Limiting factor of nitrogenase system mediating hydrogen production of Rhodobacter sphaeroides S

To explain the decrease of hydrogen production rate in the batch culture of Rhodobacter sphaeroides S, the activities of enzymes related to the TCA cycle, nitrogenase in cell-free extracts, ATP generation by chromatophores, and ferredoxin were examined at the beginning, middle and end of the hydrogen production phase of batch culture. The activities of TCA cycle enzymes, nitrogenase and ATP generation were found to remain at almost the same level throughout the culture, while ferredoxin activity decreased linearly with time. In addition, by bubbling N₂ gas into the culture broth at the end of the culture, the hydrogen production rate was restored to the initial level through the increase of the ferredoxin activity. Although the decrease of ferredoxin activity and its restoration by bubbling N₂ gas remained unexplained, ferredoxin activity was considered to be a key function in the nitrogenase system for H₂ production by this photosynthetic bacterium.     

Effects of in vivo treatments on the activity of nitrogenase isolated from Rhodospirillum rubrum

Nitrogenase activities of partially purified extracts of Rhodospirillum rubrum grown on different nitrogen sources were examined. Most of the nitrogenase from cells grown on N₂ or glutamate was in the inactive form. This form was also predominant in extracts from cells grown on limiting N₂ or glutamate plus N₂. The enzyme from cells grown with limiting NH⁺₄ was fully active. Nitrogenase displayed varying degrees of sensitivity to in vivo inhibition by NH⁺₄, depending on the culture conditions. However, addition of NH⁺₄ to the cultures prior to harvest did not change the proportion of the active form of the enzyme in extracts from that found in control samples. Several of these observations are inconsistent with the three component model of nitrogenase regulation of Yoch and Cantu (Yoch, D.C. and Cantu, M. (1980) J. Bacteriol, 142, 899–907). A regulatory system controlled by products of NH⁺₄ assimilation is suggested.     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Increase of Nitrogenase Activity in the Blue-Green Alga Nostoc muscorum (Cyanobacterium)

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under hydrogen or argon (nongrowing conditions, neither CO₂ nor N₂ or bound nitrogen present) in the light resulted in a two- to fourfold increase of light-induced hydrogen evolution and a 30% increase of acetylene reduction. Preincubation under the same gases in the dark led to a decrease of both activities. Cultivation of algae under a hydrogen-containing atmosphere (N₂, H₂, CO₂) increased neither hydrogen nor ethylene evolution by the cells. Formation of both ethylene and hydrogen is due to nitrogenase activity, which apparently was induced by the absence of N₂ or bound nitrogen and not by the presence of hydrogen. Inhibitors of protein biosynthesis prevented the increase of nitrogenase activity. Hydrogen uptake by the cells was almost unaffected under all of these conditions. With either ammonia or chloramphenicol present, nitrogenase activity decreased under growing conditions (i.e., an atmosphere of N₂ and CO₂). The kinetics of decrease were the same with ammonia or chloramphenicol, which was interpreted as being due to rapid protein breakdown with a half-life of approximately 4 h. The decay of nitrogenase activity caused by chloramphenicol could be counteracted by nitrogenase-inducing conditions, i.e., by the absence of N₂ or bound nitrogen. A cell-free system from preconditioned algae with an adenosine 5′-triphosphate-generating system exhibited the same increase or decrease of nitrogenase activity as the intact cell filaments, indicating that this effect resided in the nitrogenase complex only. We tentatively assume that not the whole nitrogenase complex, but merely a subunit or a special protein with regulatory function, is susceptible to fast turnover.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

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.     

Regulation of ammonium uptake and metabolism by nitrogen fixing bacteria. III. Clostridium pasteurianum

Addition of ammonium salts to N₂ fixing continuous cultures of Clostridium pasteurianum caused immediate stop of nitrogenase synthesis, while the levels of glutamine synthetase, glutamate dehydrogenase and asparagine synthetase remained constant. No evidence for an interconversion of the glutamine synthetase was found. The activities of glutamate synthase in crude extracts were inversely related to the nitrogenase levels. The intracellular glutamine pool rapidly expanded during nitrogenase repression and decreased as fast during derepression while the pool sizes of all other amino acids were not strongly related to the rate of nitrogenase formation. These investigations suggest glutamine as corepressor of nitrogenase synthesis.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O₂ concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters N_m, N_d, and α were derived. In both strains, nitrogenase activity (N_tot = N_m + N_d) was the highest at 39°C–42°C and at 0% O₂. The ratio N_tot/N_d was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (N_d). N_tot/N_d increased with decreasing O₂ concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (N_m). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O₂ concentration and temperature as the latter may dynamically alter the flux of O₂ into the heterocyst.     

A changed nitrogenase activity in Rhodospirillum rubrum after substitution of tungsten for molybdenum

The nitrogenase activity in Rhodospirillum rubrum was changed when the cells were made either Mo-deficient or when Mo was replaced by tungsten (W) as trace element in the growth medium: In the absence of N₂, normal Mo cells evolved H₂ (via nitrogenase) from added malate in the light faster than W cells, which in turn evolved H₂ faster than Mo-deficient cells. In the presence of N₂, on the other hand, nitrogen fixation rate in W cells was very close to the low rate found with Mo-deficient cells. Incubation after harvesting of Mo-deficient cells with 2×10^-5 M molybdate or tungstate stimulated the H₂ evolution (similarly with both trace elements) as well as the N₂ fixation (Mo was more effective than W). This indicates that the nitrogenase activity of W cells was truly caused by W and not merely by remaining traces of Mo. The ATP consumption is apparently higher with a W-containing nitrogenase than with the normal Mo-nitrogenase. Further, the affinity to N₂ of the W cells seems to be lower than with the Mo cells.     

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.     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

Utilization of simple phenolics for dinitrogen fixation by soil diazotrophic bacteria

Summary A microaerobic diazotrophic bacterium tentatively identified as aPseudomonas species was isolated from a forest soil. Its nitrogenase (C₂H₂ reduction) activity in liquid medium was significantly supported by phenolic compounds when compared with glucose-, mannitol- or malate-supported activity. The utilization of phenolics was dependent on substrate induction and the appropriate oxygen concentration. At a pO₂ of 0.05 protocatechuate was a better carbon source for N₂ fixation than glucose. In the case ofLignobacter protocatechuate was a better carbon source for N₂ fixation than glucose at pO₂ 0.2 but not at pO₂ 0.05. It is suggested that certain monomeric phenols can support nitrogenase activities in many carbon-limited soil environments.Contribution No. 1484 from the Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Canada.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.