Relation between nitrogenase synthesis and activity in a marine unicellular diazotrophic strain, Gloeothece sp. 68DGA (Cyanophyte), grown under different light/dark regimens

The relationship between the abundance of nitrogenase and its activity was studied in the marine unicellular cyanobacterium Gloeothece sp. 68DGA cultured under different light/dark regimens. The Fe‐ and MoFe‐protein of nitrogenase and nitrogen (N₂)‐fixing (acetylene reduction) activity were detected only during the dark phase when the cells were grown under a 12 h light/12 h dark cycle (12L/12D). Nitrogenase activity appeared about 4 h after entering the dark phase. Maximum nitrogenase activity occurred at around the middle of the dark phase, and the activity rapidly decreased to zero before the start of the light phase. The rapid decrease of nitrogenase activity and the Fe‐protein of nitrogenase near the end of the dark phase in 12L/12D were partly recovered by the addition of l ‐methionine‐sulfoximine, an inhibitor of glutamine synthetase. Diurnal oscillation of the abundance of nitrogenase was maintained in the first subjective dark phase (i.e. the period corresponding to the dark phase) after the cells were transferred from 12L/12D to continuous illumination. However, enzyme activity was detected only when photosynthetic oxygen (O₂) evolution was completely suppressed by reducing the light intensity or by the addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Nitrogenase always appeared in the cells about 16 h after starting the light phase, even when the 12L/12D cycle was modified by the addition or subtraction of a single 6 h period of light or dark. These results suggest the following: (i) N₂‐fixation by Gloeothece sp. 68DGA is primarily regulated by an endogenous circadian oscillator at the level of nitrogenase synthesis. (ii) The endogenous circadian rhythm resets on a shift of the timing of the light phase. (iii) Nitrogenase activity is not always reflected in the presence of nitrogenase. (iv) The activity of nitrogenase is negatively regulated by fixed nitrogen and the concentration of ambient O₂.     

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.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

光合细菌(Rhodopseudomonas capsulata)固氮活性与钼的吸收

利用紫色非硫细菌能在厌气光照下和好气黑暗下交替生长的特点和同位素~(99)Mo示踪,来探讨Rhodopseudomonas capsulata中Mo的积累与固氮酶合成的关系。 用硫酸铵和谷氨酸盐作为氮源,把Rps. capsulata置于厌气光照下生长。由于硫酸铵阻遏固氮酶,所以菌体内既无固氮酶活性也无~(99)Mo积累。而谷氨酸盐解遏固氮酶的合成,菌体则显示固氮活性并有~(99)Mo积累。 黑暗好气生长的Rps. capsulata菌体既无固氮活性,也没有~(99)Mo的积累。将这样的菌体转移到含~(99)Mo(无谷氨酸)的培养液进行光照,固氮酶活性迅速出现,同时有~(99)Mo的积累。在Rps. capsulata中钼的吸收与固氮酶的合成及活性是紧密偶联的。     

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     

浑球红假单胞菌及其谷氨酸合成酶突变株氢酶活性和合成

浑球红假单胞菌野生型菌株的氢酶表达被有机碳、氮底物所抑制。在光照和黑暗时,氧浓度变化对氢酶的作用不同,但高氧浓度都阻遏氢酶的表达。微量Ni~(2+)能专一性地促进氢酶活性,固氮酶的产氢也可以调节氢酶的表达水平。该野生菌株的GOGAT突变株缺乏固氮酶和氢酶活性,在加入谷氨酰胺合成酶抑制剂MSX后,固氮酶和氢酶以相关联的方式合成出来,固氮酶产生的氢看来诱导了氢酶的合成。然而在固氮酶不表达的情况下,外源氢也可诱导氢酶的合成。     

Hydrogenase activity in Rhodopseudomonas capsulata: relationship with nitrogenase activity.

Hydrogenase activity was found in cells of Rhodopseudomonas capsulata strain B10 cultured under a variety of growth conditions either anaerobically in the light or aerobically in the dark. The highest activities were found routinely in cells grown in the presence of H2. The hydrogenase of R. capsulata was localized in the particulate fraction of the cells. High hydrogenase activities were usually observed in cells possessing an active nitrogenase. The hydrogen produced by the nitrogenase stimulated the activity of hydrogenase in growing cells. However, the synthesis of hydrogenase was not closely linked to the synthesis of nitrogenase. Hydrogenase was present in dark-grown cultures, whereas nitrogenase synthesis was not significant in the absence of light. Unlike nitrogenase, hydrogenase was present in cultures grown on NH4+. Conditions were established which allowed the synthesis of either nitrogenase or hydrogenase by resting cells. We concluded that hydrogenase can be synthesized independently of nitrogenase.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light-dark Cycle

Nitrogenase of the non-heterocystous nitrogen-fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe-protein, Component I) and dinitrogenase reductase (Fe-protein, Component II). O. limosa was grown diazotrophically under an alternating light-dark cycle (16–8h light-dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light-dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm^?2 thin section were detected. Nitrate-grown cells did not contain nitrogenase label. Western blot analysis of the Fe-protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti-MoFe-protein antiserum revealed a single band of 56 kDa which was present throughout the light-dark cycle. Nitrate-grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light-dark cycle but that the Fe-protein is modified (inactive form) during the light period when nitrogenase activity is absent.     

Response of nitrogenase to altered carbon supply in a Frankia-Alnus incana symbiosis

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro ( Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that, in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.     

Effect of light intensity and inhibitors of nitrogen assimilation on NH4+ inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp

Nitrogenase activity in Rhodospirillum rubrum was inhibited by NH4+ more rapidly in low light than in high light. Furthermore, the nitrogenase of cells exposed to phosphorylation uncouplers was inhibited by NH4+ more rapidly than was the nitrogenase of controls without an uncoupler. These observations suggest that high levels of photosynthate inhibit the nitrogenase inactivation system. L-Methionine-DL-sulfoximine, a glutamine synthetase inhibitor, prevented NH4+ from inhibiting nitrogenase activity, which suggests that NH4+ must be processed at least to glutamine for inhibition to occur. An inhibitor of glutamate synthase activity, 6-diazo-5-oxo-L-norleucine, inhibited nitrogenase activity in the absence of NH4+, but only in cells exposed to low light. The mechanism of 6-diazo-5-oxo-L-norleucine inhibition appeared to be the same as that induced by NH4+, because nitrogenase activity could be restored in vitro by activating enzyme and Mn2+. The inhibitor data suggest that the glutamine pool or a molecule that responds to it activates the Fe protein-modifying (or protein-inactivating) system and that the accumulation of this (unidentified) molecule is retarded when the cells are exposed to high light. It was confirmed here that Anabaena nitrogenase is also inhibited by NH4+, but only when the cells are incubated under low light. This inhibition, however, unlike that in R. rubrum, could be completely reversed in high light, suggesting that the mechanisms of nitrogenase inhibition by NH4+ in these two phototrophs are different.     

KATAGNYMENE: CHARACTERIZATION OF A NOVEL MARINE DIAZOTROPH

The marine planktonic cyanobacterial genus Katagnymene was described by Lemmermann in 1900 and is found in oligotrophic tropical and subtropical oceans. The genus comprises two species, K. pelagica Lemmermann and K. spiralis Lemmermann, and both were observed in most stations sampled during a cruise in the southwest Pacific Ocean in 1998. Katagnymene is nonheterocystous and characterized by single ensheathed trichomes that do not form colonies. Acetylene reduction-GC demonstrated that natural populations of Katagnymene fixed nitrogen and that nitrogenase activity occurred exclusively in the light during a 12:12 light:dark cycle. Whole cell immunolocalization revealed that nitrogenase (the Fe protein) appeared in 7% of the total number of cells and that these were arranged in zones composed of consecutively arranged cells. At least one zone of nitrogenase-containing cells per trichome was found. Nitrogenase was present throughout both the day and night, as shown by Western blotting, and in the same percentage of cells. Ultrastructural immunolocalization on sectioned trichomes also confirmed the presence and localization of nitrogenase in Katagnymene. Cultures of Katagnymene are able to grow on nitrogen-free media and fix nitrogen only in the light. Finally, cloning and sequencing of nifH verified the diazotrophic nature of the genus Katagnymene and demonstrated a close relationship to members of the marine genus Trichodesmium.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

Boron Protection for O(2) Diffusion in Heterocysts of Anabaena sp. PCC 7119

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N₂ fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.     

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.     

Identification and study of growth and nitrogenase activity of nitrogen fixing cyanobacteria from tropical soil

Identification of cyanobacteria species has been performed on samples coming from two different harvest areas. The most important fixing belongs to Scytonema genus. The other genus identified are Nostoc and Lyngbia. Moreover, these cells are living closely with non-fixing cyanobacteria as well as with bacteria. The growth of cells as well as nitrogenase activity has been studied on a semi-axenic strain of Scytonema, a nitrogen fixing cyanobacterium, isolated from soil crusts. The cell growth is relatively show in liquid medium depleted in combined nitrogen. The growth rate increases when nitrates are supplied to cells. A release of ammonium is observed in medium during cell culture. This release exhibits several maxima and minima during cell growth. The heterocyst cells disappear within four days when filaments are growing in nitrates supplied medium. On the contrary, the heterocyst frequency increases up to more 5% in a nitrogen depleted medium. The heterocyst frequency reaches a maxima after 4 days of culture, then decreases later on. Nitrogenase activity changes during cells growth too. The maximum activity is observed after 5 to 6 days of culture to decrease after even though the cells are still in their exponential phase of growth. Nitrogenase activity increases with light intensity, what indicate a possible relation between photosynthetic and nitrogenase activities.     

A mutant of Anabaena sp. CA with oxygen-sensitive nitrogenase activity.

Studies on the O₂ protection mechanism for nitrogenase in a mutant (PM10) of $\text{Anabaena}$ sp. CA indicated that the ability to protect nitrogenase from O₂ was functionally impaired. Growth rates of PM10 were substantially improved when cells were cultured under microaerobic conditions. Nitrogenase activity was totally inhibited by exposure to O₂ for 30 min; partial restoration of activity was attained when cell suspensions were subsequently made microaerobic. Experiments in which induction of nitrogenase activity was followed indicated that the synthesis of the O₂ protection mechanism was temporally separated from synthesis of heterocysts and nitrogenase.     

Control of nitrogenase in a photosynthetic autotrophic bacterium, Ectothiorhodospira sp.

An Ectothiorhodospira species fixed nitrogen when grown as an autotroph in completely inorganic medium by using a variety of electron donors. The organism also used organic carbon sources; however, this required induction of synthesis of various enzymes, whereas the enzymes needed for autotrophic growth were synthesized constitutively. Nitrogenase induction and function were inhibited by ammonium chloride. Nitrogenase activity was dependent on light and inhibited by oxygen.     

Nitrogen fixation by Anabaena cylindrica

Summary The effects of oxygen, light and photosynthesis inhibitors on nitrogenase activities in Anabaena cylindrica batch cultures were followed as a function of time after inoculation. During the early rapid growth period the nitrogenase activities of cultures grown under air/CO₂ or N₂/CO₂ were relatively resistant to oxygen and DCMU inhibition. These cultures also exhibited oxygen-dependent nitrogenase activity in the dark of up to 50% of that measured in the light. After active growth ceased the cultures continued to slowly grow for a prolonged period of time. The nitrogenase activities of these old cultures were very sensitive to oxygen and DCMU inhibition. These cultures also had little or no dark nitrogenase activities. The photosynthesis inhibitor DBMIB was not a specific inhibitor of light-driven electron transport since it inhibited both light and dark nitrogenase activities. Nitrogenase activities induced under oxygen-free/CO₂ gas mixtures initially were significantly more sensitive to oxygen inhibition than those induced under air/CO₂. We discuss these results in relation to heterocyst function.