gelatinosus S1. First, a system for conjugative transfer of mobilizable plasmids fromEscherichia coli toRx. gelatinosus S1 was established. Secondly, optimal conditions for the transformation ofRx. gelatinosus S1 by electroporation were determined. A Δpuf strain was constructed. Complementation with thepuf operon from a wild-type strain cloned in a replicative plasmid restored photosynthetic growth. Two insertion strains were also selected. All the strains constructed were green, due to a change in carotenoid content. Characterization of these strains provides genetic evidence for a “superoperon” organization in this bacterium.