Oscillations of the NAD(P)H pool size and of the redox state of a cytochrome b during dark respiration of the blue-green alga, Anacystis nidulans

Oscillations of the oxygen uptake rate of the blue-green alga (cyanobacterium) Anacystis nidulans were induced by light pulses. The pool size of NAD(P)H and the redox state of a cytochrome b showed oscillations of similar shape and frequency. Phase diagrams revealed that these three oscillations were presumably linked. The cytochrome b should be a part of the respiratory chain of this blue-green alga. The oscillations were inducible only in a limited physiological state of the alga.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Effect of Growth Temperature on the Lipid and Fatty Acid Composition, and the Dependence on Temperature of Light-induced Redox Reactions of Cytochrome f and of Light Energy Redistribution in the Thermophilic Blue-Green Alga Synechococcus lividus

The thermophilic blue-green alga Synechococcus lividus was grown at 55 and 38 C. Arrhenius plots of the transient reduction of cytochrome during actinic illumination with light that excited both pigment systems revealed breaks near 43 and 26 C for cells grown at 55 C. In cells grown at 38 C these breaks occurred near 37 and 28 C, respectively. The shift from pigment state 1 to state 2 measured by fluorescence transients also showed characteristic breaks in the Arrhenius plots at 44 C for cells grown at 55 C and at 37 to 38 C and possibly at 25 C for cells grown at 38 C. The break points in the Arrhenius plots for the state shift as well as for the cytochrome f reduction are discussed in relation to phase transitions of thylakoid membrane lipids as studied by the temperature dependence of chlorophyll a fluorescence.     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

Chilling-Susceptibility of the Blue-Green Alga Anacystis nidulans: III. LIPID PHASE OF CYTOPLASMIC MEMBRANE

The lipid phase of cytoplasmic membrane was studied by freeze-fracture electron microscopy in the chilling-susceptible blue-green alga, Anacystis nidulans. At growth temperatures, intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane, whereas, at chilling temperatures, the fracture faces were composed of particle-free and particle-containing regions. These findings indicate that lipids of the cytoplasmic membrane were in the liquid-crystalline state at the growth temperatures and in the phase-separation state at the chilling temperatures. Temperatures for the onset of phase separation were 5 and 16°C in cells grown at 28 and 38°C, respectively.     

Plastoquinone as a common link between photosynthesis and respiration in a blue-green alga

The role of plastoquinone in a thermophilic blue-green alga, Shynechococcus sp., was studied by measuring reduction kinetics of cytochrome 553 which was oxidized with red flash preferentially exciting photosystem I. Sensitivity of the cytochrome reduction to DBMIB indicates that cytochrome 553 accepts electrons from reduced plastoquinone. Plastoquinone is in turn reduced in cells without electrons from photosystem II, since DCMU, which inhibited methyl viologen photoreduction more strongly than DBMIB, failed to affect the cytochrome reduction. Participation of cyclic electron transport around photosystem I in cytochrome reduction in the presence of DCMU was excluded, because methyl viologen and antimycin A had no effect on the cytochrome kinetics. On the other hand, electron donation from endogenous substrates to plastoquinone was suggested from decreases in rate of the cytochrome reduction by dark starvation of cells and also from restoration of fast reduction kinetics by the addition of exogenous substrates to or by reillumination of starved cells.KCN, which completely suppressed respiratory O₂-uptake, induced a marked acceleration of the cytochrome reduction in starved cells. The poison was less or not effective in stimulating the cytochrome reduction in more extensively starved or reilluminated cells.Results indicate that plastoquinone is functioning not only in the photosynthetic but also in the respiratory electron transport chain, thereby forming a common link between the two energy conservation systems of the blue-green alga.

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Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

Heterotrophic Growth of Blue-Green Algae in Dim Light

A unicellular blue-green alga, Agmenellum quadruplicatum, and a filamentous blue-green alga, Lyngbya lagerheimíi, were grown heterotrophically in dim light with glucose as major source of carbon and possibly energy. The dim-light conditions did not support autotrophic growth. The two blue-green algae appeared to have the same metabolic block, namely an incomplete tricarboxylic acid cycle, as has been found in other obligately phototrophic blue-green algae. Under dim-light conditions, glucose made a greater contribution to cell constituents (amino acids) of A. quadruplicatum and L. lagerheimii than under high-light conditions.     

The influence of organic substrates and inhibitors on the induction of a respiratory oscillation in the cyanobacterium Anacystis nidulans

The rate of respiratory oxygen uptake of the cyanobacterium (blue-green alga) Anacystis nidulans oscillated under certain physiological conditions after light pulses or after addition of sodium acetate. The oscillation started either by the photosynthetic inhibition of respiration or by the stimulation of oxygen uptake caused by sodium acetate. The photosynthetic inhibition of respiration decreased the rate of oxygen uptake to about 20% of the rate in the dark.Starved cells (48 h dark) had lost the inducibility of the oscillation. In starved as well as in non-starved cells oscillations were inducible in the presence of fructose or glucose. Well developed oscillations were not promoted further. All other substances tested as substrates did not restore the inducibility of the oscillation in starved cells. The induction of the oscillation was inhibited by iodoacetamide (0.1 mM), p-hydroxymercuribenzoate (0.1 mM) and sodium fluoride (100 mM). It is suggested that a flow from glyceraldehyde-3-phosphate to the incomplete tricarboxylic acid cycle is a prerequisite for the oscillation.     

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).     

Phycochromes b and d: their occurrence in some phycoerythrocyanin-containing blue-green algae (cyanobacteria)

Abstract
Phycochromes b and d, two types of photoreversibly photochromic pigments previously extracted from the blue-green alga Tolypothrix distorta , which contains phycoerythrocyanin, have now been found in three Anabaena strains also containing phycoerythrocyanin. Tests for the presence of phycochromes b and d in a number of blue-green algae lacking phycoerythrocyanin have been negative. The possibility that phycochrome b-type absorbance changes are due to changes in the α-subunit of phycoerythrocyanin is discussed.     

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H⁺/e⁻ stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.     

Chromatographic separation of photosystems I and II from the thylakoid membrane isolated from a thermophilic blue-green alga

The thylakoid membrane of a thermophilic blue-green alga, Synechococcussp., was separated into four chlorophyll-containing fractionsby a single chromatographic manipulation with a diethylaminoethyl-cellulosecolumn after digitonin treatment. Photosystems I and II, orchlorophyll a forms, were unevenly distributed among the fourfractions, which were designated F-1, F-2, F-3 and F-4 in theorder of elution from the column. F-1 has a simple composition of the chlorophyll a form and totallylacks photochemical activity. This fraction may be an antennachlorophyll a-protein in the blue-green alga. F-2 is rich inshorter wavelength chlorophyll a forms and shows the three-bandedfluorescence emission spectrum characteristic of photosystemII at liquid nitrogen temperature. This fraction is highly activein 2,6-dichloroindophenol photoreduction and contains one photooxidizablecytochrome b₅₅₉ per 50–100 chlorophyll a, whereas theP-700 content is as low as one P-700 per 2,000 chlorophyll a.Thus, F-2 represents photosystem II in a highly purified state.F-3 is rich in photosystem I, since this fraction is inactivein 2,6-dichloroindophenol photoreduction, and contains one P-700per 200 chlorophyll a and smaller amounts of cytochrome b₅₅₉.Longer wavelength chlorophyll a forms are abundant and a peakat 730 nm is the most prominent in the low-temperature fluorescencespectrum in this fraction. F-4, which consists of larger membranefragments shows spectral and photochemical features similarto those of F-3. (Received August 8, 1979; )     

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.     

Estimation of chlorophyll a distribution in the photosynthetic pigment systems I and II of the blue-green alga Anabaena variabilis.

Chlorophyll a distribution in pigment systems I and II was estimated with the blue-green alga Anabaena variabilis by two methods: first, with intact cells using delayed light emission as an index reaction; second, by measuring the 2,6-dichlorophenolindophenol-Hill reaction and the cytochrome c photooxidation in membrane fragments. The first estimation indicated that 0.053+/-0.014 of total chlorophyll a functions as a component of pigment system II, and the second method 0.086+/-0.012. Though the values were somewhat different in the two methods, both estimations indicated that pigment system II chlorophyll a occupies a very small fraction of total chlorophyll a.     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.     

The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions

This review focuses on the essential role played by the green alga Chlamydomonas reinhardtii in revealing both the mechanism and the physiological consequences of state transitions. Two aspects are considered. The first is the role of the cytochrome b6f complex in regulating state transitions, in light of the recently obtained 3D structure. The second is the switch between linear and cyclic electron flow that follows state transitions in Chlamydomonas. Structural and dynamic elements that might be involved in such a switch, as well as its consequences on the energetic metabolism, are discussed.     

Immunochemistry on cryptomonad biliproteins

A survey is made of the immunochemical behavior of four of the six known types of cryptomonad biliproteins: phycocyanins 612 and 645 and phycoerythrins 545 and 566. They were compared both among themselves and to selected biliproteins isolated from blue-green and red algae. All the cryptomonad biliproteins were shown to be closely related to each other by Ouchterlony double diffusion technics. An antigenic relationship among all the cryptomonad biliproteins and B-phycoerythrin (red alga) and C-phycoerythrin (blue-green alga) was established. Only a very marginal cross-reactivity was found between C-phycocyanin (blue-green algae) and the cryptomonad biliproteins. These results suggest a common ancestor for the photosynthetic units of all three biliprotein-containing phyla.