Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA

Anabaena sp. CA does not synthesize heterocysts or express nitrogenase activity when grown with nitrate as the nitrogen source. Heterocysts and nitrogenase are induced in such cultures by various tryptophan analogs. The effect does not require inhibition of de novo protein synthesis in the culture. It is restricted to tryptophan analogs only, and, more specifically, to those which can be incorporated into proteins. dl-7-Azatryptophan was effective at triggering both heterocysts and nitrogenase when incubated in the culture for only 1–2 h, even though 6–7 h was required for heterocysts to fully mature and nitrogenase activity to be expressed. Chloramphenicol completely negated this effect, supporting the idea that the analogs are either incorporated into protein themselves or trigger the synthesis of proteins which initiate complete development of mature heterocysts. Using toluene-permeabilized cells, we have shown that anthranilate synthetase, the first key enzyme in tryptophan biosynthesis, has glutamine-dependent activity. This activity can be effectively feedback inhibited by the various tryptophan analogs at concentrations which are also effective in triggering heterocyst differentiation. These data provide firm evidence for a link between tryptophan biosynthesis, nitrogenase synthesis, heterocyst differentiation, and primary ammonia assimilation.     

Cellular differentiation and nitrogenase activity in the cyanobacteriumAnabaena

Nitrogenase activity at periods of differentiation of heterocysts and akinetes was assayed by the acetylene reduction technique. There was no nitrogenase activity in ammoniumgrown, non-heterocystousAnabaena sp.; the activity appeared only after a lag-phase of about 17 – 21 h after the ammonium-grown culture had been transferred to medium free of combined nitrogen. This activity started appearing as the proheterocysts were developing to mature heterocysts. Maximum nitrogenase activity was attained with exponential phase of culture and mature heterocysts. This activity gradually decreased with the differentiation of akinetes. Only insignificant nitrogenase activity was observed in old cultures in which most cells had matured into akinetes.     

Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O₂ for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O₂ tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.     

D-erythrose supports nitrogenase activity in isolated Anabaena sp. strain 7120 heterocysts.

Among organic compounds tested for their ability to support nitrogenase activity in isolated heterocysts of Anabaena sp. strain 7120 under argon, D-erythrose (5 mM) was unique in supporting acetylene reduction at 10 times the control rates. Higher concentrations of D-erythrose exhibited substrate inhibition. At 50 kPa of H2, all concentrations of D-erythrose inhibited H2-supported acetylene reduction. The effects of D-erythrose on nitrogenase activity were explored. Erythrose enhanced 15N2 incorporation by heterocysts, but NADP+ did not enhance erythrose-supported acetylene reduction. H2 protected nitrogenase from O2 inactivation, but erythrose did not; erythrose did not counter protection by H2. Tests with inhibitors of electron transport showed that erythrose-supported acetylene reduction requires electron flow through ferredoxin, a b-type cytochrome, and a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-sensitive transfer agent whose electron flow is not mediated through the plastoquinone and Rieske iron protein.     

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.     

Effects of D-erythrose on nitrogenase activity in whole filaments of Anabaena sp. strain 7120.

D-Erythrose, which has been shown to enhance nitrogenase activity (acetylene reduction) by isolated heterocysts, was studied for its effects on nitrogenase activity and nitrite uptake by whole filaments of Anabaena sp. strain 7120. D-Erythrose had little effect on acetylene reduction in the light; however, at a concentration of 10 mM, it could restore 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea-inhibited or dark-limited levels to light-supported levels. Sucrose, glucose, or fructose did not exhibit similar effects. D-Erythrose had little effect on nitrite uptake, an indirect measure of nitrite reductase activity by nitrate-grown whole filaments. It was concluded that erythrose effects were mediated by heterocysts and were therefore specific for nitrogenase.     

Expression of Rhizobial Nitrogenase: Influence of Plant Cell-Conditioned Medium

Conditioned medium was obtained from suspension cultures of soybean (Glycine max L. Merrit) cells after incubating them for 4 to 8 days with rhizobia which were separated from the soybean cells by two dialysis bags, one within another. This conditioned medium from the plant cell side (PCM) of the two membranes was used to elicit and influence nitrogenase activity (acetylene reduction) in rhizobia. When conditions for obtaining PCM from the soybean cell suspension cultures were varied, it could be shown that freshly grown rhizobia were able to induce active compounds in the PCM. These compounds caused acetylene reduction activity in test rhizobia under conditions where control rhizobia, containing various substrates, showed little or no acetylene reduction activity. Rhizobia that were already capable of acetylene reduction could not induce such compounds in the PCM when this was included with test rhizobia. The PCM from soybean cultures was also found to aid the expression of nitrogenase activity in suspension cultures of rhizobia normally associated with either peas, lupins, broad beans, or clovers. This is the first communication indicating nitrogenase activity in freeliving cultures for various species of rhizobia.     

Heterocystless mutants of Anabaena PCC7120 with nitrogenase activity

Following NTG mutagenesis, four independent mutants of Anabaena PCC7120 defective in heterocyst differentiation were isolated. These fell into 2 distinct classes; (1) those unable to differentiate heterocysts or show whole-cell acetylene reduction activity; and (2) those unable to differentiate heterocysts but capable of microaerobic acetylene reduction. All mutants grew equally well as the wild type with added nitrogen sources and showed no apparent differences in glutamine synthetase or glutamate synthase activities compared with the wild type. The mutants of class (2) evolved H₂ only under microaerobic conditions, suggesting that H₂ is evolved via nitrogenase in Anabaena PCC7120.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Oxygen Sensitive Nitrogen-Fixing Mutants of Anabaena

Two Anabaena mutants having heterocysts but incapable of fixing molecular nitrogen in air have been isolated by using ultraviolet radiation or NTG mutagenesis. Their vegetative cells differentiated into heterocysts at a higher frequency than that of the wild type. The phenotype of the mutants is stable and a low frequence of spontaneous reversion was observed. Under microaerobic condition the mutants cells can express the genetic information which encodes nitrogenase synthesis and were capable of utilizing nitrogen for growth with a low acetylene reductiop activity. The level of nitrogenase activity was correlated reciprocally with the content of cell phycocyanin and the light intensity. Both synthesis and activity of the mutant nitrogenase were very sensitive than wild type to the oxygen in vive. Introduction of 1% O2 (v/v) into the gas phase inhibited evidently acetylene reduction. Exposure of the mutant suspension to 20% O2 (v/v) resulted in total and irreversible denaturation of nitrogenase. Withdrawing of O2 in gas phase, the nitrogenase was synthesized de nero; The synthesis process was repressed by chloramphenical or ammonia. The nitrogenase activity of mutant cells increased significantly either by nitrogen- starvating to decrease the phycocyanin content or by lowering the light intensity. Specifically, during the anaerobic induction by treating the mutants filaments with diehloromethylurea which prevents photosynthetic oxygen production, the specific activity of mutant nitrogcnase was equivalent nearly to that of wild type. The ability to reduce 2, 3, 5-triphenyltetrazolium was lower in heterocysts and vegetative cells of mutants than in that of wild type. The results suggest that the oxygen sensitivity of nitrogen fixation by heterocystous bluegreen algal mutants may be duc to the defect of some enzymic systems which might play a role in scavenging oxygen toxity, so that the process of nitrogen fixation is inhibited by the active oxygen produced by vegetative cells. The mechanism of protecting nitrogenase from oxygen damage in blue-green algae is discussed.     

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

Phosphorylation and nitrogenase activity in isolated heterocysts from Anabaena variabilis (ATCC 29413)

Adenylate-pool composition, energy charge, and nitrogenase activity were examined in isolated heterocysts from Anabaena variabilis (ATCC 29413). ATP formation was detected as a light- or oxygen-induced increase in ATP concentration. No cofactors or substrates had to be added for photophosphorylation to occur, whereas oxidative phosphorylation was dependent on hydrogen and oxygen (Knallgas reaction). The increase in ATP concentration was reflected by a decrease in AMP concentration, accompanied by small changes in ADP levels. Thus, a regulation of the adenylate pool by a myokinase (adenylate kinase) has to be assumed. Upon dark-light transitions, the energy charge in heterocysts increased from values below 0.4 to values approaching 0.8. High energy-charge values, reached in the light only, allowed for high rates of acetylene reduction in the presence of hydrogen. The increase in the energy charge in the dark to approx. 0.64 by addition of oxygen (5% ($\text{v}\text{v}$) in the presence of hydrogen) resulted in low nitrogenase activities, generally not exceeding 1–3% of the light-induced rates. In the dark, oxygen concentrations above 10% were inhibitory to both ATP formation and acetylene reduction. Increasing light intensities led to a steep increase in energy charge followed by an increase in nitrogenase activity. Plotting enzyme activity versus energy charge, a nonlinear, asymptotic relationship was observed.     

Inoculationin vitro of wheat seedlings and wheat straw cultures withBacillus azotofixans

Bacillus azotofixans is a recently described species capable of fixing molecular nitrogen efficiently.Ecological studies performed in monoxenic wheat cultures, both in 0.7% agar and in vermiculite-sand mixture, showed that no acetylene reduction occurred and that this bacteria did not grow when supplied only with the wheat plant root exudates. However, after glucose addition to the 0.7% agar cultures, acetylene reduction ability (ARA) was detected. Comparing ARA for media with glucose both with and without plants, it was observed that the plants supply some component leading to the increase of the nitrogenase activity, since the ARA doubled in the samples containing plants.In wheat straw cultures a fast growth of the bacteria was observed in the first 24 hours after inoculation, but no acetylene reduction was detected. After glucose addition to the media with and without straw, nitrogenase activity was detected.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Modes of reduction of nitrogenase in heterocysts isolated from Anabaena species

N₂ fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, d-glucose 6-phosphate, 6-phosphogluconate and dl-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

PHYSIOLOGICAL ECOLOGY OF NITROGEN FIXING BLUE-GREEN ALGAL CRUSTS IN THE UPPER-SUBALPINE LIFE ZONE1

Blue-green algal (cyanobacterial) crusts composed of nitrogen fixing Nostoc commune Voucher ex Born. et Flah. and Tolypothrix conglutinata var. colorata Ghose were studied in the upper-subalpine life zone, Mission Mountain Wilderness, Montana. Rates of ethylene production were highest in the submerged shoreline crusts, lower for exposed crusts pioneering rocky shorelines and lowest in the Carex meadow. Nitrogenase activity (acetylene reduction technique) was constant between 200–285% crust moisture content (wet/dry weight) and then rapidly declined to 0 between 200–140%. Optimal temperatures for ethylene production by illuminated cells was between 20–30° C for T. conglutinata, 20° C for N. commune and about 25° C in darkness for both species. Nitrogenase activity by T. conglutinata in culture was unaffected by repeated freeze-thaw treatments whereas N. commune was severely inhibited. In contrast, N₂-ase activity of these two species in an intact crust was unaffected by repeated freeze-thaw treatments. Application of nitrogen-free growth medium to intact crusts increased nitrogenase activity by 3.7 times implying that these were mineral deficient under field conditions. Photosynthesis was light saturated at 125 μmol-m^?2.s^?1 whereas nitrogenase activity was light independent for cells with carbohydrate reserves. When carbohydrate reserves were reduced by 8 h incubation in darkness, between 1–3 h of illumination were required to restore nitrogenase activity to 80% of the maximum rate. Biochemical pathway inhibitor studies employing DCMU, MFA, and CCCP showed that oxidative metabolism was the source of reductant for acetylene reduction. Tetrazolium precipitation in heterocysts paralleled acetylene reduction activity in the inhibitor treated cells.