Effects of long-term treatment with acetylene on nitrogen-fixing microorganisms.

Long periods of experimental incubation with acetylene led to a multifold enhancement of acetylene-reducing activity in Anabaena cylindrica, Anabaenopsis circularis, Rhodospirillum rubrum, and Azotobacter vinelandii. Rates of acetylene reduction showed a gradual increase and reached a peak after 2 to 6 h of continuous incubation under acetylene. Thereafter, enzyme activity rapidly declined. A similar enhancement of ethylene production was observed when pretreatment with acetylene was interrupted periodically by a brief exposure to ambient (or oxygen-free) atmosphere without acetylene although the decline of acetylene-reducing activity was less rapid. Pretreatment with acetylene depressed photosynthetic 14CO2 fixation and 15N2 incorporation in Anabaena cylindrica. It is concluded that assessments based on long-term experimental incubation with acetylene may grossly overestimate the actual quantities of fixed nitrogen in the field.     

Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria.

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.     

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.     

Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.     

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.     

柱孢鱼腥藻的铁蛋白与棕色固氮菌的钼铁蛋白的交叉互补作用

纯化的柱孢鱼腥藻铁蛋白能够与棕色固氮菌的钼铁蛋白有效地交叉反应,展现较高的活性。此异源交叉反应的乙炔还原比活及放氢比活,分别是蓝藻同源互补比活的83.8及66.7％。比较藻铁蛋白与菌钼铁蛋白异源交叉反应及藻固氮酶组分之间的同源反应的动力学特点时发现,铁蛋白对钼铁蛋白的最佳克分子比数前者(异源交叉反应)较后者(藻同源反应)为高,前者为5,后者为1;但反应的时间进程两者差别不大。     

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.     

Anaerobic and aerobic hydrogen gas formation by the blue-green alga Anabaena cylindrica.

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5'-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Anaerobic and Aerobic Hydrogen Gas Formation by the Blue-Green Alga Anabaena cylindrica

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5′-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

二株固氮芽孢杆菌的固氮特性研究

在无氮Ｈｉｎｏ培养基ＪＲ_１菌株的乙炔还原活性在氧浓度３％时最高,ＺＺ_１２菌株则随氧浓度上升而下降,但两菌株在空气条件下也能有效固氮;在有０．０１％酵母膏存在时,各氧气浓度菌株的乙炔还原值起伏不大。０．０２％铵盐存在时ＪＲ_１乙炔还原作用最强,但活性随硝酸盐浓度上升而一直下降;０．０２％硝酸盐时ＺＺ_１２菌株乙炔还原力最强,但其活性值随铵盐浓度递增而逐减。土壤中存在可利用糖类时ＪＲ_１和ＺＺ_１２     

几种固氮蓝藻的固氮酶活性及其某些特性

对三种固氮蓝藻:固氮鱼腥藻(水生686)、柱孢鱼腥藻和鱼腥藻7120的整细胞及无细胞抽提液的固氮酶活性,进行了比较研究。水生686的整细胞酶活虽然不低(51.9毫米乙烯峰高/光密度/30分),仅次于柱孢鱼腥藻,但其无细胞抽提液的酶活却最低。这可能与它含有大量藻胶有关。研究了Mn++、Fe++对蓝藻固氮酶的作用,以及测定其在不同酶浓度下的反应动力学表明:柱孢鱼腥藻中不存在象深红螺菌中所看到的那种激活因子。用甲苯-乙醇溶液处理藻细胞,对固氮酶作原位测定,探索了它的氧损伤及氧保护机理。       

Endorhizal and Exorhizal Acetylene-reducing Activity in a Grass (Spartina alterniflora Loisel.)-Diazotroph Association

Earlier studies indicated that bacteria responsible for nitrogenase activity of some grasses are located inside the roots. Those studies were conducted with excised roots in which a long, unexplained “lag phase” occurred before initiation of nitrogenase activity. When hydroponically maintained Spartina alterniflora Loisel. was incubated in a two-compartment system with acetylene, ethylene was produced following, at most, a 2-hour lag in both the upper (shoot) and lower (roots + water) phases. Ethylene production in the upper phase not attributable to leaf-associated acetylene-reducing activity or to diffusion of ethylene from around the roots is considered to represent “endorhizal acetylene-reducing activity,” the internally produced ethylene diffusing into the upper phase via the lacunae. Ethylene produced in the lower phase is designated “exorhizal acetylene-reducing activity.” The endorhizal acetylene-reducing activity, in comparison to exorhizal activity, was relatively insensitive to additions of HgCl₂, NH₄Cl, or carbon sources to the lower phase. Post-lag acetylene-reducing activity of roots excised from plants growing in soil responded to additions in a manner similar to that of endorhizal acetylene-reducing activity, whereas post-lag acetylene-reducing activity of rhizosphere soil responded in a manner similar to that of exorhizal acetylene-reducing activity.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

The effect of nickel on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis.

Anabaena inaequalis was sensitive to nickel ion in the order of decreasing sensitivity of growth, photosynthesis, and acetylene reduction. At a culture density of 9 x 10(4) cells per millilitre, growth after 12 days was completely inhibited by 0.125 ppm (microgram/mL) Ni2+. Nickel caused the increase of both the lag phase of growth and the culture doubling time, and caused the retardation phase to be sooner. Photosynthesis and acetylene reduction were completely inhibited by 10 and 20 ppm Ni2+, respectively, at a cell concentration of 1.3 x 10(6) cells per millilitre. Preincubation for 24 h in the presence of nickel ion significantly increased the sensitivity of photosynthesis and acetylene reduction. Under these conditions acetylene reduction was more sensitive than photosynthesis. Nickel ion reduced culture growth by 35% at a level of 0.05 ppm and inhibited that culture's acetylene-reducing ability by 29% while leaving photosynthesis unaffected. Nickel caused some damage to filament apical cells and induced pigment bleaching in aged cultures. Nickel toxicity was proposed to be due to poisoning of intracellular enzyme systems by nickel ions.     

Pleomorphism and acetylene-reducing activity of free-living rhizobia.

Cowpea-type Rhizobium sp. strain 32H1 and Rhizobium japonicum USDA 26 and 110 grown on a glutamate-mannitol-gluconate agar medium showed increases in the number of pleomorphic cells coincident with their acetylene-reducing activity. Pleomorphs appeared to be inhibited in growth nonuniformly, because acetylene-reducing cultures were mixtures of rod, branched (V, Y, and T), and other irregularly shaped cells. In contrast, strain USDA 10 consistently failed to reduce acetylene, even though it also could grow and yield pleomorphic cells under various conditions. With minimal inhibitory supplements (5 micrograms per ml of medium) of nalidixic acid and novobiocin as cell division inhibitors, an increase in pleomorphic cells was observed, but the inhibited cultures displayed lower acetylene-reducing activity. A study of pleomorphic cells derived in different ways indicated that not all pleomorphs reduce acetylene.     

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; )     

Formation of glutamine from [13n]ammonia, [13n]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica.

A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C2H4 formed per mg of heterocyst chlorphyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of [13N]NH3 and of amidelabeled [13N]glutamine form [13N]N2. A method is also described for the production of 6-mCi amounts of [13N]NH3. Isolated heterocysts formed [13N]glutamine from [13N]NH3 and glutamate, and [14C]glutamine from NH3 and [14C]glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N2 by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either [13N]glutamine or alph-[14C]ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed [14C]glutamate rapidly from alpha-[14C]ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implication of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.     

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.     

Protective mechanisms in photosynthesis of Anabaena cylindrica

The role of O₂ photoreduction was studied in intact cells of normal and photobleached Anabaena cylindrica Lemm. strain PCC 7122. We found that O₂ photoreduction represents a protective mechanism against over-reduction of the photosyn-thetic electron transport chain only in normal Anabaena cells. This protective mechanism was not functioning in photobleached cells in spite of the increased rate of photosynthetic electron flow. A new electron acceptor, the induced reversible hydrogenase, is suggested to be operating in photobleached Anabaena cylindrica .     

Salt stress sensitivity of nitrogen fixation in Enterobacter agglomerans strains

Two strains 333 to 339 of Enterobacter agglomerans were selected in the present study to evaluate the response of increasing concentrations of NaCl on growth, N(2)-fixation, and nitrogenase activity/synthesis. E. agglomerans strains 333 and 339 showed optimum growth and acetylene-reducing activity with 0.5 to 1.0% NaCl in a nitrogen-free minimal medium (NFDM) with glucose, respectively, in 28 h incubation, although both strains displayed better growth and acetylene-reducing activity with 3.0% and 2.0% NaCl after 52 h and 100 h incubation periods than the 28 h culture did. Our experiments with shiftings of salt concentrations in NFDM medium indicated that a synthesis of nitrogenase enzyme was generally more sensitive to higher concentrations of NaCl than nitrogenase activity was.