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Identification of the sources of energy for nitrogen fixation and physiological characterization of nitrogen-fixing members of a marine microbial mat community.

Experimental manipulations of a microbial mat community were performed to determine sources of energy and reductant used for nitrogen fixation and to physiologically characterize the responsible diazotrophs. The dominant photolithotrophic members of this community were nonheterocystous cyanobacteria, but other potential nitrogen-fixing microorganisms were also present. Pronounced diel variability in rates of acetylene reduction was observed, with nighttime rates a factor of three to four higher than daytime rates. Acetylene reduction measured at night was dependent upon the occurrence of oxygenic photosynthesis the preceding day; mats incubated in the dark during the daytime reduced acetylene at rates comparable to those of light-incubated mats but were not able to reduce acetylene at the normally high rates the following night. The addition of various exogenous carbon compounds to these dark-incubated mats did not elicit nighttime acetylene reduction. Nighttime acetylene reduction apparently proceeds under anoxic conditions in these mats; the highest rates of acetylene reduction occur late at night. Additions of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of oxygenic photosynthesis) to mats resulted in a pronounced stimulation of acetylene reduction during the day, but acetylene reduction the next night proceeded at greatly reduced rates (relative to untreated mats). This daytime stimulation, under the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced anoxic conditions in the experimentally treated mats, was light dependent. These results suggest that nitrogen fixation in these mats may be attributed to the activities of nonheterocystous cyanobacteria utilizing storage products of oxygenic photosynthesis under anoxic conditions at night.

Journal Article↗

Dynamics of the 'echo' effect in a phytoplankton system with nitrogen fixation.

Consideration of nitrogen fixation adds a positive nonlinear feedback to plankton ecosystem models. We investigate the consequences of this feedback for secondary phytoplankton blooms and the response of phytoplankton dynamics to physical forcing. The dynamics of phytoplankton, Trichodesmium (the nitrogen fixer), and nutrients is modeled with a system of three differential equations. The model includes two types of nonlinear interactions: the competition of phytoplankton and Trichodesmium for light, and the positive feedback resulting from Trichodesmium recycling. A typical simulation of the model in time, with forcing by a varying mixed-layer depth, reveals a clear successional sequence including a secondary or 'echo' bloom of the phytoplankton. We explain this sequence of events through the stability analysis of three different steady states of the model. Our analysis shows the existence of a critical biological parameter, the ratio of normalized growth rates, determining the occurrence of 'echo' blooms and the specific sequence of events following a physical perturbation. The interplay of positive and negative feedbacks appears essential to the timing and the type of events following such a perturbation.

Algorithms↗

Marine oscillatoria (Trichodesmium): explanation for aerobic nitrogen fixation without heterocysts.

Nitrogen fixation in marine Oscillatoria appears to be associated with differentiated cells located in the center of the colony. These central cells exhibit reduced pigmentation relative to peripherally located cells and do not incorporate 14CO2 in photosynthesis. Central cells apparently do not produce O2 which would deactivate nitrogenase. When central cells are exposed to O2 via disruption of the colonies, N2 fixation (acetylene reduction) decreases sharply even though individual trichomes remain intact. Disruption of colonies in the absence of O2 does not cause reduced nitrogenase activity. In the sea, turbulence from wave action apparently separates trichomes allowing O2 to enter thus decreasing nitrogenase activity. These observations explain how Oscillatoria is able to fix N2 without heterocysts in an aerobic environment and why it blooms virtually always occur in calm seas.

Eukaryota↗

Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids.

In vivo CO(2) fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.Forage harvest and nitrate application reduced both in vivo and in vitro CO(2) fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.The results demonstrate that CO(2) fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO(2) fixation for attached and detached alfalfa nodules at low CO(2) concentrations (0.13-0.38% CO(2)) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO(2) fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

Journal Article↗

Interaction of Azospirillum lipoferum with wheat germ agglutinin stimulates nitrogen fixation.

In vitro, the nitrogen fixation capability of A. lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect required N-acetyl-D-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

Azospirillum↗

Buckminsterfullerenes: a non-metal system for nitrogen fixation.

In all nitrogen-fixation processes known so far--including the industrial Haber-Bosch process, biological fixation by nitrogenase enzymes and previously described homogeneous synthetic systems--the direct transformation of the stable, inert dinitrogen molecule (N2) into ammonia (NH3) relies on the powerful redox properties of metals. Here we show that nitrogen fixation can also be achieved by using a non-metallic buckminsterfullerene (C60) molecule, in the form of a water-soluble C60:gamma-cyclodextrin (1:2) complex, and light under nitrogen at atmospheric pressure. This metal-free system efficiently fixes nitrogen under mild conditions by making use of the redox properties of the fullerene derivative.

Ammonia↗

Differential expression of photosynthesis and nitrogen fixation genes in the cyanobacterium Plectonema boryanum.

The filamentous non-heterocystous cyanobacterium Plectonema boryanum fixes dinitrogen at a high rate during microaerobic growth in continuous illumination by temporal separation of oxygen-evolving photosynthesis and oxygen-sensitive dinitrogen fixation. The onset of nitrogen fixation is preceded by a depression in photosynthesis that establishes a sufficiently low level of dissolved oxygen in the growth medium. A several-fold reduction in the level of transcripts coding for phycocyanin (cpcBA) and the chlorophyll a binding protein of photosystem II (psbC) and psbA accompanied the depression in photosynthetic oxygen evolution. Unlike most of the other organisms examined to date, in P. boryanum, psbC and psbD do not appear to be co-transcribed. The psbC transcripts were down-regulated several fold, while the psbD transcript declined marginally during the nitrogen fixation phase. A decrease in dissolved oxygen and a dramatic increase in the level of nifH transcripts and the enzyme activity of nitrogenase were characteristic of the nitrogen fixation phase. The level of transcript for glnA, which encodes glutamine synthetase, was not altered. Reciprocal regulation of gene expression was well orchestrated with the alternating cycles of photosynthesis and nitrogen fixation in P. boryanum.

Cyanobacteria↗

Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp. strain RF-1.

Sixteen nif and 'nif-associated' genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp. strain RF-1 have been cloned and sequenced. All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons. The nifH operon (nifH-nifD-nifK) has been reported previously. nifB, fdxN, nifS, nifU and nifP were found to be located upstream of the nifH operon. nifB-fdxN-nifS-nifU were expressed as an operon. A nifP-like gene was found to be located just upstream of nifB. nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and 'fdx' were found to be located downstream from nifK. The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB-'+ ++fdx' and span approximately 7 kb. The function of the ORF situated between nifX and nifW is not known. However, it was identified as a counterpart of ORF-2 in Anabaena sp. strain PCC 7120 based on the deduced amino acid sequence. Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifX-orf, nifW-hesA-hesB and 'fdx'-containing operons, respectively. According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h light/12 h dark regimen. The rhythm persisted after the culture was transferred to continuous illumination.

Aerobiosis↗

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw.

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10 cells per g of substrate, were evident after 4 and 5 days of incubation at 30 degrees C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO(2) production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10 cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10 cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.

Journal Article↗

Denitrification and nitrogen fixation in alaskan continental shelf sediments.

Rates of nitrogen fixation and denitrification were measured in Alaskan continental shelf sediments. In some regions, rates of nitrogen fixation and denitrification appeared to be equal; in other areas, rates were significantly different. Potential rates of denitrification were found to be limited primarily by the available nitrate substrate. Major regional differences in rates of denitrification were not statistically significant, but significant differences were found for nitrogen fixation rates in different regions of the Alaskan continental shelf. Estimated net losses of nitrogen from Bering Sea sediments were calculated as 1.8 x 10 g of N/yr. Experimental exposure of continental shelf sediments to petroleum hydrocarbons reduced rates of nitrogen fixation and denitrification in some cases but not others. Long-term exposure was necessary before a reduction in nitrogen fixation rates was observed; unamended rates of denitrification but not potential denitrification rates (NO(3) added) were depressed after exposure to hydrocarbons.

Journal Article↗

Factors influencing dark nitrogen fixation in a blue-green alga.

Nitrogen-fixing activity declines first rapidly and then more gradually when Anabaenopsis circularis is transferred from light into dark conditions. The rate and duration of dark acetylene reduction (nitrogen fixation) depend upon conditions prevailing during the preceding light period. Factors (such as light intensity, CO2 concentration, and supply of glucose), which in the light affect photosynthesis and the accumulation of reserve carbon, have a profound effect on dark nitrogen fixation. Glucose greatly promotes nitrogen fixation in the light and supports prolonged nitrogenase activity in the dark. The results suggest that heterotrophic nitrogen fixation by blue-green algae in the field may be important both under light and dark conditions.

Acetylene↗

[Influence of different ecological groups of earthworms on the intensity of nitrogen fixation].

The results of studying nitrogen fixation in organic substrates processed by different ecological groups of earthworms suggest that the earthworms Aporrectoidea caliginosa actively stimulate nonsymbiotic nitrogen fixation. In this respect, they exceed the manure worms Eizenia foetida tens of times due to the formation in the organic substrate of conditions favorable for the nitrogen fixing bacteria, and namely: low content of nitrogen easily accessible for microorganisms, changes in the structure of the microbial community of the substrate in favor of non-spore forms of bacteria, and suppression of the growth of saprophyte bacilli, the main competitors of nitrogen fixing bacteria for carbon nourishment sources.

Animals↗

Nitrogen fixation (acetylene reduction) in a salt marsh amended with sewage sludge and organic carbon and nitrogen compounds.

Seasonal distribution of nitrogen fixation by Spartina alterniflora epiphytes and in surface and soil samples was investigated in a Georgia salt marsh which was amended with sewage sludge or with glucose and/or ammonium nitrate. There was no significant difference between the rates of fixation in the unamended and sewage sludge plots. Additional perturbation experiments suggested that nitrogen addition indirectly stimulates nitrogen fixation by enhancing Spartina production and root exudation. Glucose additions, on the other hand, suppressed nitrogen fixation on a long-term basis. It is suggested that the microbial population in the soil out-competed the plants for the available nitrogen and in turn suppressed plant production and possibly root exudation. A comparison of nitrogen fixation in clipped and unclipped Spartina plots substantiated the suggestion that root exudation probably supports nitrogen fixation. Fixation in the clipped plots was significantly lower (P < 0.05) than the rates in the unclipped plots.

Journal Article↗

Effect of sulfide on nitrogen fixation in a stream sediment-water system.

Nitrogen fixation (C(2)H(2) reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C(2)H(4) production occurring in the presence of 8 mumol of S per ml of stream water. Sodium sulfide at concentrations of 16 mumol of S per ml or greater inhibited nitrogen fixation, with 64 mumol of S per ml being completely inhibitory. Sulfide at levels of 16 mumol/ml or above inhibited CO(2) production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO(2) production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 mumol of S per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H(2), H(2)S, and other sulfur species at various pH values, it appeared that H(2)S was the toxic entity and that HS was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS from H(2)S.

Journal Article↗

An alternate photosynthetic electron donor system for PSI supports light dependent nitrogen fixation in a non-heterocystous cyanobacterium, Plectonema boryanum.

Plectonema boryanum exhibits temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. During nitrogen fixation, the photosynthetic electron transport chain becomes impaired, which leads to the uncoupling of the PSII and PSI activities. A 30-40% increase in PSI activity and continuous generation of ATP through light-dependent processes seem to support the nitrogen fixation. The use of an artificial electron carrier that shuttles electrons between the plastoquinone pool and plastocyanin, bypassing cytochrome b/f complex, enhanced the photosynthetic electron transport activity five to six fold during nitrogen fixation. Measuring of full photosynthetic electron transport activity using methyl voilogen as a terminal acceptor revealed that the photosynthetic electron transport components beyond plastocyanin might be functional. Further, glycolate can act as a source of electrons for PSI for the nitrogen fixing cells, which have residual PSII activity. Under conditions when PSI becomes largely independent of PSII and glycolate provides electrons for PSI activity, the light-dependent nitrogen fixation also was stimulated by glycolate. These results suggest that during nitrogen fixation, when the photosynthetic electron transport from PSII is inhibited at the level of cytochrome b/f complex, an alternate electron donor system for PSI may be required for the cells to carry out light dependent nitrogen fixation.

Cyanobacteria↗

Marine nitrogen fixation: what's the fuss?

Biological nitrogen fixation is a much more important process in the nitrogen cycle of the oceans than previously thought. Further, nitrogen fixation may have an influence on the capacity of the oceans to sequester carbon. A greater diversity of marine nitrogen fixers has also been uncovered but their quantitative significance remains to be determined.

Biological Evolution↗

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae.

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

Bacterial Proteins↗

Nitrogen fixation and carbon metabolism in legume nodules.

A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen (ammonia) into organic compounds. The source of energy is provided in the form of photosynthates by the host plant. Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPC-mediated CO2 fixation in nodules results in the synthesis of C4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which can be transported into bacteroids with the intervention of dicarboxylate transporter (DCT) protein. PEPC has been purified from the root nodules of few legume species. Information on the relationship between nitrogen fixation and carbon metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of carbon and nitrogen metabolism in legume root nodules.

Carbon↗