Immunological characterization of nitrogenase in the filamentous non-heterocystous cyanobacterium Oscillatoria limosa

The filamentous non-heterocystous cyanobacterium Oscillatoria limosa was subjected to Western blot analyses using two antisera raised against the small subunit (Fe-protein) of the nitrogenase complex. Two polypeptides were recognized in nitrogen-fixing cultures irrespective of the antiserum used while no bands were detectable in nitrate-grown cultures. The apparent molecular weights of the two polypeptides were approximately 40.5 and 39.5 kDa respectively, with the former, probably an inactive form, dominating. In situ immunogold electron microscopy was used to reveal the cellular and subcellular localization on the Fe-protein. All cells of the trichomes of nitrogen-fixing O. limosa showed a dense label. The label was homogeneously distributed throughout the cytoplasm including the thylakoid area. Nitrate-grown cultures contained a very low label.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis This study was supported by the Swedish Natural Science Research Counsil and the M. and M. Wallenberg Fund (to B. Bergman). We are grateful to Dr. S. Nordlund (University of Stockholm, Sweden) for providing us with the antiserum of Rhodospirillum rubrum nitrogenase and to Drs. S. Reich and P. Böger (University of Konstanz, FRG) for the antiserum of Anabaena variabilis. Skilful technical assistence by K. Östlund and E. Danielsson is gratefully acknowledged. We would also like to thank M. Villbrandt (University of Oldenburg, FRG) for providing cultures of Oscillatoria limosa and Dr. P. Lindblad for valuable discussions and suggestions.To whom correspondence should be addressed.     

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.     

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     

NITROGENASE CONFINED TO RANDOMLY DISTRIBUTED TRICHOMES IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Nitrogenase reductase (Fe-protein) was detected in the marine planktonic cyanobacterium Trichodesmium. The molecular weight was about 38 kD, as shown by western blotting using anti -Rhodospirillum rubrum nitrogenase reductase antiserum. The enzyme was confined to a limited number (ca. 10–40%) of randomly distributed trichomes in the Trichodesmium colonies, as shown by immunogold localization and transmission electron microscopy. Associated microorganisms had little or no nitrogenase. Nitrogenase showed a diel cycle in localization: present throughout the cytoplasm of cells in N₂-fixing (daytime) colonies but at the periphery of non-N₂-fixing (nighttime) colonies. This structural arrangement of N₂-fixing trichomes and nitrogenase is novel and different from the previously held paradigm for this and other diazotrophic cyanobacteria.     

Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness

Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.     

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.     

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.     

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,Trichodesmium spp.

Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.     

In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413).

Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. B?ger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.     

固氮蓝藻与棕色固氮菌固氮酶组分的交叉互补功能

本文报告了藻菌之间固氮酶组分的交叉互补试验。初步结果证明:固氮蓝藻(Anabacnaazotica水生686)的钼铁蛋白与棕色固氮菌(Azotobacter vinelandii)的铁蛋白之间存在着明显的互补功能。但这种蓝藻的铁蛋白在非细胞形态下很不稳定,易于失活。本实验为不同生理类型和不同进化程度的固氮生物之间固氮酶组分的交叉互补研究提供了新的资料。       

Temporal organization of chilling resistance in cotton seedlings: effects of low temperature and relative humidity

The effect of temperature and relative humidity (RH) on the time course of the rhythmic endogenous changes of chilling resistance was studied in cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12 h. The resistant phase to 5° C, 85% RH lasted during most of the dark period while to 5° C, 100% RH it was longer and extended into the last half of the light period because a transient phase advance occurred when chilling started at the middle of the light period. Seedlings acclimated by low temperature were resistant throughout the light-dark cycle. A treatment with 100% RH before chilling to acclimated seedlings introduced a sensitive phase that corresponded to that of non-acclimated seedlings. In non-acclimated seedlings, this treatment decreased the resistance but the basic pattern of the rhythm was sustained. Changes in chilling resistance were analyzed under fluctuating temperatures and RHs, and explained taking into consideration the functioning of the circadian clock and environmental induction of resistance.Abbreviations CR chilling resistance - LDC light-dark cycle of 24 h - RH relative humidity     

Photoperiod modifies daily maps of light and dark sensitivity for N-acetyltransferase activity in pineal glands of 3-week oldGallus domesticus

Summary N-acetyltransferase (NAT) activity in pineal glands exhibits a circadian rhythm with peak activity occurring in the dark-time. We previously showed that inGallus domesticus chicks pretreated with LD12:12, NAT activity was increased by dark exposure (peak dark sensitivity occurred during the expected dark-time) or decreased by light at night (peak light sensitivity occurred early in the night during the time of dark sensitivity). In this study we mapped dark sensitivity vs time (for NAT activity increase in response to 2 h dark pulses), and light sensitivity vs time (for NAT activity decrease in response to 10 min or 30 min light pulses) over a cycle for 3-week old chicks,Gallus domesticus, pretreated with long (LD16:8) or short photoperiod (LD8:16). Sensitivity to light was increased in the second 8 h after L/D by LD8:16. Sensitivity to dark was increased in the first 8 h after L/D by LD16:8.Abbreviations LD16:8 a light-dark cycle consisting of 16 h of light alternating with 8 h of dark - LD8:16 a light-dark cycle consisting of 8 h of light alternating with 16 h of dark - DD constant dark - LL constant light - L/D lights-off - D/L lights-on - NAT pineal serotonin N-acetyltransferase - NAT activity is given in nmoles/pineal gland/h - chick used here to denote a young bird of either sex of the speciesGallus domesticus from hatching to three weeks of age     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Circadian periodicity in cockroaches altered by high frequency light-dark cycles

Summary The circadian period of the freerunning activity rhythm in the cockroach is systematically altered by high frequency light-dark cycles (HF-LD) according to the ratio of light to dark within each cycle. With a standard 10 min cycle time, brief (e.g., 0.5 min) exposure to light each cycle causes the free-running period to shorten significantly in comparison to its steady-state value in constant darkness. As the ratio of light to dark in HF-LD is increased, the period of the rhythm is progressively lengthened. These findings are discussed in terms of the general proposition that light, applied throughout the circadian cycle, predictably modifies periodicity according to the asymmetrical shape of the circadian phase response curve.Abbreviations LD light-dark cycles in which cycle length is in hours - HF-LD light-dark cycles in which cycle length is in min; period of the activity rhythm; change in period of the activity rhythm - PRC phase response curve - LL constant light