Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Effects of Aminooxyacetate and Aminoacetonitrile on Glycolate and Ammonia Release by the Cyanobacterium Anabaena cylindrica

Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH₄-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica.     

Carbamoyl Phosphate Synthetase of the Cyanobacterium Anabaena cylindrica

Carbamoyl phosphate synthetase from the cyanobacterium Anabaenacylindrica was purified by the following procedures: ammoniumsulfate fractionation, DEAE-Toyo-pearl, Affi-gel Blue, SephacrylS-300 HR, and Mono Q column chromatography. The molecular weightof the holoenzyme was estimated to be 166,000 by gel permeationchromatography. SDS-PAGE showed that the enzyme consisted oftwo subunits with molecular weights of 130,000 and 43,000. Optimal pH of this enzyme was 7.8 in HEPES buffer. Its MgATPsaturation curve was sigmoidal, yielding a Hill coefficientof 1.9 and an apparent K^m of 4.5 mM. The K^m values for glutamine,NH⁴C1 and NaHC0³ were 55 µM, 182 mM and 2.5 mM, respectively.A high concentration of K⁺ (100 mM) was required for maximumactivity. The enzyme was activated by ornithine, IMP, GMP, andGDP, and inhibited by UMP and UDP. Ornithine increased the affinityof the enzyme to ATP by acting as a positive allosteric effector,whereas UMP reduced it by acting as a negative allosteric effector. (Received December 24, 1996; Accepted April 10, 1997)     

Macromolecular Composition of Spores from the Filamentous Cyanobacterium Anabaena cylindrica

Spores were isolated from the filamentous cyanobacterium Anabaena cylindrica, and their deoxyribonucleic acid, ribonucleic acid, and protein compositions were determined.     

Differential Effects of Methionine Sulfoximine on Light and Dark Uptake of Nitrate by Anabaena cylindrica

Methionine sulfoximine (MSX), a glutamine synthetase inhibitor,suppressed the inhibitory effect of ammonia on nitrate uptakeby Anabaena cells in both the light and dark. In the light,MSX did not inhibit nitrate uptake in the absence of ammonia,but under dark conditions, MSX above 2 µM inhibited nitrateuptake. Nitrite uptake, which is not affected by ammonia ineither the light or the dark, was inhibited by MSX in the darkbut not in the light. (Received October 3, 1984; Accepted April 22, 1985)     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Expression of an Adenylate Cyclase Gene of Anabaena cylindrica in the Cyanobacterium Anabaena sp. Strain PCC 7120

A transformant of Anabaena 7120 was made by introducing a plasmidthat includes an adenylate cyclase gene of Anabaena cylindrica.Expression of this gene was driven by the bacterial tac promoter.Transformants accumulate cAMP 170 fold higher than the concentrationin the parental strain. The transformation resulted in the fragmentationof filaments in both nitrogen-replete and nitrogen-free media.It was suggested that this fragmentation caused the inhibitionof growth under nitrogen-fixing conditions. (Received December 26, 1997; Accepted April 30, 1998)     

Comparative Amperometric Study of Uptake Hydrogenase and Hydrogen Photoproduction Activities between Heterocystous Cyanobacterium Anabaena cylindrica B629 and Nonheterocystous Cyanobacterium Oscillatoria sp. Strain Miami BG7

Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N₂ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production, photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O₂ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O₂ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7.     

Influence of Cadmium on Growth and Nitrogen Metabolism of Anabaena cylindrica Lemm

A study was made of the effects of cadmium on the Cyanobacterium(blue-green alga) Anabaena cylindrica Lemm. as part of the paddy-fieldecosystem. A simple culture vessel has been designed, which allows periodicalmeasurement of growth (optical density) and nitrogenase activity(C₂H₂-C₂H₄ method). The influence of medium renewal was checked:the renewal of the medium maintained a higher growth rate andhigher nitrogen fixation ability. The cadmium effects were studied using six concentration levelsranging from 0 (control) to 2 parts 10^–6 with renewedmedia (10% every day). No significant differences could be seen up to 1 part 10^–6for nitrogenase activity and relative percentage of heterocysts(decreasing as a function of time from ±4% to ±1.5%). Inhibition of growth (OD and dry weight) was weak at 1 part10^–6 but important at 2 parts 10^–6; at this concentrationcadmium induced morphological and physiological effects: chlorosis,cellular malformations and destruction, and increase in heterocystfrequency (up to 7.72% ±0.19). The cadmium concentration factors were much lower than thosereported for other plants like Chlorella and water pests     

Phosphate Metabolism in the Cyanobacterium Anabaena doliolum Under Salt Stress

In the present study, we have investigated the effects of NaCl concentrations on the growth and phosphate metabolism of an Anabaena doliolum strain isolated from a paddy field, in order to determine the possible effects of salinization. Growth rate, chlorophyll content, and protein content decreased with increasing salt concentration in the growth medium, while carbohydrate concentration increased. Phosphate content and phosphate uptake rate decreased. There was an increase in total alkaline phosphatase activity, with an approximately 7-fold increase in extracellular activity compensating for an approximately 3-fold decrease in cell-bound activity. NaCl effects on protein and chlorophyll concentrations were greater in P-deficient medium, while presence or absence of P in the medium had little effect on cellular carbohydrate concentrations. It is concluded that growth in high salt likely leads to reduced phosphate uptake in A. doliolum.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Nitrogen fixation by Anabaena cylindrica

Summary The effects of oxygen, light and photosynthesis inhibitors on nitrogenase activities in Anabaena cylindrica batch cultures were followed as a function of time after inoculation. During the early rapid growth period the nitrogenase activities of cultures grown under air/CO₂ or N₂/CO₂ were relatively resistant to oxygen and DCMU inhibition. These cultures also exhibited oxygen-dependent nitrogenase activity in the dark of up to 50% of that measured in the light. After active growth ceased the cultures continued to slowly grow for a prolonged period of time. The nitrogenase activities of these old cultures were very sensitive to oxygen and DCMU inhibition. These cultures also had little or no dark nitrogenase activities. The photosynthesis inhibitor DBMIB was not a specific inhibitor of light-driven electron transport since it inhibited both light and dark nitrogenase activities. Nitrogenase activities induced under oxygen-free/CO₂ gas mixtures initially were significantly more sensitive to oxygen inhibition than those induced under air/CO₂. We discuss these results in relation to heterocyst function.     

Nitrogen fixation by Anabaena cylindrica

Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H₂ (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H₂ and was strongly inhibited by oxygen (50% inhibition at about 5–6% O₂). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O₂ on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO₂ fixation by vegetative cells.     

Magnetobiological Effects on a Cyanobacterium,Anabaena Doliolum

Exposure time and magnetic-pole-dependent physiological response of a cyanobacterium, Anabaena doliolumwere studied by exposing the samples to two poles [north (N) and south (S)] of a 0.3 Tesla permanent magnet for 1–6 h. Study revealed that both magnetic poles, N and S, produce different effects, depending on the length of exposure. However, physiological response of the cyanobacterium to N + S mixtures was significantly different from the response to either of the poles, N or S, suggesting a change in structural chemistry of the water or nutrient solution responsible for the magnetobiological effects.     

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     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.     

Effect of nitrate on nitrogen-fixation by the blue-green alga Anabaena cylindrica

The effect of nitrate on nitrogen-fixation in the blue-greenalga Anabaena cylindrica Lemm (Fogg strain) was investigated.At concentrations up to 2x10^–2 M, nitrate neither inhibitedthe activity of nitrogenase nor repressed its formation. Atthe late logarithmic phase, more than 50% of cell nitrogen wasprovided by nitrogen-fixation when the cells were grown in thepresence of nitrate. Ammonia at a concentration of 1x10^–3M completely repressed the formation of nitrogenase, but hadno effect on its activity. Nitrogen-fixing activity in the algavaried to a considerable extent during growth on N₂ and themaximum activity was attained at the middle logarithmic phase.However, atmospheric nitrogen did not directly affect the inductionof nitrogenase. The formation of nitrogenase in A. cylindricaappears to be controlled by the intracellular level of a certainnitrogenous metabolite. ¹ This work was supported by grant No. 38814 from the Ministryof Education. (Received January 26, 1972; )     

Effect of ammonia on nitrogen fixation by the blue-green alga Anabaena cylindrica

Ammonia at a concentration of 1 ? 10^–3M completely inhibitednitrogenase activity, as measured by acetylene reduction, inthe blue-green alga Anabaena cylindrica. Free ammonia was undetectablein cells grown either on N₂ or ammonia within the limits ofprecision of the method used. Glutamic acid formed a major aminoacid pool in N₂-grown cells, and basic amino acids, i.e. lysine,histidine and arginine were abundant in ammonia-grown cells.A 10-fold increase in the amounts of labile amino compound(s)was observed when N₂-grown cells were exposed to ammonia. When cells were incubated under anaerobic conditions, the acetylene-reducingactivity increased 2-fold or more; ammonia had no effect. Oxygenwas required for ammonia to inhibit acetylene reduction. Modes of inhibition by ammonia on acetylene reduction were comparedwith those by chloramphenicol, puromycin, cycloheximide, DCMUand CCCP. On the basis of these comparisons we concluded thatammonia not only acts as a suppressor of nitrogenase synthesisbut also inhibits acetylene-reducing activity by lowering thesupply of reductant and/or of energy for the nitrogenase system. ¹This work was supported by grant No. 38814 from the Ministryof Education. (Received July 30, 1973; )