Arachidonic acid production by the red alga Porphyridium cruentum

The single-celled alga, Porphyridium cruentum, was assessed by means of chromatographic separation and mass spectral analysis of its fatty acids to be a potentially competetive source of arachidonic (5,8,11,14-eicosatetraenoic) acid. Models for both cell growth and production of the prostaglandin precursor at various temperatures and light intensities are presented. Increasing the light intensity within the range 1700-8000 lux increases the cell growth rate without affecting the arachidonic acid yield per cell; increasing the cultivation temperature from 18 degrees C to ca. 32 degrees C lowers the yield of arachidonic acid per cell but increases the rate of its production per unit volume and time. The increase of the weight ratio of arachidonic:palmitic acids at low temperatures is interpreted as a means of controlling the microviscosities of cellular membranes. In addition, the arachidonic acid content of cells decreases with the culture's age, despite increases in unit cell dry weight. The maximum rate of 0.46 mg arachidonic acid L(-1) h(-1) was calculated by means of the model to occur at ca. 32 degrees C and 8000 lux in liquid cultures of 12 x 10(9) cells/L. Estimates of the cost of producing arachidonic acid by means of this alga range from $0.15/g to $1.00/g of arachidonic acid. Cells grown at 18 degrees C in the presence of 0.3% linoleic acid swelled and produced gorlic (13-cyclopent-2-enyltridec-6-enoic) acid and another compound not normally observed. An estimated threefold increase of arachidonic acid content also occurred, but no significant lipogenesis was induced at 23 degrees C in the presence of 1% kerosene or 0.3% palmitic, stearic, oleic, or linoleic acids.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Phototaxis of the unicellular red alga Porphyridium cruentum was studied by staining the slime tracks of individual cells as well as with the aid of a population method. Because of the increased straightness of the movement the mean linear velocity of a unilaterally illuminated population exceeds considerably that of an only photokinetically stimulated one. In white light the phototactic reaction is saturated already at 100 lx. The zero threshold lies at about 1 lx. Spectral sensitivity curves of phototaxis obtained at high photon fluence rates (>=10^–11 mol cm^–2 s^–1) display two main peaks which shift against each other at intermediate irradiances and, finally, form a single maximum in the blue range (443 nm) at low photon fluence rates (10^–12 and 10^–13 mol cm^–2 s^–1). Photon fluence rate-response curves reveal that supraoptimal irradiances decrease the phototactic reaction, especially in the range of the highest sensitivity of the cells. The action spectrum of phototaxis was calculated on the basis of the photon fluence rate-response curves. It shows a maximum at 443 nm and shoulder at 416 nm and between 467 and 477 nm. Wavelengths longer than 540 nm are phototactically inactive even at very high irradiances (25 W m^–2). Thus, this is the first phototactic action spectrum of a biliprotein-containing organism which does not indicate the participation of biliproteins in the absorption of phototactically active light. DCMU and potassium iodide have no specific effects on phototaxis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Photophobic reactions of the red alga Porphyridium cruentum have been studied by single cell observations and by population experiments with the light trap method. In white light traps photoaccumulation is saturated at about 6000 lx. Experiments with monochromatic light demonstrate the necessity of carefully separating the three basic light reactions, viz. phototaxis, photokinesis and photophobic response by an appropriate experimental set-up: In single-beam experiments trap wavelengths >695 nm cause photodispersal which is not due to photophobic entrance reactions, but is exclusively due to the positive photokinetic effect of the trap light. This photodispersal can be cancelled by a photokinetically active background light. In the short wavelength range not only photokinesis, but also phototaxis interferes with photophobic reactions thus affecting the density of photoaccumulations in the light trap. Phototactic and photokinetic interference can be avoided by a blue background light. The action spectrum measured this way indicates activity of photosystem I and photosystem II pigments in the perception of the step-down photophobic stimulus. Varying the wavelength of the background light at constant trap light absorbed mainly by photosystem I or photosystem II respectively, efficient spill-over of light energy from photosystem II to the light reaction of photosystem I could be demonstrated. From the results it is concluded that phobic reactions are induced by a decrease of the electron flow rate in the linear electron transport chain.     

A photosystem II-phycobilisome preparation from the red alga, Porphyridium cruentum: oxygen evolution, ultrastructure, and polypeptide resolution

The photosystem II-phycobilisome preparation, isolated by lauryldimethyl amine oxide treatment, had a greatly reduced chlorophyll content, with an average ratio of 90 chlorophyll a/phycobilisome as compared to approximately 1200 Chl/phycobilisome in unfractionated thylakoids. P700 was not detected in the particles. By electron microscopy the preparations were relatively homogeneous and were generally devoid of chloroplast membranes. In negatively stained preparations phycobilisome particles were seen often in clusters of two and three, probably due to retention of hydrophobic thylakoid fragments. The preparation was deficient in photosystem I chlorophyll complexes, but enriched in polypeptides of 85 to 92, approximately 43, and approximately 26 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 43- and 26-kDa polypeptides are attributable to the PS II core and the oxygen-evolving complex, respectively.     

Photomovement of the red alga Porphyridium cruentum (Ag.) naegeli I. Photokinesis

Photokinesis of the red alga Porphyridium cruentum was studied with the aid of a population method. Because of the slow spreading velocity (0.35 m/min) the duration of the experiments was 7 days in general. According to the white light illuminance-response curve the zero threshold of photokinesis lies below 10 lx and the optimum around 10,000 lx. With further increasing illuminance the photokinetic effect decreases, reaching zero at about 100,000 lx. The action spectrum indicates that the photokinetically active radiation is absorbed by photosynthetic pigments, namely the biliproteins B-phycoerythrin, R-phycocyanin and allo-phycocyanin, as well as by chlorophyl a, although the photokinetic effect of blue light is relatively low. From the action spectrum and the results of inhibitor experiments with DCMU, DBMIB and DSPD it is concluded that the photokinetic effect is due to an additional ATP supply from non-cyclic and/or pseudo-cyclic photophosphorylation to the motor apparatus.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) - DSPD Disalicylidenepropanediamine-(1,3)     

The covalent linkage of protein to carbohydrate in the extracellular protein-polysaccharide from the red alga Porphyridium cruentum.

The extracellular anionic polysaccharide isolated from cultures of a unicellular red alga, Porphyridium cruentum, contains a small amount of protein after extensive purification. The polysaccharide and protein are recovered in the same fraction after isopycnic CsCl-density-gradient centrifugation in 4M-guanidinium chloride, under conditions designed to separate proteins from polysaccharide. The peptide portion of the protein-polysaccharide is released from the polysaccharide by alkali under conditions for beta-elimination. The released peptide is non-diffusible, but in can be separated from the polysaccharide by precipitation of the polysaccharide as the cetylpyridinium complex. Under conditions for beta-elimination of certain O-glycosidic carbohydrate-protein linkages, selective destruction of serine and threonine occurs. The addition of a reducing agent to the alkali mixture produces a selective increase in alanine and alpha-aminobutyric acid. Addition of a tritiated reducing agent to the alkali mixture produces radioactive alanine and alpha-aminobutyric acid, and xylitol as the only sugar alcohol. Similar results are obtained from glycopeptides isolated from partial acid hydrolysates. A macromolecular structure of the protein-polysaccharide is suggested by a comparison of the intrinsic viscosity of material before and after treatment with alkali and proteolytic enzymes.     

Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ana A. Arteni and Lu-Ning Liu equally contributed to the work.     

Effects of light intensity on the growth rate of the red alga Porphyridium cruentum

The red alga, Porphyridium cruentum, which is one of the potential sources of arachidonic acid, was cultured in batch and continuous vessels. The growth rates in batch cultures were correlated to the mean light intensity in the vessels, and the cell concentrations in continuous cultures were estimated by those results. The yield of arachidonic acid was about 1.2 g per 10¹² cell at cell concentrations ranging from 0.5 to 1.5 × 10¹⁰ cell/l and independent of the mean light intensity.     

The kinetics of photoinhibition and its recovery in the red alga Porphyridium cruentum

When Porphyridium cruentum cells were illuminated with high fluence rate between 1900 and 4800 mol photons m^-2s^-1, a decrease in the photosynthetic activity of the cells was observed. Within the time frame of 20 min, and under the fluence rates studied, the sum of photons to be absorbed by cells (mg of chlorophyll (Chl), sufficient to initiate photoinhibition was calculated to be 9235.8 mol. The minimal specific light absorption rate to initiate photoinhibition in P. cruentum ranges between 2.29 and 4.26 mol photons s^-1 mg^-1 chl.a. There was a linear relationship between the specific rate of photoinhibition and the specific light absorption rate. A photon number of 2.56×10⁴ mol mg^-1 chl.a photoinhibited photosynthesis instantaneously. At 15°C, no photoinhibitory effect was observed at 2300 mol photons m^-2 s^-1 even after 45 min of illumination. At the other extreme of 35°C, 84% inhibition of photosynthetic activity was observed within 10 min of exposure to 2300 mol photons m^-2 s^-1. Between 20 and 30°C, the photoinhibitory effect was comparable. Photoinhibited P. cruentum cells recovered readily when transferred to low light (90 mol photons m^-2 s^-1) and darkness, and the specific rate of recovery was independent of the light intensity to which the cells were exposed, during the photoinhibitory treatment.Abbreviations Chlorophyll QL, specific light absorption rate Publication No. 28 of the Microalgal Biotechnology Laboratory     

Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis

Photosystem (PS) II particles retaining a high rate of O₂ evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20–45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-β-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e^?) mol^?1 (Chl) s^?1] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu²⁺) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl₂ and CaCl₂, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl₂, NaCl, CaCl₂, and ?-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies.     

Inhibition of the oxygen-evolving complex of photosystem II and depletion of extrinsic polypeptides by nickel

The toxic effect of Ni²⁺ on photosynthetic electron transport was studied in a photosystem II submembrane fraction. It was shown that Ni²⁺ strongly inhibits oxygen evolution in the millimolar range of concentration. The inhibition was insensitive to NaCl but significantly decreased in the presence of CaCl₂. Maximal chlorophyll fluorescence, together with variable fluorescence, maximal quantum yield of photosystem II, and flash-induced fluorescence decays were all significantly declined by Ni²⁺. Further, the extrinsic polypeptides of 16 and 24 kDa associated with the oxygen-evolving complex of photosystem II were depleted following Ni²⁺ treatment. It was deduced that interaction of Ni²⁺ with these polypeptides caused a conformational change that induced their release together with Ca²⁺ from the oxygen-evolving complex of photosystem II with consequent inhibition of the electron transport activity.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.     

Mechanism of the light state transition in photosynthesis. II. Analysis of phosphorylated polypeptides in the red alga,Porphyridium cruentum

The possible involvement of a reversible protein phosphorylation event in the regulation of excitation energy distribution was studied in the red alga Porphyridium cruentum. Whole cells were incubated in phosphate-depleted growth medium containing carrier-free [³²P]orthophosphate for several hours to label the intracellular phosphate pools, and they were then converted to State 1 or State 2 by illumination using blue or green light, respectively. The successful transition to State 1 or State 2 was verified by 77 K fluorescence spectroscopy of the chlorophyll emission and the cells were then denatured using either acetone, trichloroacetic acid or boiling detergent. The whole cell lysates were solubilized, treated with RNAase, and analyzed for phosphoproteins by SDS-polyacrylamide gel electrophoresis. At least twelve polypeptides were found to be phosphorylated but no changes in specific radioactivity of the polypeptides were detected when samples from cells in State 1 and State 2 were compared. We conclude that a reversible protein phosphorylation event is not implicated in the state transition in P. cruentum. A model is presented for the mechanism of the light state transition in organisms that contain phycobilisomes which is different from the mechanism of energy distribution proposed for higher plants.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Isolation and characterization of the oxygen-evolving photosystem II complex from the economical red alga <Emphasis Type="Italic">Bangia fusco-purpurea</Emphasis>

The highly pure and active photosystem II (PSII) complex was isolated from Bangia fusco-purpurea (Dillw) Lyngb., an important economic red alga in China, through two steps of sucrose density gradient ultracentrifugation and characterized by the room absorption and fluorescence emission spectra, DCIP (2,6-dichloroindophenol) reduction, and oxygen evolution rates. The PSII complex from B. fusco-purpurea had the characteristic absorption peaks of chlorophyll (Chl) a (436 and 676 nm) and typical fluorescence emission peak at 685 nm (Ex = 436 nm). Moreover, the acquired PSII complex displayed high oxygen evolution (139 μmol O₂/(mg Chl h) in the presence of 2.5 mM 2,6-dimethybenzoqinone as an artificial acceptor and was active in photoreduction of DCIP (2,6-dichloroindophenol) by DPC (1,5-diphenylcarbazide) at 163 U/(mg Chl a h). SDS-PAGE also suggested that the purified PSII complex contained four intrinsic proteins (D1, D2, CP43, and CP47) and four extrinsic proteins (33-kD protein, 20-kD protein, cyt c-550, and 14-kD protein).     

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.