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.     

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     

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.     

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.     

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     

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     

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     

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     

Investigations on the phototactic orientation of Anabaena variabilis

Phototaxis of the blue-green alga Anabaena variabilis was studied using both population method and observation of single trichomes by microscope. The trichomes react positively at low and negatively at high illuminance. The inversion point lies at about 1000 1x. The action spectrum of positive phototaxis indicates that the photosynthetic pigments chlorophyll a, C-phycocyanin and allo-phycocyanin are involved in the absorption of the active light. The same range of wavelengths is active in negative phototaxis, but in addition, wavelengths between 500 and 560 nm and between 700 and 750 nm are also effective. Obviously pigments of unknown chemical nature are sharing in light absorption. Two alternatives are discussed. Since inhibitors of photosynthesis such as DCMU and DBMIB do not affect phototactic orientation, a direct coupling of phototaxis with photosynthesis can be excluded.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) Presented in part at the International Symposium on Photosynthetic Prokaryotes: August 22–28, 1976, Dundee, Scotland     

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     

Hydrogen evolution by photobleached Anabaena cylindrica

We have studied the evolution of hydrogen by photobleached filaments of the heterocystous bluegreen alga Anabaena cylindrica. The photobleached cells became orange-yellow due to the heavy accumulation of carotenoids. We found that the yellow filaments produced much larger amounts of hydrogen than the normal, green ones, while the nitrogenase activity responsible for hydrogen evolution increased to a lesser extent. We suggest that a reversible hydrogenase activity induced in photobleached filaments is responsible for the excess amount of hydrogen. 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibits the hydrogen evolution of the yellow filaments which produce much more oxygen and fix less CO₂ than the green filaments. Therefore we consider the water to be a possible electron source for this hydrogenase. The low efficiency of light energy conversion (0.3%) in nitrogenase-catalyzed H₂ evolution (Laczkó, 1980 Z. Pflanzenphysiol. 100, 241–245) is increased to 1.5–2% by the appearance of the reversible hydrogenase activity.Abbreviations Chl chlorophyll - Car carotenoids - Phy phycocyanin - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - PSI photosystem I - PSII photosystem II     

Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Summary Free-living nitrogen-fixingNostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycadMacrozamia sp., were examined for the presence of an uptake hydrogenase (H₂ase) enzyme. In vivo and in vitro hydrogen uptake measurements were used to study activities and SDS-PAGE and Western immunoblots to reveal occurrence of the hydrogenase protein. Also, transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of H₂ase in theNostoc cells. In vivo measurements demonstrated an active uptake of hydrogen in both light and darkness. Light stimulated in vivo hydrogen uptake with approximately 100%, and this was further doubled by increasing the pH₂, from 56 to 208 M H₂. An in vitro hydrogen uptake of 1.1 mol H₂/ mg (protein)/h was observed when using phenazinemethosulphate as e^–-acceptor. Western immunoblots revealed that a polypeptide with a molecular weight of about 55 kDa was immunologically related to uptake H₂ase holoenzyme purified fromAlcaligenes latus. Immunolocalization demonstrated that the H₂ase protein was located both in heterocysts and vegetative cells. A higher specific labeling was associated with the cytoplasmic membranes where the vegetative cells are in contact with each other and where they actually are dividing into two vegetative cells. Using the particle analysis of an image processor, approximately equal H₂ase-gold labeling per cell area was observed in the nitrogen-fixing heterocysts compared to the photosynthetic vegetative cells. This study also shows that there was no correlation between presence of phycoerythrin and uptake H₂ase activity.Abbreviations H₂ase hydrogenase - IgG immunoglobulin G     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Presence of an N-terminal presequence in the PsaI protein of the Photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413

The psaI gene encoding the 5.2 kDa protein component (PsaI) of the photosystem I complex was cloned from the cyanobacterium Anabaena 29413. The gene is present in single copy in this cyanobacterial genome. The nucleotide sequence of a 500 bp region of the cloned DNA revealed the presence of an open reading frame encoding a 46 amino acid long polypeptide. The N-terminal 11 residues are absent in the mature polypeptide and thus represents the first identified cleavable presequence on the PsaI protein. We suggest that this presequence directs the N-terminus of the protein to the thylakoid lumen.     

High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

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.