The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

Posttranslational modification of dinitrogenase reductase in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> treated with fluoroacetate

Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate–polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.     

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5′-triphosphate and adenosine 5′-diphosphate in catalysis by nitrogenase

The electron paramagnetic resonance spectra of azoferredoxin and molybdoferredoxin, components of the nitrogenase of Clostridium pasteurianum, disappear when the proteins are oxidized by certain dyes. When molybdoferredoxin and azoferredoxin were mixed in a 1 to 2 molar ratio, the electron paramagnetic resonance spectrum of the mixture was the sum of the two spectra with the exception of a slight change in the azoferredoxin signal. Addition of magnesium ATP and dithionite to this reconstituted nitrogenase resulted in a rapid change in the spectrum of both nitrogenase components; the molybdoferredoxin spectrum at all g-values decreased with a half-life less than 70 ms to 40% of its original size whereas the azoferredoxin signal changed in shape and size with a half-life of less than 40 ms. If an ATP-generating system was added instead of MgATP so that no ADP accumulated, then the molybdoferredoxin signal almost completely disappeared and the azoferredoxin signal changed in shape and slightly in size. These changes occurred at molar ratios of molybdoferredoxin to azoferredoxin from 1:14 to 1:0.2. If the reaction was allowed to consume the reductant, then the molybdoferredoxin signal(s) was restored but the azoferredoxin signal disappeared. The signal of azoferredoxin was restored and the signal of molybdoferredoxin again disappeared on addition of more reductant. The data suggest that for nitrogenase to catalyze the reduction of substrates, the magnesium ATP-reduced azoferredoxin complex is formed first and this complex then reacts with molybdoferredoxin to allow electron flow. In addition the data suggests that the rate-limiting reaction is an ATP-mediated electron flow from azoferredoxin to molybdoferredoxin. Finally the results show that no flow of electrons from azoferredoxin or molybdoferredoxin occurs when a mixture of ADP and ATP in a molar ratio of 2:1 is added initially or is reached by conversion of ATP to ADP and inorganic phosphate during reduction of protons. A mechanism consistent with these findings is proposed.     

Glycogen content and nitrogenase activity in Anabaena variabilis

Nitrogenase (=acetylene-reducing activity) was followed during photoautotrophic growth of Anabaena variabilis (ATCC 29413). When cell density increased during growth, (1) inhibition of light-dependent activity by DCMU, an inhibitor of photosynthesis, increased, and (2) nitrogenase activity in the dark decreased. Addition of fructose stabilized dark activity and alleviated the DCMU effect in cultures of high cell density.The resistance of nitrogenase towards oxygen inactivation decreased after transfer of autotrophically grown cells into the dark at subsequent stages of increasing culture density. The inactivation was prevented by addition of fructose. Recovery of acetylene-reducing activity in the light, and in the dark with fructose present, was suppressed by ammonia or chloramphenicol. In the light, also DCMU abolished recovery.To prove whether the observed effects were related to a lack of photosynthetic storage products, glycogen of filaments was extracted and assayed enzymatically. The glycogen content of cells was highest 10 h after inoculation, while light-dependent nitrogenase activity was at its maximum about 24 h after inoculation. Glycogen decreased markedly as growth proceeded and dropped sharply when the cells were transferred to darkness. Thus, when C-supply (by photosynthesis or added fructose) was not effective, the glycogen content of filaments determined the activity of nitrogenase and its stability against oxygen. In cells lacking glycogen, nitrogenase activity recovered only when carbohydrates were supplied by exogenously added fructose or by photosynthesis.Abbreviations Chl chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Mechanism of nitrogenase switch-off by oxygen.

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of hydrogen-dependent nitrogenase activity by adenylates and electron flow in heterocysts of Anabaena variabilis (ATCC 29413)

Hydrogen-dependent nitrogenase activity was studied in heterocysts, isolated from the filamentous cyanobacterium Anabaena variabilis (ATCC 29413). Hydrogen provides reductant and ATP for nitrogenase via linear electron flow through Photosystem I. This allows for regulation of nitrogenase activity by controlling the turnover of the photosystem. When nitrogenase activity was varied by changing either the light intensity or the supply of reductant (i.e., hydrogen) or by inhibition of photosynthetic electron transport by DBMIB, no rate-dependent changes in cellular ATP concentrations were observed. This homeostasis of ATP was perturbed by addition of metronidazole, acting as alternative electron sink to nitrogenase, and by uncoupling agents like FCCP, gramicidin and nigericin. Valinomycin (in presence of KCl) exerted little effect on nitrogenase activity and adenylate pool composition. Metronidazole increased and uncoupling agents decreased cellular ATP concentration, ATP/ADP ratio and energy charge. Inhibition of nitrogenase activity by metronidazole was caused by reductant limitation; inhibition by uncoupling agents was due to energy limitation. Control exerted on nitrogenase activity by ATP (energy limitation) was more pronounced at high rates of electron flow to nitrogenase than during reductant limitation. When cellular ATP synthesis was suboptimal due to partial uncoupling, the connection of phosphorylation and nitrogenase activity by electron transport allowed for homeostasis of ATP also at a lowered cellular concentration.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.     

Limiting factor of nitrogenase system mediating hydrogen production of Rhodobacter sphaeroides S

To explain the decrease of hydrogen production rate in the batch culture of Rhodobacter sphaeroides S, the activities of enzymes related to the TCA cycle, nitrogenase in cell-free extracts, ATP generation by chromatophores, and ferredoxin were examined at the beginning, middle and end of the hydrogen production phase of batch culture. The activities of TCA cycle enzymes, nitrogenase and ATP generation were found to remain at almost the same level throughout the culture, while ferredoxin activity decreased linearly with time. In addition, by bubbling N₂ gas into the culture broth at the end of the culture, the hydrogen production rate was restored to the initial level through the increase of the ferredoxin activity. Although the decrease of ferredoxin activity and its restoration by bubbling N₂ gas remained unexplained, ferredoxin activity was considered to be a key function in the nitrogenase system for H₂ production by this photosynthetic bacterium.     

Studies on the effect of NAD(H) on nitrogenase activity in Rhodospirillum rubrum

The effect of NAD(P) and analogs of this nucleotide on nitrogenase activity in Rhodospirillum rubrum has been studied. Addition of NAD⁺ to nitrogen fixing Rsp. rubrum leads to inhibition of nitrogenase. NADP⁺ has the same effect but NADH or analogs modified in the nicotinamide portion do not cause inhibition. In contrast to ammonium ions, addition of NAD⁺ leads to inhibition of nitrogenase in cells that have been N-starved under argon. The inhibitory effect of NAD⁺ is more pronounced at lower light intensities. Addition of NAD⁺ also leads to inhibition of glutamine synthetase, a phenomenon also occurring when “switchoff” is produced by the addition of effectors such as ammonium ions or glutamine. It is also shown that NAD⁺ is taken up by Rsp. rubrum cells.     

The regulation of ferredoxin-dependent nitrogenase activity in Rhodospirillum rubrum and Rhodopseudomonas capsulata

Abstract The regulatory properties of Rhodospirillum rubrum nitrogenase reduced by either the endogenous electron donor (ferredoxin) or an artificial donor (dithionite) were examined. The nitrogenase obtained from glutamate-grown cells required activating enzyme for maximum activity with either reductant. The activating enzyme requirement of ferredoxin-dependent nitrogenase activity implies a physiological significance of the activating enzyme in R. rubrum. Rhodopseudomonas capsulata nitrogenase also required activating enzyme when dithionite was the reductant, but there appeared to be no activating enzyme requirement with ferredoxin as the reductant. Because the catalytic activity of the enzyme was very low under these conditions, the physiological significance of activating enzyme in this organism remains in question.     

Classes of Anabaena variabilis mutants with oxygen-sensitive nitrogenase activity.

Mutants of Anabaena variabilis deficient in the envelope glycolipids of heterocysts have no or very low nitrogenase activity when assayed aerobically. Revertants capable of aerobic growth on N2 have increased quantities of these glycolipids. Among mutants which require fixed nitrogen for growth in air and which have a normal complement of glycolipids, one expresses high nitrogenase activity at low oxygen tension. Three others show high nitrogenase activity only in the presence of dithionite and are therefore impaired in electron transfer.     

The role of cellulose-decomposing fungi in nitrogenase activity ofAzotobacter chroococcum

Azotobacter chroococcum was grown on cultures containing five carbon sources alone and also in co-cultures with three cellulolytic fungi (Aspergillus niger, Penicillium funiculosum andTrichoderma harzianum). In the absence of fungal species, nitrogenase activity was relatively low. The best nitrogenase activity was recorded in cultures containing faba bean straw followed by that in cultures having wheat straw, sugar cane leaves, carboxymethyl cellulose (CMC) or cellulose. In co-cultures with fungi,Azotobacter showed substantial nitrogenase activity on all tested substrates.Azotobacter —Trichoderma association showed the highest nitrogenase activity.     

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.     

Purification of azotophore membranes containing the nitrogenase from Azotobacter vinelandii

Azotophore membranes containing nitrogenase have been purified in high yield from $\text{A. vinelandii}$ by differential and sucrose density gradient sedimentation. The purified preparations appeared as uniform vesicular membranes of 40–75 nm diameter containing the majority of the nitrogenase activity from these cells and were readily separated from the intracytoplasmic membranes containing the cytochromes of the respiratory electron transfer system. The yield and specific activity of azotophores from cells broken by mechanical or osmotic treatment were similar.     

Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

Effects of water stress on nitrogenase activity in Abuts incana

Tolerance to water stress was studied in plants of grey alder, Alnus incana (L.) Moench, grown in a climate chamber in pots of sand supplied with a nitrogen-free nutrient solution. The plants were subjected to a single drying and recovery cycle, during which acetylene reduction, transpiration and stomatal resistance were measured. At different stress levels the plants were placed in a closed system to equilibrate the water potential in the plant-soil system. The water potential of the plants was determined, after which they were watered and their recovery studied. Nitrogenase activity showed low tolerance to water deficit. At moderate stress (−0.6 to −0.8 MPa) acetylene reduction was reduced by half, and at more severe stress, (< −1 MPa) activity was near zero. There was a rapid decrease in nitrogenase activity coincident with stomatal closure, which indicates a continuous need for photoassimilates for nitrogenase activity. Nodules or nitrogenase activity seemed to be weak sinks for assimilates compared with root pressure bleeding. Measurements of nitrogenase activity in root nodule homogenates supplied with ATP and reductant suggested a loss of active nitrogenase in the nodules in response to water stress. The recovery from moderate stress or long dark treatment took several days, and recovery from severe stress took still longer. Shortage of assimilates and disturbances in oxygen and nitrogen balances in the nodules are discussed as reasons for the reduced nitrogenase activity in response to water stress.     

The activity of two forms of nitrogenase from Rhodopseudomonas capsulata in the presence of different electron donors

Abstract The active form of Rhodopseudomonas capsulata nitrogenase is active in vitro when dithionite or ferredoxins from this bacterium are used as electron donors. The presence of the activating nitrogenase enzyme and Mn²⁺ ions is needed for functioning of the inactive form of Rh. capsulata nitrogenase in vitro with the use of dithionite as an electron donor. The use of Rh. capsulata ferredoxins as electron donors in vitro makes the inactive form of nitrogenase fully active as is the case in vivo.     

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.     

Diurnal Nitrogenase Modification in the Cyanobacterium Anabaena variabilis*

The nitrogen-fixing cyanobacterium Anabaena variabilis (ATCC 29413) was cultivated as continuous culture under a 12 h: 12 h light-dark cycle. In the light, photosynthetic activity resulted in a continuous increase in cellular glycogen content, followed by an almost complete dissimilation of the polysaccharide during the dark period. Nitrogenase activity, assayed by the acetylene reduction technique, was low at the end of the dark period and increased quickly upon illumination to reach a maximum after 4 to 6 h of light. The activity rapidly declined after darkening the culture. Increase and decrease of activity were accompanied by a change in the electrophoretic mobility of the Fe-protein of nitrogenase (dinitrogenase reductase) indicative of enzyme modification being involved in the diurnal control of nitrogenase activity. Modification and demodification of the Fe-protein were not coupled to the cell cycle since they followed darkening and illumination when the light or dark periods were changed. Addition of fructose increased nitrogenase activity even in darkness and caused demodification of the Fe-protein. Ammonium chloride supplied at the onset of illumination slowed down the increase of nitrogenase activity. A delayed inhibition of the enzyme was accompanied by partial Feprotein modification only. The reaction was completed after transfer to darkness. The function of enzyme modification in maintaining a constant C: N ratio is discussed and a dominating role of carbohydrate supply in this regulation is indicated by the reported findings.