Chantal Astier

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.

State 1-state 2 adaptation in the cyanobacteria Synechocystis PCC 6714 wild type and Synechocystis PCC 6803 wild type and phycocyanin-less mutant

The mechanism of excitation energy distribution between the two photosystems (state transitions) is studied in Synechocystis 6714 wild type and in wild type and a mutant lacking phycocyanin of Synechocystis 6803. (i) Measurements of fluorescence transients and spectra demonstrate that state transitions in these cyanobacteria are controlled by changes in the efficiency of energy transfer from PS II to PS I (spillover) rather than by changes in association of the phycobilisomes to PS II (mobile antenna model). (ii) Ultrastructural study (freeze-fracture) shows that in the mutant the alignment of the PS II associated EF particles is prevalent in state 1 while the conversion to state 2 results in randomization of the EF particle distribution, as already observed in the wild type (Olive et al. 1986). In the mutant, the distance between the EF particle rows is smaller than in the wild type, probably because of the reduced size of the phycobilisomes. Since a parallel increase of spillover is not observed we suggest that the probability of excitation transfer between PS II units and between PS II and PS I depends on the mutual orientation of the photosystems rather than on their distance. (iii) Measurements of the redox state of the plastoquinone pool in state 1 obtained by PS I illumination and in state 2 obtained by various treatments (darkness, anaerobiosis and starvation) show that the plastoquinone pool is oxidized in state 1 and reduced in state 2 except in starved cells where it is still oxidized. In the latter case, no important decrease of ATP was observed. Thus, we propose that in Synechocystis the primary control of the state transitions is the redox state of a component of the cytochrome b6/f complex rather than that of the plastoquinone pool.

Ultrastructure and light adaptation of phycobilisome mutants of Synechocystis PCC 6803

Abstract We have performed functional and ultrastructural characterization of Synechocystis PCC 6803 wild type and of two mutants, the first one (PMB11) lacking phycocyanin and still possessing the core of the phycobilisome and the second one (PΔE) lacking phycocyanin and Lcm (the core-membrane linker) and totally devoid of assembled phycobilisome structure. In the three strains, the state 1-state 2 transition occurred and was accompanied by ultrastructural changes. In state 1, an increased percentage of PS II-associated EF particles were aligned in rows compared to state 2 where more EF particles were randomly distributed in the membrane. A spill-over model is suggested by our ultrastructural and spectrophotometric results.© 1997 Elsevier Science B.V. All rights reserved.

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.

Randomization of the EF particles in thylakoid membranes of Synechocystis 6714 upon transition from state I to state II

In the cyanobacterium Synechocystis 6714 we show that changes in light energy distribution are controlled by the redox state of the PQ pool and we report on the deep ultrastructural modifications caused by these changes. We conclude that State I‐State II transitions correspond to an increased energy transfer between PS II and PS I through a randomization of the EF particles of the thylakoid membranes. This reorganization could involve a change in the interaction between PS II centers and the phycobilisomes.

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.

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D1 protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D1 in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D1 and 5% sensitive D1 were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D1. This enhancement is specifically due to high light and not to the decrease of D1 concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.

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.

Reversible and irreversible photoinhibition in herbicide-resistant mutants of Synechocystis 6714

Abstract The behaviour of two herbicide-resistant mutants of Synechocystis 6714, DCMU-IIB and Az-V, were compared to the wild type during various times of exposure to high light intensity (photoinhibition). The kinetics of the loss of variable fluorescence were similar in the three strains. However, Az-V cells lost the ability to recover Photosystem II activity more rapidly than wild-type and DCMU-IIB cells. Radiolabeling experiments showed that the turnover of D1 is similar in wild type and Az-V. Partial reactions of electron flow through Photosystem II were measured on thylakoids isolated from cells withdrawn at different times of photoinhibition. The decrease of oxygen evolution using DCBQ (electron acceptor after QB), presented the same kinetics in wild type and Az-V. In contrast, the kinetics of decrease of oxygen evolution with silicomolybdate were faster in Az-V than in wild type. Our results support the hypothesis that the QB site of reaction center II is the initial target of damage by photoinhibition. This damage can be reversed by de novo synthesis of D1 and D2 proteins. The reversible inhibition is followed by a more extensive degradation of the core complex RC II. This more extensive degradation is irreversible and is characterized by a decrease of energy transfer from the phycobilisomes to the Photosystem II, and incapacity to perform charge separation. Due to a higher instability of their core complex II the second, irreversible step of degradation happens more rapidly with Az-V mutant cells than with wild-type cells.

Regulation of Photosynthesis Genes in Rubrivivax gelatinosus: Transcription Factor PpsR Is Involved in both Negative and Positive Control

Induction of biosynthesis of the photosystem in anoxygenic photosynthetic bacteria occurs when the oxygen concentration drops. Control of this induction takes place primarily at the transcriptional level, with photosynthesis genes expressed preferentially under anaerobic conditions. Here, we report analysis of the transcriptional control of two photosynthesis promoters, pucBA and crtI, by the PpsR factor in Rubrivivax gelatinosus. This was accomplished by analyzing the photosystem production in the wild type and in the PPSRK (ppsR::Km) mutant grown under anaerobic and semiaerobic conditions and by assessing the beta-galactosidase activity of lacZ transcriptionally fused to promoters possessing the putative PpsR-binding consensus sequences. It was found that under semiaerobic conditions, inactivation of the ppsR gene resulted in overproduction of carotenoid and bacteriochlorophyll pigments, while the production of LH2 was drastically reduced. The beta-galactosidase activity showed that, in contrast to what has been found previously for Rhodobacter species, PpsR acts in R. gelatinosus as an aerobic repressor of the crtI gene while it acts as an activator for the expression of pucBA. Inspection of the putative PpsR-binding consensus sequences revealed significant differences that may explain the different levels of expression of the two genes studied.