Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis

Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystis sp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtained pgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.     

Localisation and interactions of the Vipp1 protein in cyanobacteria

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C‐terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live‐cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull‐downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high‐light exposure. These include not only photosynthetic and stress‐related proteins but also RNA‐processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress‐induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.     

Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme

Nostoc   punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc . A protocol for isolating thylakoid membranes was developed to examine the biochemical and biophysical aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N.   punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analysed by measuring oxygen evolution activity, fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centres were active in water oxidation in the final membrane preparation. Analysis of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b ₆/ f could be observed. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.     

A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced gene expression

In Synechocystis sp. strain PCC 6803, over 450 genes are upregulated following transfer of the cells from a high (1–5% CO₂ in air, HC) to a low level of CO₂ (as in air or lower, LC). This includes sbtA , ndhF3 and cmpA involved in inorganic carbon (Ci) uptake. Earlier studies implicated NdhR in the regulation of LC-induced genes but there are indications that additional components are involved. Following extraction of proteins from cells grown under HC and (NH4)₂SO₄ fractionation, we have identified LexA and two AbrB-like proteins, Sll0359 and Sll0822, which bind to a fragment of the sbtA promoter. Using extracts prepared from LC-grown cells, Sll0822 did not bind to the sbtA promoter despite its presence in the cells, suggesting that it may serve as a repressor of LC-induced genes. This is supported by the fact that sbtA , ndhF3 and cmpA normally expressed only under LC in the wild-type are transcribed under both HC and LC in a Δ sll0822 mutant. When grown under HC this mutant exhibits an elevated apparent photosynthetic affinity to Ci, typically observed in the wild-type only under LC. Clearly, expression of genes essential for Ci uptake was sufficient to raise the apparent photosynthetic affinity for external Ci.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275-4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.     

Electron transport routes in whole cells of Synechocystis sp. strain PCC 6803: the role of the cytochrome bd-type oxidase

The plastoquinone pool is the central switching point of both respiratory and photosynthetic electron transport in cyanobacteria. Its redox state can be monitored noninvasively in whole cells using chlorophyll fluorescence induction, avoiding possible artifacts associated with thylakoid membrane preparations. This method was applied to cells of Synechocystis sp. PCC 6803 to study respiratory reactions involving the plastoquinone pool. The role of the respiratory oxidases known from the genomic sequence of Synechocystis sp. PCC 6803 was investigated by a combined strategy using inhibitors and deletion strains that lack one or more of these oxidases. The putative quinol oxidase of the cytochrome bd-type was shown to participate in electron transport in thylakoid membranes. The activity of this enzyme in thylakoids was strongly dependent on culture conditions; it was increased under conditions where the activity of the cytochrome b(6)f complex alone may be insufficient for preventing over-reduction of the PQ pool. In contrast, no indication of quinol oxidase activity in thylakoids was found for a second alternative oxidase encoded by the ctaII genes.     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on L-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. L-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.     

The long-term responses of the photosynthetic proton circuit to drought

Proton motive force (pmf) across thylakoid membranes is not only for harnessing solar energy for photosynthetic CO₂ fixation, but also for triggering feedback regulation of photosystem II antenna. The mechanisms for balancing these two roles of the proton circuit under the long-term environmental stress, such as prolonged drought, have been poorly understood. In this study, we report on the response of wild watermelon thylakoid 'proton circuit' to drought stress using both in vivo spectroscopy and molecular analyses of the representative photosynthetic components. Although drought stress led to enhanced proton flux via a ∼34% increase in cyclic electron flow around photosystem I (PS I), an observed ∼fivefold decrease in proton conductivity, g_H⁺, across thylakoid membranes suggested that decreased ATP synthase activity was the major factor for sustaining elevated q_E. Western blotting analyses revealed that ATP synthase content decreased significantly, suggesting that quantitative control of the complex plays a pivotal role in down-regulation of g_H⁺. The expression level of cytochrome b ₆ f complex – another key control point in photosynthesis – also declined, probably to prevent excess-reduction of PS I electron acceptors. We conclude that plant acclimation to long-term environmental stress involves global changes in the photosynthetic proton circuit, in which ATP synthase represents the key control point for regulating the relationship between electron transfer and pmf.     

集胞藻PCC 6803高产精氨酸藻株的紫外诱变选育

精氨酸在医药和食品工业上具有广泛用途。集胞藻PCC 6803是单细胞蓝藻, 能利用工业废气(主要成分是氮氧化物NO_x)与水反应生成的硝酸盐和亚硝酸盐合成氨基酸等化合物, 因而选育高产精氨酸藻株, 不仅能提高精氨酸产量, 而且能去除工业废气中的NO_x, 具有潜在的应用前景。研究在集胞藻PCC 6803中利用紫外诱变, 筛选抗0.8 g/L D-精氨酸和抗0.2 g/L 6-氮尿嘧啶的突变株, 选育到了一株精氨酸产量显著提高的突变株#13807-111-55, 它每OD₇₃₀值细胞的胞外精氨酸产量相比出发株提高了62.3倍, 达到(0.76±0.1) mg/(L·OD₇₃₀), 总精氨酸产量相比出发株提高了6.0倍, 达到(0.82±0.08) mg/(L·OD₇₃₀)。该突变株每OD₇₃₀值细胞的胞外精氨酸产量明显高于胞内, 表明该突变藻株是精氨酸分泌型, 因而具有潜在的应用前景。     

Mutants of the cyanobacterium Synechocystis PCC6803 impaired in respiration and unable to tolerate high salt concentrations

Abstract The cyanobacterium Synechocystis PCC6803, when challenged with a salt stress (0.5 M), increased its respiratory and cytochrome c oxidase activities. Spontaneous mutants impaired in respiration could be isolated through their incapacity to tolerate salt stress. Mutants from one class, among two classes obtained, were deficient for respiratory capacity and cytochrome c oxidase activity. The latter enzyme exhibited similar kinetic modifications, but no change in the M _r of subunits I and II, in the complexes present in the cytoplasmic and thylakoid membranes.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.