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Resonance Raman spectra of phycocyanin, allophycocyanin and phycobilisomes from blue-green alga Anacystis nidulans.

Resonance Raman spectra of native C-phycocyanin, allophycocyanin and whole, intact phycobilisomes from the blue-green alga Anacystis nidulans (Synechococcus 6301) are reported. A tentative assignment for the more prominent resonance Raman bands is suggested. The possibly sensitive regions for inter-chromophore interactions in the case of phycobilisomes are also discussed.

Cyanobacteria↗

The role of glutathione and ascorbate in hydroperoxide removal in cyanobacteria.

The antioxidative potential of cyanobacteria to scavenge hydroperoxides formed as by-products of photosynthetic activity was investigated in Nostoc muscorum 7119 and Synechococcus 6311. These cells contained a high concentration of glutathione, 2-5 mM, and a low concentration of ascorbate, 20-100 uM. No glutathione peroxidase was detected while the activity of ascorbate peroxidase was high, reacting with hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide. Dehydroascorbate reductase was active in recycling ascorbate and glutathione reductase regenerated glutathione from glutathione disulphide. The activity of these antioxidative enzymes in the cyanobacteria was sufficient to remove between 60-230 nmoles H2O2 .mg protein-1 min-1. It is suggested that in cyanobacteria an effective reaction sequence for removal of hydroperoxides involves ascorbate peroxidase and recycling of glutathione and ascorbate.

Ascorbate Peroxidases↗

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes.

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans-membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelength is obtained with valinomycin-induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.

Adenosine Triphosphatases↗

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing.

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.

Amino Acid Sequence↗

N-terminal sequencing of low-molecular-mass components in cyanobacterial photosystem II core complex. Two components correspond to unidentified open reading frames of plant chloroplast DNA.

We recently reported the presence of several low-molecular-mass protein components in the PS II O2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus [(1989) FEBS Lett. 244, 391-396]. Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing. There were two components in the 4.7 kDa region, both having a blocked N-terminus. One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts. The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II. The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.

Amino Acid Sequence↗

Effect of formate on Mössbauer parameters of the non-heme iron of PS II particles of cyanobacteria.

Mössbauer spectra were measured for PSII particles having an active water-splitting system. The particles were isolated from the thermophilic cyanobacterium Synechococcus elongatus enriched in 57Fe. The Mössbauer resonance absorption spectrum is a superposition of 3 doublets with the following quadrupole splitting and chemical shift: 1, delta = 0.40, delta = 0.85; II, delta = 1.35, delta = 2.35; III, delta = 0.25, delta = 1.65. The delta and delta values of doublets I, II, III are characteristic of proteins with iron-sulphur center, non-heme iron of the reaction center of higher plants and of the oxidized cytochrome b-559. Treatment with sodium formate to remove bicarbonate affects only the doublet of non-heme iron, causing its quadrupole splitting to reduce to 1.75 and the chemical shift to reduce to 0.90. After washing out the formate, the Mössbauer spectrum of non-heme iron is restored. The data suggest that bicarbonate is a ligand for the non-heme iron of the reaction center of cyanobacteria.

Bicarbonates↗

Identities of four low-molecular-mass subunits of the photosystem I complex from Anabaena variabilis ATCC 29413. Evidence for the presence of the psaI gene product in a cyanobacterial complex.

Photosystem I (PSI) complex of Anabaena variabilis ATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis. N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined. Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes. The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively. The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria.

Amino Acid Sequence↗

Cyanobacterial thylakoid membrane proteins are reversibly phosphorylated under plastoquinone-reducing conditions in vitro.

Reversible, light-dependent protein phosphorylation was observed in isolated thylakoid membranes of the cyanobacterium Synechococcus 6301. A polypeptide of 15 kDa in particular was phosphorylated under plastoquinone-reducing conditions and was not phosphorylated under plastoquinone-oxidising conditions. Phosphorylation and dephosphorylation reactions involving this and several other membrane polypeptides showed sensitivity to inhibitors of protein kinases and phosphatases. Changes in phosphorylation state correlated with changes in low temperature fluorescence emission characteristic of changes in excitation energy distribution between the photosystems. The 15 kDa phosphopolypeptide is likely to be involved directly in light state adaptations in cyanobacteria.

Cyanobacteria↗

Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme.

The first committed step in the biosynthetic pathway of carotenoids in plants and algae is the conversion of geranylgeranyl pyrophosphate (GGPP) to prephytoene pyrophosphate (PPPP), which is converted to phytoene. We have cloned the gene pys that encodes the enzyme phytoene synthase in the cyanobacterium Synechococcus PCC7942. The co-expression of pys in cells of Escherichia coli together with the gene crtE from Erwinia uredovora, which encodes geranylgeranyl pyrophosphate synthase, resulted in accumulation of phytoene. This result indicates that phytoene synthase is a single polypeptide enzyme that catalyzes the 2-step reaction from GGPP to phytoene. The deduced amino acid sequence of pys is highly conserved with that of pTOM5, a tomato cDNA that is differentially expressed during fruit ripening. These findings suggest that pTOM5 encodes phytoene synthase in tomato.

Alkyl and Aryl Transferases↗

Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.

Amino Acid Sequence↗

Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene.

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

Carotenoids↗

Asymmetrical core structure in phycobilisomes of the cyanobacterium Synechocystis 6701.

The light-harvesting complex of cyanobacteria and red algae, the phycobilisome, has two structural domains, the core and the rods. Both contain biliproteins and linker peptides. The core contains the site of attachment to the thylakoid membrane and the energy transfer link between the phycobilisome and chlorophyll. There are also six rod-binding sites in the membrane-distal periphery of the core. The structure of phycobilisomes in the cyanobacterium Synechococcus 6301 was studied by Glazer, who proposed a model for the internal organization of the bicylindrical core. In the construction of that model, it was necessary to make arbitrary decisions between two possible locations for one of the trimeric protein complexes within a core cylinder and between two possible orientations of the basal core cylinders relative to one another. We isolated the tricylindrical cores from an ultraviolet-light-induced mutant of the cyanobacterium Synechocystis 6701 and obtained, by partial dissociation, a unique core substructure that maintained some contacts between the two basal cylinders. From its structure and spectral properties, we conclude that this particle is a central core substructure that resulted from dissociation of the two layers of peripheral trimers in the intact core. The compositions of this particle and the dissociated trimers were inconsistent with the proposed location of one of the trimers in the 6301 core model, but supported the placement of that trimer in the alternative position within the basal core cylinder. Rod-binding sites within the central core substructure were studied by partial dissociation of the short-rod phycobilisomes from another mutant of 6701. This dissociation generated particles that were interpreted as being central core substructures with the two basal rods attached. The appearance of these particles in the electron microscope suggested that both basal rods would be localized towards the same side of the intact core. Such an asymmetrical arrangement of basal rods is supported by previously published edge-views of intact cores with basal rods from strain 6701. These observations suggest a parallel arrangement of the basal cylinders with respect to each other, creating an asymmetrical core. A phycobilisome model was constructed that incorporated core asymmetry. This model predicts the energy transfer pathways from the basal and upper rods to specific trimers in the core.

Cyanobacteria↗

Influence of cypermethrin and fenvalerate on a green alga and three cyanobacteria isolated from soil.

The effects of two pyrethroid insecticides, cypermethrin and fenvalerate, on a green alga (Scenedesmus bijugatus) and three species of cyanobacteria (Synechococcus elongatus, Nostoc linckia, and Phormidium tenue), all isolated from a black cotton soil, were studied using either cell number or chlorophyll a as toxicity criterion. All the four species were either unaffected or stimulated at 5 micrograms ml-1. Of the two insecticides, cypermethrin, at 10 to 50 micrograms ml-1, inhibited S. bijugatus while these concentrations stimulated or only slightly inhibited the growth of S. elongatus. There was a significant inhibition in the growth of S. bijugatus and stimulation in S. elongatus with 10 to 50 micrograms ml-1 fenvalerate. The growth of N. linckia was enhanced by both insecticides while P. tenue was significantly affected.

Chlorophyll↗

Purification of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium.

Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein. The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside. The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.4 M and within 10 min. The FPLC fractions were analyzed for protein and pigment contents and by polypeptide composition. The purest fraction is essentially free of pigments and has a high specific ATP hydrolysis activity (about 1.6 mumol ATP X min-1 X mg protein-1) which is sensitive to N,N'-dicyclohexylcarbodiimide.

Adenosine Triphosphatases↗

Cloning and expression in Escherichia coli of the gene coding for phytoene synthase from the cyanobacterium Synechocystis sp. PCC6803.

The gene coding for the carotenoid biosynthesis enzyme phytoene synthase (pys) has been cloned from the unicellular cyanobacterium Synechocystis sp. PCC6803. The gene has been functionally expressed in Escherichia coli, where it directs the biosynthesis of phytoene from geranylgeranyl pyrophosphate (GGPP). Analysis of the sequence of the Synechocystis pys protein deduced from the gene sequence shows that it is highly homologous to the tomato and Synechococcus phytoene synthases and shows conserved domains with other bacterial phytoene synthase enzymes. The pys gene starts 60 nucleotides downstream of the pds gene (which codes for phytoene desaturase). However, it seems to be transcribed mainly from its own promoters, because insertions that disrupt the pds gene do not affect significantly the expression of the pys gene.

Alkyl and Aryl Transferases↗

Nucleotide sequence of the nar beta gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea.

The nucleotide sequence of the structural gene of nitrate reductase (n ar beta) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The nar beta gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases.

Alcaligenes↗

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119.

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.

Amino Acid Sequence↗

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.

Biotechnology↗