Alanine dehydrogenase of the N2-fixing blue-green alga, Anabaena cylindrica.

The L-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270,000, has 6 sub-units, each of molecular weight approximately 43,000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The Kampp for NAD+ is 14 muM and 60 muM at low and high NAD concentrations respectively.     

ATP pools and transients in the blue-green alga,Anabaena cylindrica

Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla ^-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total photophosphorylation, cyclic photophosphorylation and oxidative phosphorylation. The alga can maintain its ATP pool by switching rapidly from one of these phosphorylating mechanisms to another depending on the environmental conditions. At each switch-over there is a transient drop in the ATP pool for a few seconds. On switching to conditions where only substrate level phosphorylation operates, the ATP pool falls immediately, but takes several hours to recover. The apparent rates of ATP synthesis by total photophosphorylation and by cyclic photophosphorylation are both much higher (210±30 and 250±13 moles ATP mg chla ^-1 h^-1 respectively) than the apparent rate of ATP synthesis by oxidative phosphorylation (22±3 moles ATP mg chla ^-1 h^-1). In long term experiments the ATP pool is maintained when total photophosphorylation is operating. It cannot be maintained in the long term by cyclic photophosphorylation alone in the absence of photosystem II activity or endogenous carbon compounds, or by oxidative phosphorylation in the absence of endogenous carbon compounds. Measurements of ATP, ADP and AMP show that the total pool of adenylates is similar in the light and in the dark in the short term. There is only limited production of ATP under dark anaerobic conditions when glycolysis and substrate phosphorylation can operate which suggests that these processes are of limited significance in providing ATP in Anabaena cylindrica.Abbreviations ADP adenosine 5-diphosphate - AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)1,1-dimethyl urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PEP phosphoenolpyruvate     

The utilization of molecular hydrogen by the blue-green alga Anabaena cylindrica

The blue-green alga Anabaena cylindrica is found to consume molecular hydrogen in a hydrogenase dependent reaction. This hydrogen uptake proceeds in the dark and is strictly dependent on oxygen, thus representing a Knallgas reactions. Its rate is almost as high as that of the endogenous respiration in Anabaena. Studies with inhibitors reveal that hydrogen is utilized via the complete respiratory chain providing additional energy for the alga. CO plus C₂H₂ completely block the Knallgas reaction which explains the previously reported considerable increase in the total H₂ formation representing the difference between the nitrogenase-dependent H₂-evolution and the reutilization of the gas catalysed by the hydrogenase in intact Anabaena.H₂ is able to support the C₂H₂-reduction in the dark in a reaction again strictly dependent on oxygen. Moreover, H₂ is also consumed in experiments carried out under far red light and in the presence of dichlorophenyl-dimenthyl-urea (DCMU) where the energy for nitrogen fixation is no longer provided by respiration but by cyclic photophosphorylation. Under these conditions, H₂ is found to supply electrons for the formation of C₂H₄ from C₂H₂ in a reaction no longer dependent on the presence of oxygen. Moreover, in these experiments, the presence of H₂ stabilizes the C₂H₂-reduction activity against the deleterious effect of oxygen.Thus, this communication provides evidence for a triplicate function of the H₂-uptake catalysed by hydrogenase in intact Anabaena which is (a) to provide energy by the Knallgas reaction, (b) to supply reducing equivalents for nitrogenase, (c) to protect nitrogenase from damage by oxygen.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea - DNP 2-4-dinitrophenol - FCCP carbonylcyanid-p-trifluormethoxyphenyl-hydrazone(=p-CF₃-CCP) - Chl chlorophyll     

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.     

Light-induced accumulation of lactate and succinate in Anabaena cylindrica

In the cyanobacterium Anabaena cylindrica lactate accumulated in large amounts when the cells were exposed to light. The presence or absence of oxygen, or a change in CO₂ concentration did not affect the lactate accumulation. The cellular succinate level also increased in the light when CO₂ was supplied at the high concentration of 1%. 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor of photosynthetic electron flow, inhibited the increase in the concentration of lactate and succinate. Photosynthesis is a prerequisite for the increase of these organic acids. Thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase, inhibited the increase of succinate, suggesting that the succinate is formed via fumarate by the reverse of reactions of tricarboxylic acid (TCA) cycle. Upon addition of ammonium to the cell suspension in the light under high CO₂ concentration, the increases in the concentrations of lactate and succinate were inhibited while those of glutamine, glutamate and aspartate were stimulated. Ammonium apparently changed the products of metabolism of pyruvate and oxaloacetate from lactate and succinate to amino acids.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - TTFA thenoyltrifluoroacetone - PCA perchloric acid     

Cellular compartmentation of photosynthetic and photorespiratory enzymes in the heterocystous cyanobacterium Anabaena cylindrica

Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO₂ fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P d-ribulose 5-phosphate - RuBP d-ribulose 1,5-bisphosphate - DCPIP 2,6-dichlorophenolindophenol - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate     

Heterocyst and akinete induction with altered pattern in Anabaena cylindrica,caused by neo-peptone

Neo-peptone B119 (Difco) was found to have a significant effect on differentiation of heterocysts and akinetes in Anabaena cylindrica. On adding neopeptone (0.4 g/l) to exponential phase culture of A. cylindrica, the following effects were observed (i) increased heterocyst frequency with altered heterocyst spacing and presence of double and multiple heterocysts after 24 h in cultures grown on N-free medium, (ii) induction of regular pattern of heterocysts after 48 h, in culture grown on medium supplemented with NH₄Cl, (iii) induction of pro-akinetes after 48 h in both N-free and ammonium-grown cultures. The higher concentrations of neo-peptone were lytic to A. cylindrica, and, its lytic and inductive effects could be decreased by acid hydrolysis or supplementation of NH₄Cl. Gel-filtration of neo-peptone showed that the inductive as well as the lytic effect was associated with some active factor(s) with molecular weight between 10,000–20,000. The retention of the inductive effect on autoclavation but its loss on trypsin digestion suggested that active factor(s) may be heat stable polypeptide(s). The heterocyst induction by active factor(s) decreased and akinete induction increased with increasing culture age. The pro-akinetes induced during exponential phase divided before maturation, while those induced during late exponential phase, could achieve full maturity. Growth and nitrogenase activity was unaffected while there was an increase in mean cell length on treatment of A. cylindrica with active factor(s) from neo-peptone, indicating that the effect may be mediated through cell division process(es).Abbreviations used N Nitrogen - chl chlorophyll     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Fructose supports glycogen accumulation,heterocysts differentiation,N2 fixation and growth of the isolated cyanobiont Anabaena azollae

Cells of the cyanobiont Anabaena azollae isolated from the water fern Azolla filiculoides were found to take up and utilize fructose in the light for mixotrophic growth. Fructose was favored by the cyanobiont as a substrate over sucrose and glucose. Cell growth in the presence of 8 mM fructose led to glycogen accumulation in the cells which approached 20% of the cell dry weight within 2 to 3 days, followed by reduction of glycogen content during the fourth day. Glucose-6-phosphate dehydrogenase activity was increased 5–6-fold in the fructose grown cells from the third day of growth onwards. The frequency of heterocysts in fructose-grown cells increased from 6 to 18%, and acetylene reduction by nitrogenase was increased 3-fold in the presence of fructose as compared with control cells, with maximum values observed between the third and fifth day of mixotrophic growth. Fructose-supported growth yielded a 2–4-fold increase in cell dry weight over controls.It is suggested that fructose-supported development and growth of the cyanobiont in batch cultures may resemble its mixotrophic growth and development in situ in the leaf cavity of the host fern Azolla.Abbreviation G6PDH glucose-6-phosphate dehydrogenase     

Comparison of Photosystem-I and Photosystem-II activities of spheroplasts from normal and photobleached Anabaena cylindrica

The analysis of photochemical activities of Photosystem I and Photosystem II in spheroplasts from normal and photobleached Anabaena cylindrica showed an increase in Photosystem II activity relative to Photosystem I in photobleached cells. We suggest that the reasons for this modification in photochemical activity are, (i) a rearrangement of pigments between the two photosystems, and (ii) improved functional condition of the photosynthetic units in Photosystem II.Abbreviations PSI Photosystem I - PSII Photosystem II - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - MV methylviologen - DCPIP 1,6-dichlorphenol indophenol - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - FeCN Ferricyanide - APC anophycocyanin - PC phycocyanin     

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O₂ production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.     

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.     

Utility of phyllosphere N2-fixing micro-organisms in the improvement of crop growth

Summary The beneficial effect of spraying some highly active phyllosphere N₂-fixing microorganisms on high and low yielding cultivars of rice plants as compared with that of urea applied at different doses are described. The dry weight, N-content, 1000 grain weight, and yield were remarkably increased in all cases with the application of phyllosphere microorganisms. The performance of two isolates KUP₄ and KUPBR₂ with IR-8 and IR-26 rice, was better than that of 52 kg urea-N per hectare. IR-579 rice leaves in association with some phyllosphere bacteria reduced acetylene at the rate of 664–816 nmoles/g leaf/h. In IR-26 rice the effect of application of bacterial suspension at three phases of plant growth corresponded very well with that of urea application in three split doses under identical conditions. Recommended fertilizer rates produced the same yield as the half dose plus bacterial spray in the cultivars Pankaj and Rupsail. Fertilizer application in Pankaj and Rupsail rice reduced nitrogenase activity and the beneficial effects of phyllosphere N₂-fixation was reduced by 40–55%.     

Solubilization of nitrate reductase from the blue-green alga Anabaena cylindrica

Nitrate reductase was solubilized and purified from Anabaenacylindrica by Triton X-100 treatment of particulate preparationsfollowed by adsorption on calcium phosphate gel. Reduced methylviologen, FAD or FMN, but not ferredoxin, served as an effectiveelectron donor for the nitrate reduction by solubilized nitratereductase. ¹This work was supported by a grant (4061) from the Ministryof Education (Received June 25, 1970; )     

The occurrence of the hydrogenase in some blue-green algae

Several blue-green algae were surveyed for the occurrence of the hydrogenase which was assayed by the oxyhydrogen or Knallgas reaction in the intact organisms. In aerobically grown cultures, the reaction was detectable in Anabaena cylindrica, Nostoc muscorum and in two Anabaena variabilis species, whereas virtually no activity was observed in Anacystis nidulans and Cyanophora paradoxa. In these latter two algae, the reaction was, however, found after growth under molecular hydrogen for several days, which drastically increased the activity levels with all the algae tested. In the nitrogen fixing species, the activity of the Knallgas reaction was enhanced when all combined nitrogen was omitted from the media. H₂ and hydrogenase could not significantly support the CO₂-fixation in photoreduction experiments with all blue-green algae investigated here. Hydrogenase was assayed by the dithionite and methyl viologen dependent evolution of hydrogen and was found to be present with essentially the same specific activity levels in preparations of both heterocysts and vegetative cells from Anabaena cylindrica. Na₂S₂O₄ as well as H₂ supported the C₂H₂-reduction of the isolated heterocysts. The H₂-dependent C₂H₂-reduction did not require the presence of oxygen but was strictly light-dependent where H₂ served as an electron donor to photosystem I of these cells. It is concluded that hydrogen can be utilized by two different pathways in blue-green algae.Abbreviations Chl chlrophyll - CP creatine phosphate - CP kinase creatine phosphokinase - DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea     

Isolation of gas vacuole-less mutants of the blue-green alga Anabaenopsis raciborskii

From a bloom forming blue-green alga, Anabaenopsis raciborskii, spontaneous mutants, which had lost the ability to form gas vacuoles have been isolated; the mutant frequency was 4.8×10^-3. The filaments of gas vacuole-less mutants settled at the bottom of flasks in liquid culture media unlike the parent alga. The growth and nitrogen fixation were comparatively poor in the mutants.