Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

5-hydroxylysine, an analogue of glutamate and lysine, causes $\text{NH}{}_4{}^{\mathrm{+}}$ production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of $\text{NH}{}_4{}^{\mathrm{+}}$ on heterocyst production and C₂H₂ reduction and in $\text{NH}{}_4{}^{\mathrm{+}}\text{-grown}$ cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary $\text{NH}{}_4{}^{\mathrm{+}}$ assimilation in A. cylindrica, that $\text{NH}{}_4{}^{\mathrm{+}}$ alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both.     

Mutants of Anabaena cylindrica altered in heterocyst spacing

Nitrosoguanidine induced mutants of Anabaena cylindrica have been obtained, which are altered in heterocyst spacing. In the wild type organism the pattern is composed of single intercalary heterocysts. The mutant patterns fall into several classes: those with only terminal heterocysts, with both terminal and intercalary heterocysts, with groups of heterocysts and those totally lacking heterocysts. The mutants are described in detail, and the various pattern modifications are interpreted in terms of a model we have proposed.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

Emerson enhancement of carbon fixation but not of acetylene reduction (nitrogenase activity) in Anabaena cylindrica

Summary Enhancement of carbon fixation was demonstrated in the bluegreen alga, Anabaena cylindrica, grown in either aerobic or microaerobic conditions. Under identical conditions no enhancement of acetylene reduction was observed. Light absorbed by photosystem I supported relatively more acetylene reduction than carbon fixation. No competition between the two processes was observed under light-limiting conditions. The findings suggest that carbon fixation and acetylene reduction may depend on different pools of reductant and ATP. When aerobically grown cells were placed in the dark or at limiting light intensities, acetylene reduction was higher in air than under argon. In contrast, carbon fixation was lower in air than in argon.     

Effects of amino acids on methionine-sulfoximine-induced heterocyst formation in Anabaena

Heterocyst development in ammonia-grown cultures of Anabaena variabilis and Anabaena 7120 was fully induced by the amino-acid analog methionine sulfoximine (MSO) at concentrations of 0.5–1.0 M. Glutamine, glutamate, aspartate, and alanine at 0.5 mM blocked the induction of heterocysts by MSO in A. variabilis. With Anabaena 7120, glutamine and glutamate were fully effective and alanine partially effective in preventing MSO-induced heterocyst formation. In MSO-treated algae, glutamine synthetase activity was reduced to less than 15% of control values within 4–6 h. Inactivation of the enzyme was prevented by all four amino acids tested.     

Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica.

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaema cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective election donors to nitro genase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum.     

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.     

Effects of adenine nucleotides and phosphate on adenosine triphosphate sulphurylase from Anabaena cylindrica.

Production of adenosine 5'-[35S]sulphatophosphate by a partially purified ATP sulphurylase from Anabaena cylindrica was inhibited by AMP, ADP and P1. Decreases in enzyme activity in the presence of these inhibitors were reversed by increasing the concentrations of ATP. The adenine nucleotides inhibited the enzyme competitively with respect to ATP. In the presence of P1, ATP showed a positive co-operative effect on enzyme activity. The inhibition by P1 was enhanced by increasing concentrations of MG2+. The effects of the adenine nucleotides and the interaction of P1 and Mg2+ on ATP sulphurylase activity are discussed in relation to the regulation of sulphate assimilation via the energy metabolism of the alga.     

Aluminum Effects on Uptake and Metabolism of Phosphorus by the Cyanobacterium Anabaena cylindrica

Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Preor post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AIPO₄ never exceeded 10% of the total phosphate concentration. The uptake of ³²P-phosphorus is not disturbed by aluminum either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica.     

Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica.

Glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from Anabaena cylindrica was inhibited by alanine, glycine, serine and aspartate. The effects of alanine and serine were uncompetitive with respect to glutamate, while those of glycine and asparatate were uncompetitive with respect to glutamate, while those of glycine and aspartate were non-competitive and mixed type respectively. Different pairs of amino acids and their various combinations caused a cumulative inhibition of the enzyme activity. Glutamine synthetase was also inhibited by ADP and AMP and both nucleotides affected the enzyme competitively with respect to ATP and non-competitively for glutamate. Inorganic pyrophosphate, between 2 and 3 mM, produced a very pronounced inhibiton of enzyme activity. The inhibition by PPi was uncompetitive for ATP. Various combinations of the adenine nucleotides, PPi and Pi exerted a cumulative inhibitory effect on the enzyme activity, as did the amino acids, in different combinations with either adenine nucleotides, PPi or Pi. The effects of the adenine nucleotides and the amino acids were more pronounced at higher concentrations of ammonia. Except for serine similar responses of these effectors were obtained with increasing concentrations of Mg2+. It is proposed that changes in the free concentrations of Mg2+ are important in energy-dependent regulation of the enzyme activity in this alga.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis.

Mutants of the cyanobacterium Anabaena variabilis that were capable of increased uptake of glutamine, as compared with that in the parental strains, were isolated. Growth of these mutants and their parental strains was measured in media containing N2, ammonia, or glutamine as a source of nitrogen. All strains grew well with any one of these sources of fixed nitrogen. Much of the glutamine taken up by the cells was converted to glutamate. The concentrations of glutamine, glutamate, arginine, ornithine, and citrulline in free amino acid pools in glutamine-grown cells were high compared with the concentrations of these amino acids in ammonia-grown or N2-grown cells. All strains capable of heterocyst differentiation, including a strain which produced nonfunctional heterocysts, grew and formed heterocysts in the presence of glutamine. However, nitrogenase activity was repressed in glutamine-grown cells. Glutamine may not be the molecule directly responsible for repression of the differentiation of heterocysts.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

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