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)     

Glucolipid Synthesis Activities in Cytoplasmic and Thylakoid Membranes from the Cyanobacterium Anacystis nidulans

Cytoplasmic membranes (plasma membranes), thylakoid membranesand cell walls prepared from the cyanobacterium, Anacystis nidulans,were compared for UDP-glucose: l,2-diacylglycerol glucosyltransferaseactivity. When 1,2-dipalmitoylglycerol was added as a glucosylacceptor, both cytoplasmic membranes and thylakoid membranesincorporated glucose from UDP-glucose into monoglucosyl diacylglycerol,but the cell walls containing the outer membranes did not. Thecytoplasmic membranes incorporated about twice as much glucoseas the thylakoid membranes on a protein basis. These observationssuggest that in A. nidulans the UDP-glucose: 1,2-diacylglycerolglucosyltransferase participating in glucolipid biosynthesisis located in both cytoplasmic and thylakoid membranes, butnot in the outer membrane. ¹Solar Energy Research Group, The Institute of Physical andChemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan. (Received November 21, 1985; Accepted January 27, 1986)     

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.     

Thermal Analysis of Membrane Lipids from the Blue-Green Algae Anacystis nidulans and Anabaena variabilis

Differential scanning calorimetry was used on aqueous dispersionsof lipids extracted from the blue-green algae Anacystis nidulansand Anabaena variabilis. The reversible curves, exothermic oncooling and endothermic on heating, indicated that thermotrophicphase transition of the membrane lipids took place over a widerange of temperatures. These results suggest that the lipidsof the thylakoid membranes in these algae were in the liquidcrystalline state at the growth temperature, entered the phaseseparation state at about room temperature and went into thegel state below the freezing point. The temperature for theonset of phase separation was dependent on both the growth temperatureand the growth stage of the culture. ³Present address: Solar Energy Research Group, Institute ofPhysical and Chemical Research (RIKEN), Wako-shi, Saitama 351,Japan. (Received December 20, 1982; Accepted March 24, 1983)     

Phase Transition Temperatures Determined for Chloroplast Thylakoid Membranes and for Liposomes Prepared from Charged Lipids Extracted from Thylakoid Membranes of Higher Plants

Lipid phase separation temperatures of intact thylakoid membranesfrom a number of chilling sensitive plants were measured usingchlorophyll a as the intrinsic fluorescent probe. The phospho-and sulfolipids were extracted from the thylakoid lamellae ofthese plants and purified by silicic acid column and thin layerchromatographies. These separated lipids were eluted and recombinedto give a total charged anionic thylakoid lipid fraction thatwas used to prepare liposomes containing purified chlorophylla as the fluorescent probe. The phase separation temperaturesof these liposomes were compared to phase separation temperaturesin intact thylakoid membranes isolated from the same plants. The chilling-sensitive plants—corn, pepper, tomato andwater hyacinth — showed phase separation temperaturesranging from 9 to 19°C for both the liposomes and the thylakoidmembranes. In addition, low temperature phase separations wereseen from –21 to –27°C. Mimulus, which is notas chilling sensitive as the former plants, had a phase separationtemperature near 0 to 2.5°C and at –27°C. In general,there was a good agreement between the phase separation temperaturesof intact thylakoids and the purified anionic lipid fractionextracted from these thylakoids. Similar results were obtained using either trans-parinaric acidor chlorophyll a as the fluorescent probe in liposomes madefrom anionic thylakoid lipids or in liposomes prepared frompure dimyristoyl phosphatidyl choline, distearoyl phosphatidylcholine, or mixtures of equal amounts of these phospholipids. ¹ CIW-DPB Publication # 728. ³ Present address: Laboratory of Experimental Physics, Departmentof Biophysics, State University of Utrecht, Princetonplein 5,Utrecht, The Netherlands. (Received January 18, 1981; Accepted July 2, 1981)     

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.     

Primary Role of the Cytoplasmic Membrane in Thermal Acclimation Evidenced in Nitrate-Starved Cells of the Blue-Green Alga, Anacystis nidulans

The lipid phase transition of the cytoplasmic membrane and the chilling susceptibility were studied in nitrate-starved Anacystis nidulans cells. Nitrate starvation resulted in the disappearance of the thylakoid membrane system, without any effect on chilling susceptibility. The chilling susceptibility of the algal cells depended on the growth temperature. Temperatures of lipid phase transitions of the cytoplasmic membranes were detected by chilling-induced spectral changes in the carotenoid region, in vivo. These values were identical to those of cultures containing intact thylakoid systems. Our results suggest that cytoplasmic membrane plays a determinative role in the thermal acclimation of the alga cells.     

Changes in the Polypeptide Composition of the Cytoplasmic Membrane in the Cyanobacterium Anacystis nidulans during Adaptation to Low CO2 Conditions

When cells of Anacystis nidulans grown under high CO₂ conditions(3%) were transferred to low CO₂ conditions (0.05%), their abilityto transport extracellular inorganic carbon (C_i) into the cellsincreased severalfold. There was a marked increase of 42-kDapolypeptide in the cytoplasmic membranes during the adaptationto low CO₂ conditions, while no changes were observed in thepolypeptide compositions of the thylakoid membranes and cellwalls. The results suggested that the increase of the 42-kDapolypeptide during adaptation is involved in the increased abilityto transport C_i (Received January 28, 1985; Accepted May 30, 1985)     

Isolation and Characterization of the Cytoplasmic Membranes from the Blue-Green Alga (Cyanobacterium) Anacystis nidulans

Cells of the blue-green alga (cyanobacterium) Anacyslis nidulanswere disintegrated, and their thylakoid membranes and cytoplasmicmembranes were isolated by floatation centrifugation on a sucrosedensity gradient. Electron micrographs revealed that the cytoplasmicmembranes formed single closed vesicles having diameters of200–400 nm. These membranes contained xanthophylls asthe major constituent pigments and rß-carotene andchlorophyll a as very minor ones. The major peaks in their absorptionspectra were due to carotenoids at 435, 455 and 487 nm, witha minor one due to chlorophyll a at 673 nm. These findings areconsistent with the yellow color of the cytoplasmic membranes.The absorption spectrum of the membranes in the carotenoid regionwas markedly affected by temperature: with a decrease in temperature,the peaks at 455 and 487 nm diminished and a new peak appearedat 390 nm. (Received February 12, 1983; Accepted June 20, 1983)     

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)     

Temperature dependence of chlorophyll {alpha} fluorescence in lettuce and spinach chloroplasts at sub-zero temperatures

The temperature dependence of chlorophyll fluorescence wasmeasured in spinach and lettuce chloroplasts at sub-zero temperaturesin the presence of 50% ethylene glycol. In the presence of 5mM Mg²⁺, a fluorescence maximum appeared at –31?C in boththe spinach and lettuce chloroplasts, while in the presenceof only 5 mM Na⁺ as cations the maximum shifted to –20?Cin the spinach chloroplasts and to –11?C in the lettucechloroplasts. Since the occurrence of a maximum in the temperatureversus fluorescence curve is an indication for the transitionof the physical phase of thylakoid membrane lipids between theliquid crystalline and the phase-separation state (16, 18),these findings suggest that the (major) phase transition ofmembrane lipids occurs at these low temperatures in chloroplastsof higher plants and also that the phase transition temperatureis markedly lowered by the presence of divalent cations. Ethylene glycol at a concentration of 50% had almost no effecton the temperature dependence of chlorophyll fluorescence ina lamellar membrane preparation of Anabaena variabilis. In awater suspension of dimyristoylphosphatidylcholine, the additionof ethylene glycol to 50% did not alter the characteristic featureof the temperature dependence of fluorescence of 1-anilinonaphthalene-8-sulfonate.These findings suggest that 50% ethylene glycol does not affectthe temperature of the transition of the physical phase of membranelipids. ¹ C.I.W.-D.P.B. Publication No. 592. ² Present Address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Hongo 113, Tokyo, Japan. (Received June 22, 1977; )     

Studies on the Hill reaction of membrane fragments of blue-green algae V. A correlation between the solubilization of a photochemically active chromoprotein (ACP) and the inactivation of photosystem II activity in membrane fragments of Anabaena cylindrica

The relationship between a photochemically active chromoprotein(ACP) (cf. ref. 1) and photosystem II was investigated withmembrane fragments of Anabaena cylindrica, A. variabilis andP. boryaman. ACP was solubilized from membrane fragments of A. cylindricabut not from those of A. variabilis or P. boryanum, when themembrane fragments had been incubated in a dilute buffer andhad lost their Hill or photosystem II activity. In A. cylindrica,ACP-solubilization always occurred, independent of photosystemII inactivation, on incubation of the membrane fragments inmedia without PEG. However, the amount of ACP solubilizationaccompanying photosystem II inactivation was twice that withoutphotosystem II inactivation. The increase in ACP solubilizationaccompanying photosystem II inactivation. The kinetics resembledthose for the decrease in 695 nm fluorescence emitted by membranefragments at — 196?C (cf. 2). The ACP solubilized independent of photosystem II inactivationwas assumed to have been released during disruption of intactcells in the preparation of membrane fragments. The slow ACPsolubilization upon the inactivation of photosystem II was attributedto the pigment being bound to membranes. We assume that thephoto-reactive component of ACP, P690 (cf. 3, 4), is releasedfrom the membranes during photosystem II inactivation, and thatP690 is a component of photosystem II which emits the 695 nmfluorescence at — 196?C. (Received March 22, 1974; )     

Ontogenetic Change of the PSI- and PSII-Distribution in the Thylakoid System of Euglena gracilis

The thylakoid membranes of isolated Euglena chloroplasts wereseparated into two fractions by aqueous two-phase-partitioning(mixture of dextran 500 and poly(ethylene glycol) 4000) followingpress disruption. These two fractions differ in many respectsduring most of the cell cycle of this alga in comparison withthe thylakoid characteristics of higher plants or green algae.The amount of thylakoid membranes with separation characteristicscomparable with inside-out-vesicles of higher plant chloroplastschanges depending on the cell cycle stage of Euglena gracilis.Photosystems II and I are not restricted to one fraction. Boththylakoid membrane fractions evolve oxygen photosynthetically.When chloroplast differentiation in Euglena gracilis is complete(i.e. at the end of the light-time) the composition and thephotosynthetic efficiency of the two thylakoid fractions aregenerally equal. Photosystem I-related LHCI is present in bothfractions. Photosystem II-related CP29, however, was only detectedin unfractionated thylakoid membranes. The implications forthylakoid organization in Euglena chloroplasts are discussed. Key words: Euglena gracilis, photosystem I, photosystem II, stacking, thylakoids     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

Streptomycin- and Penicillin-induced Inhibition of Growth and Pigment Production in Blue-green Algae and Production of Strains of Anacystis nidulans Resistant to these Antibiotics

Relatively low concentrations of streptomycin inhibited thegrowth of Anacystis nidulans and Anabaena variabilis. The productionof chlorophyll, carotenoid, and phycocyanin pigments in thesealgae was significantly but reversibly inhibited by streptomycin.Strains of Anacystis nidulans that are resistant to streptomycinand penicillin were produced by serially subculturing the algain gradually increasing concentrations of the respective antibiotic.These strains seem stable. Evidence which suggests that theresistant strains originate primarily by mutation is presented.     

Spectral microscopic imaging of heterocysts and vegetative cells in two filamentous cyanobacteria based on spontaneous Raman scattering and photoluminescence by 976 nm excitation

Photosynthetic pigment-protein complexes are highly concentrated in thylakoid membranes of chloroplasts and cyanobacteria that emit strong autofluorescence (mainly 600–800?nm). In Raman scattering microscopy that enables imaging of pigment concentrations of thylakoid membranes, near infrared laser excitation at 1064?nm or visible laser excitation at 488–532?nm has been often employed in order to avoid the autofluorescence. Here we explored a new approach to Raman imaging of thylakoid membranes by using excitation wavelength of 976?nm. Two types of differentiated cells, heterocysts and vegetative cells, in two diazotrophic filamentous cyanobacteria, Anabaena variabilis, and Rivularia M-261, were characterized. Relative Raman scattering intensities of phycobilisomes of the heterocyst in comparison with the nearest vegetative cells of Rivularia remained at a significantly higher level than those of A. variabilis. It was also found that the 976?nm excitation induces photoluminescence around 1017–1175?nm from the two cyanobacteria, green alga (Parachlorella kessleri) and plant (Arabidopsis thaliana). We propose that this photoluminescence can be used as an index of concentration of chlorophyll a that has relatively small Raman scattering cross-sections. The Rivularia heterocysts that we analyzed were clearly classified into at least two subgroups based on the Chla-associated photoluminescence and carotenoid Raman bands, indicating two physiologically distinct states in the development or aging of the terminal heterocyst.     

Relationship between growth temperature of Anacystis nidulans and phase transition temperature of its thylakoid membranes

The temperatures of the lipid phase transition at which the solid phase disappears were determined by using the X-ray diffraction method in thylakoid membranes of the blue-green alga, Anacystis nidulans. The temperatures were determined as 26 and 16°C for cells grown at 38 and 28°C, respectively.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer