A model of the phototactic reaction chain of the cyanobacterium Anabaena variabilis

The hypothesis has been proposed that in Anabaena variabilis the phototactic reaction sign is regulated by an unknown reaction sign reversal generator which is controlled by the intracellular level of singlet molecular oxygen (¹O₂). This hypothesis is supported by the following findings presented in this paper: Gassing with N₂ and Ar shifts the phototactic transition point at which the positive reaction becomes negative to higher fluence rates. Surprisingly this is true also for gassing with molecular oxygen ³O₂. Since ¹O₂ is produced in photosynthesis, the availability of external molecular oxygen seems not to be important. Apparently, a stream of any gas which is fast enough to remove ¹O₂ from the surface of the Anabaena trichomes decreases the internal ¹O₂ concentration and this way acts on the reaction sign reversal generator. Moreover, several carotenoids such as the water-soluble crocetin and preparations of solubilized -carotene, canthaxanthine and the C₃₀-ester ethyl--apo-8-carotenoate shift the transition point of phototaxis to higher fluence rates by about one order of magnitude. Several tested furan derivatives, such as dimethylfuran, diphenylisobenzofuran, and furfuryl ethanol, are either cytotoxic or not water-soluble at the concentrations necessary for an effective ¹O₂ quenching. Based one these results a model of the phototactic reaction chain of A. variabilis is proposed.Abbreviations DABCO 1,4-diazabicyclo(2.2.2)octane - DMF dimethylfuran - DPBF diphenylisobenzofuran Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis

The fluence rate dependence of the photobleaching in the cyanobacterium Anabaena variabilis was studied under physiological conditions. According to the in-vivo absorption spectra measured every day during the 5 d exposition the phycobiliproteins are more sensitive to high fluence rates than chlorophyll a. The carotenoids are least sensitive, so that a relative, but not an absolute increase in the carotenoid content occurred. At very high fluence rates exceeding about 50 Wm^-2 white light the organisms were photokilled after 5 d of irradiation. Measurements of the nitrate concentrations during the experiments have shown that nitrate was not the limiting factor in these experiments. Analysis of the photobleaching kinetics at 13.5 Wm^-2 white light revealed that after about 8 d the contents of all the pigments studied have reached a new, constant level. After exposure of the photobleached cyanobacteria to low irradiances repigmentation occurred. Thus, photobleaching is a light adaptation process and not simply a photodamage phenomenon. Studying the wavelength dependence of photobleaching at a constant photon fluence rate of 4·10^-8 mol cm^-2 s^-1 we found that the photobleaching of both phycobiliproteins and chlorophyll a was exclusively caused by wavelengths absorbed by the phycobiliproteins, mainly phycoerythrocaynin, and red light absorbed by short wavelength chlorophyll. Wavelengths <520 nm were ineffective.     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Isolation of mutants of the cyanobacterium,Anabaena variabilis,impaired in photoautotrophy

Mutants ofAnabaena variabilis Kütz. that have a decreased ability to grow photoautotrophically have been isolated by a modification of the techniques used to isolate auxotrophic mutants of that filamentous cyanobacterium, and have been stably propagated. Three mutants have a reduced content of phycocyanin and, as determined by in situ assays of partial reaction sequences of photosynthesis, an impairment in photosystem II. Three other strains, all of which appear to have a normal complement of carotenoids when grown heterotrophically, are sensitive to light.Abbreviations Used TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid, sodium salt - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, sodium salt - MV methylviologen - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DAB 3,3-diaminobenzidine - P-BQ p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fecy K-ferricyanide - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Reversible hydrogenase in Anabaena variabilis ATCC 29413

The presence and localization of a reversible hydrogenase in non-N₂-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO₂ for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s).     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at –35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)₆]^3- added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A₂₇₈/A₅₉₇=1.14. This procedure is faster and the yield higher than for previous procedures.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - PC plastocyanin     

Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis the dependence of photoinhibition on fluence rate, duration and wavelength of irradiation were studied by measurements of oxygen production and fluorescence emission spectra. The analysis of the photosynthetic activity revealed that photoinhibition affects exclusively photosystem II (PS II), whereas photosystem I (PS I) remained largely unimpaired. Furthermore, PS II fluorescence emission decreased much faster in bleached than in unbleached controls.Studying the wavelength dependence of photoinhibition it was found that only radiation between 520 and 680 nm causes photoinhibition. This is about the same range of wavelengths which causes photobleaching. Fluorescence emission spectra of samples exposed to high fluence rates of 582 and 662 nm, respectively, essentially agree with those samples exposed to high fluence rates of white light, whereas the fluorescence emission spectra of samples exposed to blue light resemble those exposed to dim white light.NaN₃, a substance which prevents photobleaching, inhibits the photosynthetic O₂ production of Anabaena and, hence, enhances the photoinhibitory effect.     

Die-offs of the blue-green alga,Anabaena variabilis,in fish ponds

Dense populations of the blue-green alga, Anabaena variabilis, frequently develop in fish ponds at Auburn, Alabama, during windy weather in March and early April. Massive die-offs of this alga can be expected when it forms surface scums during prolonged periods of calm, clear, warm weather between mid April and mid May. Dissolved oxygen concentrations decline following die-offs and fish kills may result.     

Effects of gassing,pH, quenching agents and photodynamically active compounds on photobleaching and photoinhibition of the cyanobacterium Anabaena variabilis

The findings presented in this paper support the suggestion that in the cyanobacterium Anabaena variabilis photobleaching is the result of an increased intracellular level of singlet molecular oxygen, whereas photoinhibition is controlled by a different molecular mechanism. Photobleaching of Anabaena trichomes can be prevented effectively by gassing with argon, nitrogen and carbon dioxide as well as by treatment with the ¹O₂ quenchers sodium azide and crocetin, and finally, with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, photodynamically active compounds, capable of ¹O₂ generation, increase photobleaching drastically. Thus, photobleaching is probably caused by singlet molecular oxygen. Photoinhibition studied with the aid of the fluorescence induction was not prevented by most of the treatments which prevent photobleaching. Therefore, different control mechanisms have to be assumed for this process.Abbreviations DABCO 1,4-diazabicyclo(2,2,2)octane - DBMIB dibromothymoquinone = (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - C-PC C-phycocyanin - Chl a chlorophyll a - LFE low fluence rate exposure - HFE high fluence rate exposure     

The regulation of aromatic amino acid biosynthesis in amino acid liberating mutant strains of Anabaena variabilis

Mutant strains of Anabaena variabilis which are resistant to the tryptophan analogue, 6-fluorotryptophan, liberated a wide range of amino acids although none liberated tryptophan in detectable quantities. Four strains (FT-7, FT-8, FT-9, FT-10) produced predominantly alanine together with small amounts of phenylalamine and tyrosine, strain FT-2 liberated mainly phenylalanine and tyrosine and strain FT-6 liberated mainly glutamate, NH ₄ ⁺ and several unidentified ninhydrin-positive compounds. Two forms of 3-deoxy-D-arbinoheptulosonate 7-phosphate (DAHP) synthase were identified in the parent strain, a tyrosine-sensitive form and a phenylalanine-sensitive form. In strains FT-2 and FT-6 the phenylalanine-sensitive enzyme was not detected and in strain FT-7 it was apparently deregulated with respect to inhibition by phenylalanine. No deregulation of anthranilate synthase was observed but mutant strains were found to have higher specific activities of this enzyme than the parent strain.Abbreviations chla chlorophyll a - 6-FT 6-fluorotryptophan - DAHP 3-deoxy-D-arabinoheptulosonate 7-phosphate - PEP phosphoenolpyruvate     

Fructose utilization by the cyanobacterium Anabaena variabilis studied using whole filaments and isolated heterocysts

We have investigated the utilization of [¹⁴C]-fructose by whole filaments and isolated heterocysts of Anabaena variabilis ATCC 29413, a strain which is capable of fructose-dependent heterotrophic growth. The experimental conditions were chosen such that both transport and subsequent metabolism were studied. The apparent K_m for fructose was 60 mM, close to the results of previous studies. Rates of fructose utilization were the same in light and darkness. When photosynthetic CO₂ fixation was possible, almost all the label appeared as cell-carbon. In darkness or in the presence of DCMU appreciable amounts of label were released as CO₂. Isolated heterocysts with high rates of endogenous metabolism were not capable of utilizing added fructose at significant rates. The effects of oxygen concentration on the metabolism of added fructose in darkness showed that uptake was saturated at low pO₂ values. Increasing the pO₂ values lead to an increase in the ratio between the lable released as CO₂ and that recovred as cell-carbon. These results suggest that fructose is taken up only by the vegetative cells but carbon derived from added fructose can be released as CO₂ as a result of respiration in the heterocysts. Fructose utilization was inhibited by uncouplers. The greatest inhibition was found when both (delta) (psi) and (delta) pH were abolished. High concentrations of erythrose inhibited fructose utilization. None of the other potential analogs tested had any effect.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.     

Green flagellar autofluorescence in brown algal swarmers and their phototactic responses

A flavin-like green autofluorescent substance is noticed to occur in one of the flagella of flagellated cells in the Phaeophyceae, Chrysophyceae, Synurophyceae, Xanthophyceae and Prymnesiophyceae. In the phaeophycean swarmers the autofluorescence occurs in the posterior flagellum throughout its length. It is considered to be involved in the photoreception of phototaxis, since it almost always occurs in the swarmers which have a flagellar swelling and stigma and show phototaxis. In the phaeophycean swarmers, the stigma is shown to act as a concave reflector mirror focusing the reflection light onto the flagellar swelling. In the action spectrum studies, phaeophycean swarmers showed phototaxis between 370 and 520 nm, having two major peaks at 420 or 430 nm and 450 or 460 nm. Their responses were true phototactic and not photophobic. Rotation of the swarmer was shown to be essential in the photoreception ofEctocarpus gametes. Recipient of the Botanical Society Award for Young Scientists, 1991.     

Hydrogen metabolism in isolated heterocysts of Anabaena 7120

Isolated heterocysts of Anabaena 7120 evolve H₂ in an ATP-dependent nitrogenase-catalyzed process that is inhibited by N₂ and C₂H₂. Heterocysts have an active uptake hydrogenase that only requires an electron acceptor of positive redox potential, e.g., methylene blue, dichlorophenolindophenol or potassium ferricyanide. O₂ supplied at low partial pressures is a very effective physiological oxidant for H₂ uptake. High concentrations of O₂ are inhibitory to H₂ uptake. The oxyhydrogen reaction in heterocysts appears to be mediated by a cytochrome-cytochrome oxidase system, and it supports ATP synthesis via oxidative phosphorylation. Attempts to demonstrate acetylene reduction in isolated heterocysts employing H₂ as an electron donor were unsuccessful. It is suggested that the uptake hydrogenase functions to conserve reductant that otherwise would be dissipated via nitrogenase-catalyzed H₂ evolution.     

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