Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP⁺ or ferricyanide. P/2e⁻ ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.

Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C₅₅₄ and C₅₄₉ and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C₅₅₄ or plastocyanin, and may be a coupling factor.

Analysis of the well-washed membranes by low temperature (77°K) difference spectrophotometry reveals the presence of cytochrome b₆ and a bound form of cytochrome C₅₅₄ in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C₅₅₄, relative to cytochrome b₆ is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C₅₅₄, which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b₆: chlorophyll a was found to be 1:500.

Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C₅₄₉.

     

Enhanced Model for Photophobic Responses of the Blue-Green Alga, Phormidium uncinatum

An improved computer-based mathematical model of photophobicresponses in the blue-green alga, Phormidium uncinatum, hasbeen used to simulate the behavior of the alga under variouslight and dark conditions. Under high light intensities, themodel predicts the induction of step-up phobic responses. Thisnovel result could be varified experimentally in vivo. Appropriatefeatures have been incorporated to simulate the effects of ionophoresand the application of external electrical fields; results ofthe computer simulation and in vivo experiments are compared.Several parameters of the model have been varied to specifythe range in which phobic responses are permitted. These parametersallow conclusions to be drawn on the functional correlates inthe living organism. (Received July 13, 1982; Accepted October 9, 1982)     

Preparation of Metabolically Active Staphylococcus aureus Protoplasts by Use of the Aeromonas hydrophila Lytic Enzyme

Stable, metabolically active protoplasts of Staphylococcus aureus have been prepared by the use of a staphylolytic enzyme produced by Aeromonas hydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids, protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37 M sodium succinate, or 0.37 M sodium sulfate, have been shown to be comparable with the same parameters in intact cells under the same conditions.     

Cylindrical Phycobilisomes from a Blue-Green Alga, Anabaena variabilis

Cylindrical 52.5-nm-long phycobilisomes were observed in Anabaenavariabilis, differing from the generally accepted hemidiscoidalmorphology. The central part of such a phycobilisome has a network-likefine structure of slightly greater diameter (16 nm) than theconnected end parts of stacked-disc structure (12 nm in diameter).On the basis of this morphology, the molecular mass of thisphycobilisome was calculated to be 3.27?10⁶, about 60% of whichis accounted for by phycocyanin with the rest being due to allophycocyanin.Separately prepared 23 S allophycocyanin particles with a molecularmass of 1.13?10⁶ have the dimensions (16?23 nm) and network-likefine structure similar to the central part of phycobilisomes,while an aggregate form of phycocyanin (18 S) has a fine structureof stacked discs similar to the connecting end part of phycobilisomes,suggesting that the central part constitutes the core at whichthese phycobilisomes attach to the thylakoid membranes. (Received June 5, 1982; Accepted September 21, 1982)     

Myxococcal predation of the cyanobacterium Phormidium luridum in aqueous environments

Two strains of Myxococcus xanthus, and a strain of Myxococcus fulvus were compared with respect to their ability to entrap and lyse trichomes of the cyanobacterium Phormidium luridum var. olivaceae. All of these isolates form colonial aggregates and spherules in either axenic culture with a tryptone-salts medium or in a mixed culture with viable cyanobacterial cells as the sole source of nutrients. Light microscopy showed evidence of swarming activity on the surface of all three myxococci with the accompanying formation of fruiting structures. Extended incubation of mixed cultures showed the myxococci to be capable of long-term control of the cyanobacterial population with predator-prey population cycling occurring on average every 9 days. Serial transfer of mixed cultures into either fresh autotrophic medium or cyanobacterial cultures of 10⁷ per ml showed the persistence of predatory activity. Myxococcal densities were shown to return repeatedly to initial virulent levels. Predator inoculum levels could be reduced to 50 cells per 100 ml in a cyanobacterial culture of 10⁷ per ml. These in vitro data enhance the potential of the myxococcus predatory colony as a biological control agent for in situ cyanobacteria.     

Crystalline Ferredoxin from a Blue-Green Alga, Nostoc sp.

Ferredoxin isolated from a blue-green alga, Nostoc sp., was purified and crystallized. The absorption spectrum of Nostoc ferredoxin had, in the oxidized state, peaks at 276, 331, 423, and 470 nm, a pattern characteristic of chloroplast-type ferredoxin. The 423:276 absorption ratio was 0.57. The midpoint oxidation-reduction potential of Nostoc ferredoxin was found to be –406 mV, at pH 7.5. Nostoc ferredoxin mediated the photoreduction of NADP by isolated Nostoc chromato-phores and spinach chloroplasts from which the native ferredoxin was removed. The molar ratio of Nostoc ferredoxin to chlorophyll a was about 1:50, a ratio higher than usually found in photosynthetic cells. The possible evolutionary significance of the properties of Nostoc ferredoxin compared with those of ferredoxins from other photosynthetic organisms is discussed.     

The photochemical and fluorescence properties of whole cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum.

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured. Photosynthesis (O2 evolution) in whole cells; Hill reaction (O2 evolution) with Fe(CN)63- and NADP as electron acceptors (Photosystem II and photosystem II + Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern. On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90% (10%) of the chlorophyll a, 90% (10%) of the carotenoids and 15% (85%) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments; they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction. The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20--40%) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion. The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component. The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

Phosphorus-Limited Growth of a Green Alga and a Blue-Green Alga

The phosphorus-limited growth kinetics of the chlorophyte Scenedesmus quadricauda and the cyanophyte Synechococcus Nägeli were studied by using batch and continuous culturing techniques. The steady-state phosphate transport capability and the phosphorus storage capacity is higher in S. Nägeli than in S. quadricauda. Synechococcus Nägeli can also deplete phosphate to much lower levels than can S. quadricauda. These results, along with their morphological characteristics, were used to construct partial physiological profiles for each organism. The profiles indicate that this unicellular cyanophyte (cyanobacterium) is better suited for growth in phosphorus-limited oligotrophic niches than is this chlorophyte (green alga).     

钝顶螺旋藻（Spirulina platensis）藻胆体Langmuir-Blodgett膜

从钝顶螺旋藻中分离制备完整藻胆体 ,然后滴加于空气 水界面上 ,应用LB膜技术制备藻胆体LB膜。结果表明 ,藻胆体在空气 水界面上具有很好的成膜性能。将藻胆体LB单层膜转移到刚揭开的云母表面 ,喷一层金 ,然后用扫描隧道显微镜观察。结果表明 ,藻胆体在Langmuir Blodgett膜中的排列方式与其在体内类囊体膜表面的排列方式类似 ,一排排聚集在一起 ,然后排列成膜。藻胆体的“核”吸附在云母表面 ,而藻胆体的“杆”伸向外面。由于钝顶螺旋藻易于规模化培养 ,藻胆体容易批量制备 ,加之藻胆体具有的独特的光物理、光化学特性和良好的成膜性能 ,以及本身就是纳米量级的颗粒 (5 0 70nm) ,预示着藻胆体在纳米光电子器件中具有很好的应用前景。     

Purification and Properties of Glycogen Isolated from a Blue-Green Alga, Nostoc muscorum

Alpha granules were isolated from a blue-green alga, Nostoc muscorum, in large quantities and high purity by sucrose density gradient centrifugation. Sodium deoxycholate was used to eliminate membrane contamination. Isolated alpha granules from this species have average dimension of 31 nm in width and 65 nm in length. Each alpha granule consists of two equal parts. Attempts to dissociate the intact granule under mild conditions into relatively large subunits did not succeed. Analytical centrifugation of the alpha granules yields a sedimentation coefficient of 265S (S(20, w)). Chemical analysis reveals that alpha granules contain highly branched polyglucosyl units with short external chain lengths.     

Endogenous Dark Respiration of the Blue-Green Alga, Plectonema boryanum

The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested.     

Phycochrome d, a New Photochromic Pigment from the Blue-Green Alga, Tolypothrix distorta

A new reversibly photochromic pigment, phycochrome d, has been found in extracts of the blue-green alga Tolypothrix distorta. This phycochrome exhibits an absorbance increase in the red region (maximum at about 650 nm) when irradiated with 650 nm light, and a corresponding absorbance decrease when irradiated with 610 nm light. The absorbance difference spectrum and action spectra for in vitro conversions were determined.     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum

The presence of Ca²⁺ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca²⁺ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl₂ in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca²⁺. This indicates that Ca²⁺ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca²⁺, discounting fatty acid mediation of the effects of Ca²⁺. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca²⁺.     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum.

The presence of Ca2+ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca2+ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl2 in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca2+. This indicates that Ca2+ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca2+, discounting fatty acid mediation of the effects of Ca2+. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca2+.     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.