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.     

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.     

Simultaneous heterolactic and acetate fermentation in the marine cyanobacterium Oscillatoria limosa incubated anaerobically in the dark

The marine nitrogen-fixing cyanobacterium Oscillatoria limosa, strain 23 (Oldenburg) was investigated with respect to its dark anaerobic metabolism. As soon as the cells were incubated anaerobically in the dark, they started to ferment. Glycogen was presumably degraded via the heterolactic fermentative pathway. Glycogen-glucose was degraded to equimolar amounts of lactate, ethanol and carbon dioxide. The disaccharide trehalose, which serves as an osmoprotectant in O. limosa, was also catabolized. Most probably, this compound was fermented almost exclusively to acetate. Some hydrogen was produced as well. In the presence of elemental sulfur, fermentative hydrogen production ceased and sulfide was produced instead. The presence of elemental sulfur had no effect on the amounts and ratios of the fermentation products produced.     

Diurnal and seasonal variations of nitrogen fixation and photosynthesis in cyanobacterial mats

In situ measurements of nitrogenase activity and photosynthesis were performed simultaneously in cyanobacterial mats of intertidal sand flats of the Southern North Sea. Two types of cyanobacterial mats, which differed in species composition and biomass content, were investigated. The measurements were done monthly during 3 years to detect seasonal variations of nitrogen fixation and photosynthesis. Diurnal variations were investigated as well. The results showed that (i) freshly colonized sediment with the cyanobacteriumOscillatoria limosa as the dominant organism revealed the highest specific nitrogenase activities (ii) nitrogenase activities were highest in spring and summer, when mat development was initiated and (iii) diurnal fluctuations of nitrogenase activity indicated that it occurred temporally separated from oxygenic photosynthesis.     

Verteilung und Aufteilung der DNS bei einigen Cyanophyceen,festgestellt durch ihre DAPI-Fluoreszenz

The DNA of Blue-Green Algae may be specifically and most efficiently elucidated by the combined use of the fluorochrome DAPI and DNAse treatment. InChroococcus turgidus andSynechococcus aeruginosus it forms a network which usually is positioned at the periphery of the centroplasm. At the beginning of cell division it seems to be invaginated by the ingrowing cell wall. Later it must be distributed by other means because two equal parts are connected by a number of fine and straight DNA threads. InOscillatoria limosa usually all parts of the centroplasm are interspersed with the DNA network. In surface view, the DNA occasionally seems to consist of a number of independant rods, but in reality these are connected to the general network. Division apparantly occurs by ingrowth of the crosswall and usually results in equal daughter nuclear equivalents although occasionally they are unequal. Four strains ofMicrocoleus vaginatus were found to have nuclear equivalents of the same general appearance as inOscillatoria limosa.

     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Microautoradiographic studies of the role of mesophyll and bundle tissues of the Ricinus cotyledon in sucrose uptake

The aim of the study was to show which tissues and cell types of the cotyledon of Ricinus communis L. are responsible for uptake of sucrose by H⁺-sucrose symport. The cotyledons were incubated in labelled sucrose for up to 20 min and then the amount of radioactivity in each cell type of the cotyledon was assessed by microautoradiography. It was found that 50% of the label was present in the spongy mesophyll, and 10–15% was in the bundles, the epidermal layers and the palisade parenchyma. The sieve tubes contained only 2–3% of the label. The addition of sucrose to cotyledons depolarized the membrane of spongy-mesophyll cells by 33 mV. Therefore, it was concluded that the previously found H⁺-sucrose symport is at least partly located at the spongy mesophyll. No precursor-like behaviour of the label in mesophyll or bundle-sheath cells was observed in pulse-chase experiments, which indicates a direct uptake of sucrose by the sieve tube-companion cell complex from the apoplast.This work was funded by Deutsche Forschungsgemeinschaft. The valuable help by Ina Möller, Elke Schmidt, Christian Schobert (all from Bayreuth, FRG), Dr. Dieter Gradmann (Göttingen, FRG), Dr. Jörg Tittor (MPI Biochemie München, FRG), Dr. Pavlovkin (Plant Pathology, Bratislava), Dr. K. Köhler (Botany Department Würzburg, FRG) and the intense discussions with Dr. Enno Brinkmann (Bayreuth) are gratefully acknowledged. The technical assistance by Beatrix Tannhäuser-Hofmann and Hildegard Stork was of great help for this work.     

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.     

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.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Cellulose Decomposition under Nitrogen Deficiency by Bacteria Isolated from the Intestines of Phytophagous Vertebrates

A nitrogen-fixing strain identified as Klebsiella pneumonia 402-2 and two endoglucanase-synthesizing Bacillus strains were isolated from the intestines of phytophagous animals. One of the Bacillus strains was identified as Bacillus subtilis GL. Klebsiella pneumoniae 402-2 increased the endoglucanase activity of both Bacillusstrains in mixed cultures.The data on the taxonomic position of strains 402-2 and GL and on the nitrogen-fixing capacity of strain 402-2 were confirmed by sequencing and analyzing their 16S rRNA genes and by amplifying the nitrogenase gene nifH.     

Relationship between nitrogen fixation and nitrate metabolism in the Nodularia strains M1 and M2

Nitrogen fixation and nitrate-reduction activities were determined in photoautotrophic cultures of two wild-type strains of cyanobacterium Nodularia, spp. M1 and M2. Air could support growth of the two strains at a similar rate in the presence or absence of exogenous nitrate, ammonium and/or bicarbonate. Nitrogenase activity in air-grown cultures varied with culture age, and totally disappeared after 6 h of darkness. Recovery took place upon culture re-illumination. Ammonium at a concentration of 1 mM resulted in the total disappearance of nitrogenase activity and of heterocysts. In contrast, 20 mM nitrate hardly affected nitrogenase activity and heterocyst formation after ten generations. Under the same conditions, either ammonium or nitrate completely abolished nitrogenase activity and heterocyst formation in Anabaena sp. PCC 7119, a typical heterocystous strain. The inefficiency of nitrate in inhibiting nitrogen fixation in Nodularia M1 and M2 seemed to be caused by a low nitrate-reductase activity, and not by an impairment of nitrate-uptake activity. On the other hand, the presence of nitrate was not required for uptake activity to be expressed in Nodularia.Abbreviation NR nitrate reductase We thank C. Fernández-Cabrera (Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain) for technical assistance, and Dr. G. Pérez-Silva (CSIC) for his collaboration in the Anabaena NR assays. This work was supported by grants from Spanish CI-CyT (PB 87-0204 and PB 92-0497).     

Occurrence of nitrogen fixation among Vibrio spp.

Virtually all Vibrio spp. known and available in culture collections and several newly isolated Vibrio sp. were tested for their ability to fix molecular nitrogen, using the acetylene reduction technique, the fixation of the heavy isotope ¹⁵N, and by growth on media devoid of combined nitrogen. Among the 27 species tested, four, including V. diazotrophicus, proved to be nitrogenase-positive. The potential of nitrogen fixation was now also discovered in V. natriegens, V. pelagius and V. cincinnatiensis. Among the 9 newly isolated strains, 4 were nitrogenase-positive. These strains were classified as V. diazotrophicus on the basis of DNA homology studies. Nitrogenase was only induced during growth under anaerobic conditions. Dissolved oxygen as low as 1 M inhibited nitrogenase completely. This inhibition at low oxygen concentration, however, was reversible. 50–100 M dissolved oxygen inhibited nitrogenase irreversibly.This work was carried out at Geomicrobiology Division, the University of Oldenburg, FRG     

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。       

Localization of nitrogenase in vesicles of Frankia sp. Cc1.17 by immunogoldlabelling on ultrathin cryosections

Immunogoldlabelling on ultrathin cryosections of Frankia sp. Cc1.17 showed specific labelling of nitrogenase in the spherical cells called vesicles. No label was found in the hyphae in any cells grown on a medium with combined nitrogen, nor in those to which no specific antiserum was added. Similar results were obtained with cultures grown under high (20%) and low (2%) oxygen tension in the gas phase.Abbreviations BSA Bovine serum albumin fraction V (Sigma) - PBS phosphate buffered saline. Phosphate buffer 0.1 M, 8 g NaCl/1, 0.2 g KCl/1 - PIPES Piperazine-1,4-bis(ethane sulphonic acid)     

Immunogold localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in the nitrogen-fixing cyanobacterium,Plectonema boryanum

Summary Antiserum against the Calvin cycle enzyme, ribulose-1,5-bisphosphate carobxylase/oxygenase (RuBisCO), was used in conjunction with colloidal gold to localize RuBisCO in nitrogen-fixing (fix+) and nonfixing (fix–)Plectonema boryanum cells. RuBisCO antiserum consistently labeled the cytoplasm and polyhedral bodies (carboxysomes) in both fix+ and fix– cells. Through morphometry, it was determined that significantly less gold label (indicative of RuBisCO) was present in fix+ cells. This decreased RuBisCO content correlated with a decrease in net photosynthetic oxygen evolution also observed in fix+P. boryanum.Abbreviations RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase - fix+ nitrogen-fixing - fix– nonfixing     

The effect of the dissolved oxygen concentration and anabolic limitations on the behaviour of Rhizobium ORS571 in chemostat cultures

Chemostat cultures of Rhizobium ORS571 limited by the supply of oxygen or an anabolic substrate contained poly--hydroxybutyrate (PHB). Low amounts of PHB (about 10%) were present in ammonia- or nitrate-limited cultures; higher amounts were found in Mg⁺⁺-limited cultures (about 20%) and in oxygen-limited nitrogen-fixing cultures (37%). A method is described to calculate Y_ATP values (g PHB-free biomass · mol^-1 ATP) from the Y_succ values (g dry wt·mol^-1 succinate) measured. Y_succ and Y_ATP values in cultures limited by the supply of an anabolic substrate and in the oxygen-limited ammonia-assimilating culture were much lower than the values found in the PHB-free succinate-limited cultures. This shows that uncoupling of growth and energy production occurred. Therefore, H₂/N₂ ratio (mol hydrogen formed per mol nitrogen fixed) in nitrogen-fixing cultures could not be calculated from the comparison of the Y_ATP value found in the nitrogen-fixing culture and the value found in the corresponding ammonia-assimilating culture.Although the optimal dissolved oxygen concentration (d.o.c.) for nitrogen-fixing cultures of Rhizobium ORS571 is 5 or 10 M, nitrogen-fixing cultures could be obtained up to a d.o.c. of 40 M. Not only nitrogenase but also hydrogenase was active at this d.o.c. However, accumulation of PHB (10%) may indicate that cultures grown at unfavourable oxygen concentrations (15–40 M O₂) were N-limited rather than energy-limited, which may be the result of partial inactivation or repression of nitrogenase at a higher d.o.c.