Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803

α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.     

Photosystem II antenna modules CP43 and CP47 do not form a stable ‘no reaction centre complex’ in the cyanobacterium Synechocystis sp. PCC 6803

Photosynthesis Research - The repair of photosystem II is a key mechanism that keeps the light reactions of oxygenic photosynthesis functional. During this process, the PSII central subunit D1 is...     

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme ⁶-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding ⁶-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking ⁶-desaturase. Expression of the Synechocystis ⁶-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis ⁶-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a ¹²-desaturase gene linked to the ⁶-desaturase gene. Expression of Synechocystis ⁶-and ¹²-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid.     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

Changes in the antenna size of Photosystem I and Photosystem II in Synechococcus sp. strain PCC 7942 grown in the presence of SANDOZ 9785 — a Photosystem II inhibitor

SANDOZ 9785, also known as BASF 13.338, is a pyridazinone derivative that inhibits Photosystem II (PS II) activity leading to an imbalance in the rate of electron transport through the photosystems. Synechococcus sp. strain PCC 7942 cells grown in the presence of sublethal concentration of SANDOZ 9785 (SAN 9785) for 48 hours exhibited a 20% decrease in Chl a per cell. However, no changes were observed in the content of phycocyanin per cell, the size of the phycobilisomes or in the PS II:PS I ratio. From an estimate of PS II electron transport rate under varying light intensities and spectral qualities and analysis of room temperature Chl a fluorescence induction, it was deduced that growth of Synechococcus PCC 7942 in the presence of SAN 9785 leads to a redistribution of excitation energy in favour of PS II. Though the redistribution appears to be primarily caused by changes affecting the Chl a antenna of PS II, the extent of energetic coupling between phycobilisomes and PS II is also enhanced in SAN 9785 grown Synechococcus PCC 7942 cells. There was a reduction in the effective size of PS I antenna based on measurement of P700 photooxidation kinetics. These results indicate that when PS II is partially inhibited, the structure of photosynthetic apparatus alters to redistribute the excitation energy in favour of PS II so that the efficiency of utilization of light energy by the two photosystems is optimized. Our results suggest that under the conditions used, drastic structural changes are not essential for redistribution of excitation energy between the photosystems.Abbreviations APC Allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophyenyl)-1,1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - F_o fluorescence when all the reaction centres are open - f_m fluorescence yield when all the reaction centres are closed - F_v variable chlorophyll fluorescence - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic Acid - I₅₀ concentration that causes 50% inhibition in activity - MV methyl viologen - pBQ para benzoquinone - PBS phycobilisome - PC phycocyanin - PS I, PS II Photosystem I, Photosystem II - P700 reaction centre Chl a of PS I - SAN 9785 SANDOZ 9785 i.e. 4-chloro-5-dimethylamino-2-phenyl-3 (2H) pyridazinone, also known as BASF 13.338     

Synechocystis sp. PCC 6803 — a useful tool in the study of the genetics of cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 was the first phototrophic organism to be fully sequenced. The genomic sequence has revealed the structure of the genome and its gene constituents (3167 genes), as well as the relative map positions of each gene. The functions of nearly half of the genes has been deduced using similarity searches. The genome sequence has also allowed for the implementation of systematic strategies to study gene function and the mechanisms of gene regulation on a genome-wide level. Two genome databases, CyanoBase and CyanoMutants, have been established and act as a central repository for information on gene structure and gene function, respectively. As a result of the genome sequencing and the establishment of these databases, Synechocystis sp. PCC 6803 provides an extremely versatile and easy model to study the genetic systems of photosynthetic organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancing photo-catalytic production of organic acids in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC,a strain incapable of glycogen storage

A key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo-catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha-ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo-catalytic state, and characterizes the metabolic adaptability of the photo-catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo-catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo-catalytic state. Reversal to growth state is observed with re-addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha-ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions.     

Partial conservation of the 5′ ndhE-psaC-ndhD 3′ gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5 ndhE-psaC-ndhD3 gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5 portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3 portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.     

Redox Regulation of Glycogen Biosynthesis in the Cyanobacterium Synechocystis sp. PCC 6803： Analysis of the AGP and Glycogen Synthases

Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase （AGP） and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin （TrxA）, suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence （C45, C185, C320, and C337）, they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.     

Effect of green light on the amount and activity of NDH-1–PSI supercomplex in Synechocystis sp. strain PCC 6803

Photosynthetica - Cyanobacterial NDH-1 interacts with PSI to form NDH-1–PSI supercomplex. CpcG2, a linker protein for the PSI-specific peripheral antenna CpcG2-phycobilisome, is essential for...     

The D1-D61N mutation in Synechocystis sp. PCC 6803 allows the observation of pH-sensitive intermediates in the formation and release of O₂ from photosystem II

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.     

Alternative Cyanide-sensitive Oxidase Interacting with Photosynthesis in Synechocystis PCC6803. Ancestor of the Terminal Oxidase of Chlororespiration?

Influence of respiration on photosynthesis in Synechocystis PCC6803 was studied by measuring the redox transients of cytochrome f (cyt f) upon excitation of the cells with repetitive single turnover flashes. Upon the addition of KCN the flash-induced oxidation of cyt f was increased and the rereduction of cyt f+ was accelerated. Dependence of these effects on the concentration of KCN clearly demonstrated the existence of two cyanide-sensitive oxidases interacting with photosynthesis: cyt aa3, which was sensitive to low concentrations of cyanide, and an alternative oxidase, which could be suppressed by using 1 mM KCN. The interaction between the photosynthetic and the respiratory electron transport chains was regulated mainly by the activity of the alternative cyanide-sensitive oxidase. The oxidative pathway involving the alternative cyanide-sensitive oxidase was insensitive to salicyl hydroxamic acid and azide. The close resemblance of the inhibition pattern reported here and that described for chlororespiration in algae and higher plants strongly suggest that an oxidase of the same type as the alternative cyanide-sensitive oxidase of cyanobacteria functions as a terminal oxidase in chloroplasts.     

Analysis of proteins involved in the production of MAA?s in two Cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica

Mycosporine- like amino acids (MAAs) are small (<400Da), colourless, water soluble compounds composed of cyclohexenone or cyclohexinimine chromophere conjugated with the nitrogen substituent of amino acid or its amino alcohol. These compounds are known for their UV- absorbing role in various organisms and seem to have evolutionary significance. The biosynthesis of MAAs is presumed to occur via the first part of shikimate pathway. In the present work two cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica were tested for their ability to synthesize MAAs and protein involved in the production of MAAs. It was found that protein sequence 3-phosphoshikimate 1-carboxyvinyltransferase is involved in producing mycosporine glycine in Synechocystis PCC 6803 and 3-dehydroquinate synthase is involved for producing shinorine in Anabaena cylindrica. Phylogenetic and bioinformatic analysis of Mycosporine like amino acid producing protein sequence of both cyanobacterial species Synechocystis PCC 6803 and Anabaena cylindrica provide a useful framework to understand the relationship of the different forms and how they have evolved from a common ancestor. These products seem to be conserved but the residues are prone to variation which might be due the fact that different cyanobacteria show different physiological process in response of Ultraviolet stress.     

Heterogeneous Rieske proteins in the cytochrome b6f complex of Synechocystis PCC6803?

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b(6)f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b(6)f complexes in Synechocystis and suggests still to be established alternative electron transport routes.