Facultative anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica.

An isolate from H2S-rich layers of the Solar Lake, the cyanobacterium Oscillatoria limnetica, exhibits both oxygenic and anoxygenic photosynthesis. It can use Na2S as an electron donor for CO2 photoassimilation (photosystem I supplies the energy) in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 700-nm light. A stoichiometric ratio of approximately 2 is observed between the Na2S consumed and the photoassimilated CO2. The anoxygenic phototrophic capability of this cyanobacterium explains its growth in nature in high sulfide concentrations and indicates a selective advantage.     

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H₂O₂ sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - SOD superoxide dismutase     

Induction of anaerobic, photoautotrophic growth in the cyanobacterium Oscillatoria limnetica.

Anaerobic photoautotrophic growth of the cyanobacterium Oscillatoria limnetica was demonstrated under nitrogen in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (5micron), a constant concentration of Na2S (2.5 mM), and constant pH (7.3). The photoanaerobic growth rate (2 days doubling time) was similar to that obtained under oxygenic photoautotrophic growth conditions. The potential of oxygenic photosynthesis is constitutive in the cells; that of anoxygenic photosynthesis is rapidly (2 h) induced in the presence of Na2S in the light in a process requiring protein synthesis. The facultative anaerobic phototrophic growth physiology exhibited by O. limnetica would seem to represent an intermediate physiological pattern between the obligate anaerobic one of photosynthetic bacteria and the oxygenic one of eucaryotic algae.     

Na-Dithionite Promotes Photosynthetic Sulfide Utilization by the Cyanobacterium Oscillatoria limnetica

The light- and sulfide-dependent induction process leading to photosystem I-mediated sulfide utilization by Oscillatoria limnetica, for either H₂ evolution or CO₂ photoassimilation, was studied. The identical dependence on pH of the lag length, the inhibition of leucine incorporation and final H₂S concentration imply that the latter exerts a deleterious effect on nonadapted cells.

Na-dithionite (Na₂S₂O₄), Na-sulfite (Na₂SO₃), or ethanol cannot serve as photosynthetic electron donors. However, when these compounds were added to the sulfide-containing system, the need for induction was eliminated. At pH 6.9, in the presence of 3.5 millimolar sulfide, these substances (at concentrations of 10 millimolar, 5 millimolar, or 0.4 molar, respectively) completely abolished the delay preceding sulfide-dependent H₂ evolution. It is suggested that all three compounds expose a site capable of directly accepting sulfide electrons.

Only dithionite could adapt the cells to sulfide utilization on its own. Sulfite or ethanol acted only in the presence of sulfide. It is implied that this specific activity of dithionite is related to its characteristic low redox potential.

Sulfide-dependent H₂ evolution was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but was inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, in the presence as well as in the absence of dithionite. In both cases, therefore, the plastoquinone was implied in the electron transport from sulfide.

     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid     

Sulfide-dependent photosynthetic electron flow coupled to proton translocation in thylakoids of the cyanobacterium Oscillatoria limnetica

Light-induced proton translocation coupled to sulfide-dependent electron transport has been studied in isolated thylakoids of the cyanobacterium Oscillatoria limnetica. The thylakoids are obtained by osmotic shock of washed spheroplasts, prepared with glycine-betaine as the osmotic stabilizer. 13C NMR studies suggests that betaine is the major osmoregulator in O. limnetica. Thylakoid preparations obtained from both sulfide-induced anoxygenic cells and noninduced oxygenic cells are capable of proton pumping coupled to phenazinemethosulfate-mediated cyclic electron flow. However, only in the induced thylakoids can sulfide-dependent proton gradient (delta pH) formation be measured, using either NADP or methyl viologen as the terminal acceptor. Sulfide-dependent delta pH formation correlates with a high-affinity electron donation site (apparent Km 44 microM at pH 7.9). This site is not lost upon washing of the thylakoids. In addition, both sulfide-dependent electron transport and delta pH formation are sensitive to inhibitors of the cytochrome b6f complex such as 2-n-nonyl-4-hydroxyquinoline-N-oxide, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, or stigmatellin. Sulfide-dependent NADP photoreduction of low affinity (which does not saturate by as much as 7 mM sulfide) is detected in both induced and noninduced thylakoids, but this activity is insensitive to the inhibitors and is not coupled to proton transport. It is suggested that the adaptation of O. limnetica to anoxygenic photosynthesis involves the induction of a thylakoid factor(s) which creates a high-affinity site for sulfide, and the transfer of its electrons via the cytochrome b6f complex, coupled to proton translocation.     

Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation

The cyanobacterium Oscillatoria limnetica, capable of anoxygenic photosynthesis in the light with sulfide as electron donor can anaerobically break down its intracellular polyglucose in the dark. In the absence of elemental sulfur, the organism carries out lactate fermentation; in its presence, anaerobic respiration occurs in which sulfur is reduced to sulfide. Induction of anoxygenic photosynthesis or synthesis of new proteins is not necessary for either process. Cells adapted in the dark to sulfur reduction are capable of anoxygenic photosynthesis during a subsequent light period, unless protein synthesis has been inhibited during the dark incubation period.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP Carbonylcyanide p-trifluoromethoxyphenylhydrazone - mgat milligramatom - OD optical density     

Sulfide-induced sulfide-quinone reductase activity in thylakoids of Oscillatoria limnetica

Sulfide-dependent partial electron-transport reactions were studied in thylakoids isolated from cells of the cyanobacterium Oscillatoria limnetica, which had been induced to perform sulfide-driven anoxygenic photosynthesis. It was found that these thylakoids have the capacity to catalyze electron transfer, from sulfide to externally added quinones, in the dark. Assay conditions were developed to measure the reaction either as quinone-dependent sulfide oxidation (colorimetrically) or as sulfide-dependent quinone reduction (by UV dual-wavelength spectrophotometry). The main features of this reaction are as follows. (i) It is exclusively catalyzed by thylakoids of sulfide-induced cells. Noninduced thylakoids lack this reaction. (ii) Plastoquinone-1 or -2 are equally good substrates. Ubiquinone-1 and duroquinone yield somewhat slower rates. (iii) The apparent Km for plastoquinone-1 was 32 microM and for sulfide about 4 microM. Maximal rates (at 25 degrees C) were about 75 mumol of quinone reduced per mg of chlorophyll.h. (iv) The reaction was not affected by extensive washes of the membranes. (v) Unlike sulfide-dependent NADP photoreduction activity of these thylakoids, which is sensitive to all the specific inhibitors of the cytochrome b6f complex, the new dark reaction exhibited differential sensitivity to these inhibitors. 2-n-Nonyl-4-hydroxyquinoline-N-oxide was the most potent inhibitor of both light and dark reactions, working at submicromolar concentrations. 5-n-Undecyl-6-hydroxy-4,7-dioxobenzothiazole also inhibited the two reactions to a similar extent, but at 10 times higher concentrations than 2-n-nonyl-4-hydroxyquinoline-N-oxide. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone, 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether, and stigmatellin had no effect on the dark reaction at concentrations sufficient to fully inhibit the light reaction from sulfide. We propose that the sulfide-induced factor which enables the use of sulfide as the electron donor for anoxygenic photosynthesis in Oscillatria limnetica is a membrane-bound sulfide-quinone reductase. Its site of interaction is suggested to be either the cytochrome b6 (at the Qc quinone binding site or the bH site) or the plastoquinone pool. The analogy to other anoxygenic photosynthetic systems is discussed.     

UVC-induced lysis and detritus production of Oscillatoria limnetica in a two-stage continuous-flow system

In order to study in model systems the role of heterotrophicmicro-organisms in the aquatic microbial food web, a naturalfood source consisting of senescent primary producer cells isindispensable. A two-stage continuous-flow system with the abilityto produce detritus continuously is presented. In the firststage, a primary producer, i.e. the cyanobacterium Oscillatorialimnetica, was cultured. The second stage was fed with UVC-killedprimary producer cells from the first stage. A control secondstage received living primary producer cells. Both second stageswere filled with prefitered lake water in order to provide anatural inoculum of bacteria. During a 2 month experiment, thesystem was tested for its ability to produce detritus (firststage of detritus, i.e. lysis of the cell material). Chlorophylla concentrations proved to be adequate for calculating specificlysis rates. Very high specific lysis rates were found afterkilling the cells with UVC as compared to the control stage.     

Ferredoxin from the cyanobacterium Oscillatoria agardhii

Oscillatoria agardhii contained a single ferredoxin. It was a [2Fe-2S] protein of MW 11 075, with a midpoint redox potential (? 380 mV) characteristic of ferredoxins from non-nitrogen-fixing cyanobacteria and different from that of the nitrogen-fixing Oscillatoria limnetica.     

Geosmin production in the cyanobacterium Oscillatoria brevis

The cyanobacterium Oscillatoria brevis (Kütz.) Gom., strain NIVA CYA 7, was used to investigate how geosmin production is related to the synthesis of chlorophyll a, phycobiliproteins and β-carotene under nitrogen (NH ₄ ⁺ ) and light limiting conditions. Chemostat samples were used to inoculate batch cultures that were treated with inhibitors of isoprenoid synthesis, norflurazon and dimethazone, and gabaculine that inhibits tetrapyrrole synthesis. Dimethazone decreased and norflurazon increased geosmin production under light limited conditions, as was expected due to their sites of action in the isoprenoid pathway. This effect was not so pronounced in nitrogen limited cultures due to the additional effect of increasing nitrogen deficiency during the experimental period. Norflurazon was the only inhibitor that uncoupled geosmin production completely from β-carotene formation which indicates a strikt coupling between geosmin and β-carotene biosynthesis. From the observed increase of geosmin production relative to pigment synthesis after norflurazon treatment it was suggested that isoprenoid precursors are directed to geosmin synthesis when the demand for pigment precursors is very low. Within the framework of this study the data strongly support the hypothesis of geosmin formation via the isoprenoid pathway in Oscillatoria brevis as was found for actinomycetes. This research was performed at the Department of Microbiology, University of Amsterdam, with financial support provided by the royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

A phycourobilin-containing phycoerythrin from the cyanobacterium Oscillatoria sp.

A filamentous cyanobacterium Oscillatoria sp. was isolated from a thermal spring of the Kamchatka peninsula. It contained a phycoerythrin unusual for cyanobacteria in that it had a phycourobilin prosthetic group. The absorption spectrum of the native purified phycoerythrin displayed maxima at 498 and 567 nm. The phycoerythrin comprised - and -subunits of molecular weights 18,700 and 19,800, respectively, in 1:1 stoichiometry. Polyacrylamide gel isoelectric focusing revealed one protein band at pI 4.6. The - and -subunits differed in their chromophore composition and content: -subunit carried two phycoerythrobilins while the -subunit had three phycoerythrobilins and one phycourobilin. The chromophore composition of all known phycoerythrins of cyanobacteria and red algae were compared, and on the basis of this comparative study designations C₁- to C₅-phycoerythrin were proposed for cyanobacterial red pigments.     

Sulfide and pH effects on variable fluorescence of photosystem II in two strains of the cyanobacterium Oscillatoria amphigranulata

Changes in fluorescence of photosystem II (PS II) chlorophyll were used to monitor the in vivo effects of sulfide and pH on photosynthesis by the cyanobacterium Oscillatoria amphigranulata. O. amphigranulata is capable of both oxygenic photosynthesis and sulfide dependent anoxygenic photosynthesis. A genetic variant of O. amphigranulata which photosynthesizes oxygenically at normal rates, but is incapable of anoxygenic photosynthesis and cannot tolerate sulfide, was also used to explore the mode of action of sulfide. In vivo fluorescence responses of PS II chlorophyll in the first few seconds of exposure to light (Kautsky transients) reflected the electrochemical states of PS II and associated electron donors and acceptors. Kautsky transients showed a distinct difference between PS II of the wild type and the variant, but sulfide lowered fluorescence in both. Kautsky transients with sulfide were similar to transients with addition of NH₂OH, NH₄ ⁺ or HCN, indicating sulfide interacts with a protein on the donor side of PS II. The fluorescence steady-state (after 2 min) was measured in the presence of sulfide, cyanide and ammonium with pH ranging from 7.2–8.7. Sulfide and cyanide had the most impact at pH 7.2, ammonium at pH 8.7. This suggests that the uncharged forms (HCN, NH₃ and H₂S) had the strongest effect on PS II, possibly because of increased membrane permeability.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethyl-urea - Oa-wt Oscillatoria amphigranulata (wild type) - Oa-2 Oscillatoria amphigranulata (genetic variant)     

Unsaturated fatty acid composition and biosynthesis in Oscillatoria limnetica and other cyanobacteria

A number of cyanobacteria showing a high degree of adaptation to life under reduced oxygen tensions as witnessed by their potency of facultative anoxygenic CO₂ photoassimilation with sulfide as electron donor were found to lack polyunsaturated fatty acids in their lipids. Lack of polyunsaturated fatty acids was found in representatives of different taxonomic groups. One of the strains lacking polyenoic acids was Oscillatoria limnetica, which can alternatively grow acrobically or anaerobically with sulfide as electron donor. This organism was found to synthesize monounsaturated fatty acids by desaturation of their saturated counterparts, in the presence as well as in the absence of molecular oxygen.Abbreviations ACP Acyl carrier protein - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea     

Anoxygenic photosynthetic hydrogen production and electron transport in the cyanobacterium oscillatoria limnetica

The induction of anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica by sulfide was shown to involve the synthesis of a sulfide oxidizing factor; this factor, partly adsorbed on the thylakoid membrane, can be recovered in the soluble phase and is active also on membranes from oxygenically grown cells. The factor is required for sulfide dependent light-induced hydrogen evolution. It accelerates electron transport from sulfide to the electron donor of photosystem I, P700, in membranes from cells in which anoxygenic photosynthesis is induced. The plastiquinone analogue DBMIB does not inhibit electron transport to P700 but accelerates it. The analogue might promote cyclic electron transport involving P700, thus preventing electrons to reach hydrogenase.Abbrevlations chl chlorophyll - DBMIB 3,5-dibromo-3-methyl-6-isopropyl-8-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediamine tetraacetate - FAD flavin adenine dinucleotide - HEPES N-2-hydroxyethyl-piperazine N-2-ethane sulfonic acid - PS photosystem Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Feeding in Daphnia galeata on Oscillatoria limnetica and on Detritus Derived from It

Oscillatoria spp. are generally very abundant in many shallow,eutrophic lakes in the Netherlands. However, this is less truefor Daphnia galeata. The main aim of this study was to investigatewhether the edibility of live Oscillatoria limnetica by Daphniagaleata, and of the detritus derived from this filamentous cyanobacterium,was, among others, an important limiting factor for the daphnids.We measured the consumption and assimilation rates of Daphniausing dual-labelling radio-tracer techniques (¹⁴C and ³²P) tolabel separately the live Oscillatoria filaments and detritusprepared from these filaments. The two food types were mixedin different proportions, and both the food ingestion and foodincorporation rates by daphnids were measured. The main findingsof this study were that specific clearance rates of Daphniaon shorter Oscillatoria filaments were significantly higherthan on the longer filaments, in other words the weight-specificingestion rates were higher on the shorter Oscillatoria filamentsthan on the longer filaments from the batch cultures. The longerOscillatoria filaments are more likely to clump and, therefore,are more liable to be rejected by Daphnia during the food collectionand ingestion processes. The shorter filaments, in comparison,are apparently less prone to clumping and, therefore, are clearedby the daphnids at higher rates than the longer filaments. Feedingthe daphnids on double-labelled (¹⁴C and ³²P) shorter filamentsrevealed that the assimilationefficiency of shorter Oscillatoriafilaments was generally higher for P than for C, probably becauseof a high C/P ratio of Oscillatoria. Daphnia (0.75–1.85mm in size) fed significantly better on dead (detritus) Oscillatoriafilaments than on live Oscillatoria filaments, even if the relativeproportion of detritus in the food was only about one-quarterthat of the live filaments. This preference for detritus overlive Oscillatoria, as indicated by Chesson's selectivity coefficienta, was apparently a passive process, rather than a case of activefood selection. This ‘selective’ feeding on detrituswas apparently facilitated by the relatively greater rejectionof the live filaments than the detrital filaments. At very highfood concentrations (15–25 mg C l^–1), with the shareof the live Oscillatoria filaments 2 to 2.5 times greater thanthat of the detritus, the ingestion rates increased proportionallyless with increase in daphnid size than was expected on thebasis of the allometric relationship between the length andweight of these animals. This implies that the larger animalshad greater interfering effects of the Oscillatoria filamentson the food collection and ingestion processes.     

Immunological characterization of nitrogenase in the filamentous non-heterocystous cyanobacterium Oscillatoria limosa

The filamentous non-heterocystous cyanobacterium Oscillatoria limosa was subjected to Western blot analyses using two antisera raised against the small subunit (Fe-protein) of the nitrogenase complex. Two polypeptides were recognized in nitrogen-fixing cultures irrespective of the antiserum used while no bands were detectable in nitrate-grown cultures. The apparent molecular weights of the two polypeptides were approximately 40.5 and 39.5 kDa respectively, with the former, probably an inactive form, dominating. In situ immunogold electron microscopy was used to reveal the cellular and subcellular localization on the Fe-protein. All cells of the trichomes of nitrogen-fixing O. limosa showed a dense label. The label was homogeneously distributed throughout the cytoplasm including the thylakoid area. Nitrate-grown cultures contained a very low label.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis This study was supported by the Swedish Natural Science Research Counsil and the M. and M. Wallenberg Fund (to B. Bergman). We are grateful to Dr. S. Nordlund (University of Stockholm, Sweden) for providing us with the antiserum of Rhodospirillum rubrum nitrogenase and to Drs. S. Reich and P. Böger (University of Konstanz, FRG) for the antiserum of Anabaena variabilis. Skilful technical assistence by K. Östlund and E. Danielsson is gratefully acknowledged. We would also like to thank M. Villbrandt (University of Oldenburg, FRG) for providing cultures of Oscillatoria limosa and Dr. P. Lindblad for valuable discussions and suggestions.To whom correspondence should be addressed.     

Hydrogen metabolism in the facultative anoxygenic cyanobacteria (blue-green algae) Oscillatoria limnetica and Aphanothece halophytica

Two facultative anoxygenic photoautotrophic cyanobacteria, Oscillatoria limnetica and Aphanothece halophytica were found capable of CO₂ photoassimilation using molecular hydrogen as electron donor in a photosystem I driven reaction. A. halophytica was also capable of evolving hydrogen from Na-dithionite reduced methylviologen in a light independent reaction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DSPD Disallcylidenepropanediamine - FCCP Carbonylcyanide p-trifluoromethoxyphenyl hydrazone - Tricine N-tris(hydroxy methyl)-methylglycine     

Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

The filamentous, non-heterocytous, nitrogen-fixing cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions nitrogenase activity is exclusively present during the dark period. However, if anaerobic conditions were applied during the dark period, two activity maxima were observed. A relatively small activity peak occurred during the first few hours of the dark period and a high peak as soon as the light was switched on. A low activity remained during the second half of the dark period. This pattern of acetylene reduction in Oscillatoria agrees well with the field data on nitrogen fixation [Stal, L.J. and Krumbein, W.E. (1984), Mar. Biol. 82, 217–224].     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.