Induction of a nitrogenase activity rhythm inSynechococcus and the protection of its nitrogenase against photosynthetic oxygen

All oxygen levels are detrimental to the nitrogenase activity ofSynechococcus RF-1 cells. In continuous light, cultures maintain a high dissolved oxygen concentration and a continuous but usually low rate of nitrogenase activity.Cultures adapted to a light-dark regimen will reduce acetylene almost exclusively during the dark periods. When switched to continuous light, they continue to exhibit a diurnal rhythm in nitrogenase activity. While in continuous light, each upsurge of nitrogenase activity coincides with a marked drop in the net oxygen production rate; this drop is due largely to a concomitant increase in the dark respiration rate of the culture.The endogenous nitrogenase activity rhythm can be induced in continuous light by periodically lowering the oxygen concentration of the culture by either bubbling nitrogen through it or by treating the culture with 3(3,4-dichlorophenol)-1,1-dimethylurea (DCMU or diuron).     

Circadian rhythm mutants of the prokaryoticSynechococcus RF-1

Synechococcus RF-1 established circadian rhythms in nitrogen fixation and leucine uptake when growing in a diurnal light/dark regimen. The rhythms persisted in subsequent uniform light/light conditions. In order to analyze the circadian rhythm at the genetic level, mutants were induced by N-methyl-N-nitro-N-nitrosoguanidine and then isolated by procedures with the circadian nitrogen-fixing rhythm as a selecton marker. Characterization of the mutants with respect to the circadian rhythm indicated that some mutants were abnormal only in the nitrogen-fixing rhythm, while some simultaneously lost the ability to establish the nitrogen-fixing and leucine-uptake rhythms. The physiological properties of the circadian rhythm were compared. The genetic potential of the mutants that were abnormal in both rhythms is emphasized.     

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.     

Temporal separation of nitrogen fixation and photosynthesis in the filamentous,non-heterocystous cyanobacterium Oscillatoria sp.

When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C₂H₄ and 1.4 mg O₂ mg Chl a ^-1·h^-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C₂H₄ mg Chl a ^-1·h^-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.     

Effect of light nitrogenase function and synthesis in Rhodopseudomonas capsulata.

The metabolic versatility of the purple nonsulfur photosynethetic bacterial permits the expression of either a phototrophic or a dark aerobic mode of growth. These organism also possess nitrogenase activity which may function under semiaerboic conditions. On the basis of these important properties, the light dependence of nitrogenase function and synthesis in Rhodopseudomonas capsulata was investigated. Nitrogenase activity was strictly dependent on light; no activity was observed in the dark, even when energy (ATP) was supplied by oxidative phosphorylation. It was concluded that the low-potential reducing agent required by the nitrogenase-catalyzed reaction could only be generated by a photochemical reaction. Nitrogenase biosynthesis was also largely dependent on light; however, a small amount of synthesis was observed in resting cells incubated in the dark. Resting cells prepared from dark-grown cultures synthesized nitrogenase at high rates upon illumination. The highest stability of nitrogenase in these resting cells was observed when suspensions were exposed to a diurnal pattern of illumination rather than continuous light. Although nitrogenase function and synthesis are closely coupled to photosynthetic activity, the biosyntheses of bacteriochorophyll and nitrogenase are independent of each other and are most probably subject to different regulatory mechanisms by light.     

Priority of light/dark entrainment over temperature in setting the circadian rhythms of the prokaryote Synechococcus RF-1

Light/dark (L/D) and temperature are two major factors in the entrainment of circadian rhythms. The input pathways of these two environmental factors for the entrainment of circadian rhythms in Synechococcus RF-1 are different since the overt rhythms in mutant CR-1, one of the circadian-rhythm mutants of Synechococcus RF-1, could be established by temperature cycles but not by L/D. Therefore, it was of interest to investigate the phases of Synechococcus RF-1 cells entrained simultaneously by L/D and temperature. The circadian rhythms of nitrogenase activity and protein synthesis in RF-1 cells entrained by L/D, and by lowered or raised temperatures differed in their peaks of activity. Comparison of the phases of RF-1 cells entrained by L/D and temperature independently, and by L/D and temperature simultaneously indicated that L/D entrainment has priority over the temperature effect. Received: 8 February 1999 / Accepted: 1 April 1999     

Calcium is required for the increase of dark respiration during diurnal nitrogen fixation by Synechococcus RF-1

Under 12/12 h light/dark cycles, 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) added at the start of the dark period, inhibited the increase of dark respiration which was associated with nitrogen fixation in Synechococcus RF-1. Twenty-five millimolar NaNO₃ added 30 min before the start of dark period suppressed this respiratory increase. If 1.25 mM CaCl₂ was added to the EGTA-treated sample from 3 to at least 10 h later in the dark period, a quick rise in respiratory rate was observed. This rise was also reduced by 25 mM NaNO₃. Extracellular Ca²⁺ appears to be required for the increase in dark respiration associated with the rhythmic appearance of nitrogenase activity in the dark cycle.     

H2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions

Rhodospirillum rubrum is able to produce H₂ during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H₂ production by the nitrogenase could be partially inhibited by N₂. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP chloramphenicol - DSM Deutsche Sammlung von Mikroorganismen, Göttingen - FHL formate hydrogen lyase - O.D optical density - PFL pyruvate formate lyase     

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     

Cyclic variations of photosynthetic activity under nitrogen fixing conditions in Synchococcus RF-1

When nitrogen fixing cell cultures of Synechococcus RF-1 were subjected to an alternating lightdark regime (12 h:12 h), a cyclic decrease in the photosynthetic oxygen evolution potential was observed during the dark periods. This rhythm of net photosynthesis rate was maintained for at least two days after transition to continuous light. The decrease in net photosynthesis was accompanied by a stimulation of dark respiration. However, the magnitude of oxygen uptake was considerably smaller than the observed decrease in oxygen evolution. The photosynthetic activity of cells taken from the dark period was characterized by (i) a significantly lower quantum yield and (ii) a strong reduction in the light-saturated rate of photosynthesis. Growing the cultures on nitrate or under continuous light completely suppressed this rhythm. Protein synthesis was not necessary for the recovery of the light-saturated rate of photosynthesis during the light period. The cellular content of chlorophyll a and of phycobiliproteins did not vary between light and dark period, indicating that quantitative changes in the composition of the photosynthetic apparatus are not the basis for the observed oscillations. Regulatory modifications of the photosynthetic efficiency are proposed as an adaptation mechanism to adjust the intracellular oxygen concentration to the needs for nitrogenase activity.Abbreviation Chl chlorophyll     

On the role of oxygen for nitrogen fixation in the marine cyanobacterium Trichodesmium sp

The marine, non-heterocystous, filamentous cyanobacterium Trichodesmium shows a distinct diurnal pattern of nitrogenase activity. In an attempt to reveal the factors that control this pattern, a series of measurements were carried out using online acetylene reduction assay. Light response curves of nitrogenase were recorded applying various concentrations of oxygen. The effect of oxygen depended on the irradiance applied. Above a photon irradiance of 16 mumol m(-2) s(-1) nitrogenase activity was highest under anoxic conditions. Below this irradiance the presence of oxygen was required to achieve highest nitrogenase activity and in the dark 5% oxygen was optimal. At any oxygen concentration a photon irradiance of 100 mumol m(-2) s(-1) was saturating. When Trichodesmium was incubated in the dark, nitrogenase activity gradually decreased and this decline was higher at higher levels of oxygen. The activity recovered when the cells were subsequently incubated in the light. This recovery depended on oxygenic photosynthesis because it did not occur in the presence of DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Recovery of nitrogenase activity in the light was faster at low oxygen concentrations. The results showed that under aerobic conditions nitrogenase activity was limited by the availability of reducing equivalents suggesting a competition for electrons between nitrogenase and respiration.     

Induction and modification of dinitrogenase reductase in the unicellular cyanobacterium Synechocystis BO 8402

Oxygen is an important regulatory factor of nitrogenase induced in a unicellular cyanobacterium, Synechocystis BO 8402, during nitrogen starvation. Synthesis of the enzyme is limited by the efficiency of the cells to remove oxygen by respiration, supported by hydrogenases and, in the light, by inhibition of photosynthesis. With a polyclonal antibody against dinitrogenase reductase (the Fe protein of nitrogenase) a single polypeptide is detected, indicative of an active dimeric enzyme in dense cell suspensions. Inhibition of nitrogenase by addition of oxygen is accompanied by the appearance of a second polypeptide of the Fe protein having a 1.5 kDa higher molecular weight. This disappears upon removal of oxygen from the gas phase while nitrogenase activity is restored. No protein synthesis is required indicating that a fraction of the existing polypeptides is reversibly modified in response to oxygen. After induction of nitrogenase activity in dilute culture suspensions, both forms of the Fe-protein are found in variable amounts possibly due to oxygen contamination during the experiment.Abbreviations CAM chloramphenicol - Chl chlorophyll a - CHO carbohydrates - DCMU 3,4-dichlorophenyl-1,1-dimethylurea (diuron) - kDa kilodalton - SDS sodium dodecylsulphate     

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。     

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.     

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.