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Influence of sewage discharge on nitrogen fixation and nitrogen flux from coral reefs in Kaneohe Bay, Hawaii.

Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N2 were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats.

Acetylene

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells.

Bacterial Proteins

Kinetic model of a determinate legume root nodule reveals plant metabolic characteristics for more efficient nitrogen fixation symbiosis.

While nitrogen fertilizers are widely used in agricultural production, their application incurs significant environmental and energetic costs. In contrast, some crops are less dependent on these fertilizers because they engage in symbioses with rhizobia, nitrogen-fixing bacteria that provide ammonium to the plant in exchange for carbon. However, the carbon cost associated with nitrogen fixation can negatively impact crop yields. Improving the efficiency of this metabolic process could alleviate this impact on crop productivity. Mathematical models can help us quantitatively explore metabolic behavior and identify potential targets for metabolic engineering. In this work, we developed a kinetic model of determinate root nodule metabolism, where this symbiotic exchange of carbon from the plant and nitrogen from the bacteria occurs. We used this model to evaluate how the predicted metabolic behavior differs between inefficient and efficient nodules, and to identify potential engineering targets for improving nitrogen fixation efficiency and rate. We show that the enzymes phosphoenolpyruvate carboxylase and pyruvate kinase have significant influence on the predicted rate and efficiency of nitrogen fixation, especially when their expression is varied in combination with oxidative Pentose Phosphate Pathway enzymes like glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. The model predicts that pairing a 3-fold decrease in glucose-6-phosphate dehydrogenase activity along with either a 3-fold increase in phosphoenolpyruvate carboxylase activity or decrease in pyruvate kinase activity could increase nitrogen fixation rate by 8.82% while improving nitrogen fixation efficiency by 10.99%.

Enzyme kinetics

Effect of ruthenium on nitrogen fixation by some nitrogen fixers.

The effect of ruthenium chloride in the culture media on the nitrogen-fixing ability of the three nitrogen fixers (unidentified species of Azotobacter, designated here as D3, B3, and B), isolated from Allahabad soil, was studied. It was observed that the nitrogen-fixing ability of the organisms is much increased in presence of 25-75 micro M concentration of ruthenium chloride in the culture media, while sugar consumption remains more or less steady. Also, if mg nitrogen fixed/g carbon consumed in two culture media with successive increasing concentrations of ruthenium chloride is compared by calculating the difference in increase of the amount of nitrogen fixed and carbon consumed in these two culture media, it was observed that high amounts of nitrogen are fixed by B3 and B between 25-50 micro M, and between 50-75 micro M concentration of ruthenium chloride by D3.

Azotobacter

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

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

Regulation of nitrogen fixation in Rhizobium sp.

Regulation of nitrogen fixation by ammonium and glutamate was examined in Rhizobium sp. 32H1 growing in defined liquid media. Whereas nitrogenase synthesis in Klebsiella pneunoniae is normally completely repressed during growth on NH4+, nitrogenase activity was detected in cultures of Rhizobium sp. grown with excess NH4+. However, an "ammonium effect" on activity was invariably observed in cultures grown on NH4+ as sole nitrogen source; the nitrogenase activity was, depending on conditions, 14 to 36% of that of comparable glutamate-grown cultures. Glutamate inhibited utilization of exogenous NH4+ and, in one of two procedures described, glutamate partially alleviated the ammonium effect on nitrogenase activity. NH4+, apparently produced from N2, was excreted into the culture medium when growth was initiated on glutamate, but not when NH4+ was thesole source of fixed nitrogen for growth. These findings are discussed in relation to nitrogen fixation by Rhizobium bacteroids.

Ammonium Sulfate

Nitrogen fixation in the Rhode River estuary of Chesapeake Bay.

The distribution, seasonal variation, origin, and significance of biological nitrogen fixation has been determined for a Chesapeake Bay estuary using the acetylene reduction technique. Studies over a 15-month period have shown that nitrogen fixation occurs predominantly in the surface intertidal (marsh) and subtidal sediments. Negligible activity was found in surface waters. A marked seasonal variation in nitrogen fixation was observed for intertidal sediments incubated at a standard 20 degrees C. Average rates of about 37 and 12 ng N/g dry sediment per hour were observed in the late fall months of 1972 and 1973, respectively, and less than or equal to 5 ng N/g dry sediment per hour during other seaons. Peaks of activity were lowered considerably when samples were incubated at ambient temperatures (in situ). Activity in the subtidal sediments was low (less than or equal to 6.8 ng N/g dry sediment per hour but showed a similar (approximately twofold) seasonal variation in nitrogen fixation potential. Light-dark and substrate addition experiments suggest that heterotrophic bacteria are the principal agents for nitrogen fixation in sediments. Integrated estimates of nitrogen fixation in the estuary indicate that biological fixation probably accounts for less than 5% of the total influx of nitrogen into the system. Rates of activity in the intertidal sediments are insufficient to account for the high productivity of marine angiosperms found in the marsh.

Acetylene

Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors.

Many photosynthetic bacteria from aquatic and terrestrial habitats reduce atmospheric dinitrogen to ammonia. The synthesis of proteins required for nitrogen fixation in these microorganisms is repressed by fixed nitrogen or oxygen. Studies on the purple non-sulphur phototroph Rhodobacter capsulatus have helped to clarify this transcriptional control and to define the factors involved in this regulation. The molecular mechanisms by which the nitrogen and oxygen status of the cell are relayed into nif gene expression or repression involve many trans- and cis-acting factors. The roles of these factors in the nif regulatory cascade of R. capsulatus are summarized. Two levels of control are present. The first level of control involves the nitrogen sensing circuitry in which at least four proteins act in a cascade. Upon nitrogen deficiency, genes involved in the second level of control are transcriptionally activated. These genes encode regulatory proteins that subsequently activate transcription of all other nif genes under anaerobic conditions. The R. capsulatus cascade is compared to the nif regulatory cascade in Klebsiella pneumoniae, highlighting both common and unique aspects.

Bacteria

[Nonsymbiotic nitrogen fixation in the podzolic soils of the Kola Peninsula].

Non-symbiotic nitrogen fixation was studied in virgin and cultivated podzol soils of the Kola Peninsula by the acetylene and isotope techniques. The activity of nitrogen fixation varied sharply in cultivated soils due to degradation of plant residues and the action of plant root secretion. No significant changes were observed in the activity of nitrogen fixation within the vegetative period in virgin soils. Less than 1 kg of nitrogen per hectare was accumulated in soils as a result of its non-symbiotic fixation during the vegetative periods of 1976--1977.

Aerobiosis

Involvement of the cytoplasmic membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids.

1. The nitrogen-fixing efficiency of freshly prepared suspensions of Rhizobium leguminosarum bacteroids from pea root nodules was considerably enhanced by addition of bovine serum albumin. Evidence was found that during preparation of bacteroids the cell membrane is exposed to the uncoupling effect of free fatty acids and to plant phospholipase D activity. Both effects could be counteracted by bovine serum albumin. 2. A technique was developed by which concentrations of free O2 and nitrogenase activity could be measured simultaneously under conditions of steady-state respiration. By means of this system it could be shown that in contrast to previous claims, high ATP/ADP ratios can be achieved in bacteroids even with a high concentration of O2 in the medium. 3. Nitrogen fixation was found to be controlled by the ATP/ADP ratio, the generation of reducing equivalents and the switch-off phenomenon. It was demonstrated that the generation of reducing equivalents for nitrogenase is regulated by the energized state and the integrity of the bacteroid cell membrane. The data indicate that the process of aerobic nitrogen fixation in R. leguminosarum bacteroids resembles that of Azotobacter vinelandii.

Adenosine Diphosphate

Intensity of nitrogen fixation in yellow lupine in presence of different doses of mineral nitrogen.

The effect of different doses of mineral nitrogen on the nitrogen-fixing activity of lupine nodules during vegetation was studied. It was shown that 0.5 of the dose of mineral nitrogen had an inhibitory effect only in the initial period of development of the nodules, whereas full and double doses of mineral nitrogen inhibited nitrogen fixation throughout the period of vegetation. Supplements of mineral nitrogen switch the lupine nutrition from the symbiotic to the autotrophic type, which is accompanied by a decrease in the nitrogen-fixing activity in the nodules and by an increase in the nitrate-reducing capacity of the roots and leaves.

Fabaceae

Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus.

Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen. The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined. It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms. The R. capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon. beta-Galactosidase expression from an R. capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions. R. capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R. capsulatus nif genes. Repression of nif genes in response to oxygen was still present in R. capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit.

Base Sequence

Nitrogen fixation associated with grasses in Oregon.

Nitrogen fixation associated with both natural grasslands and grain crops of Oregon was studied using the acetylene-reduction assay. A number of the grasses collected has some acetylene-reducing activity. Agrostis tenuis Sibth. had substantially greater activity than any of the other species, with a mean rate estimated at 37 g N2 fixed per hectare per day. Assuming 100 days of activity, about 3 kg of N2 would be fixed per hectare per year. This quantity of nitrogen may be important in the maintenance of this species under natural conditions. Nitrogen-fixing microorganisms were isolated from the root surfaces of some of the grasses. Cultures of Bacillus macerans, Bacillus polymyxa, and Enterobacter cloacoa were isolated from wheat roots as were two cultures which have not been assigned a specific taxonomic classification. Strains of N2-fixing Bacillus species and Gram-negative aerobic bacteria were isolated from Festuca and Agrostis.

Acetylene

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

Ammonia

Nitrogen fixation by Spirillum sp. from rice roots.

Nitrogen fixation by Spirillum sp. obtained from the roots of rice plants grown at different levels of combined nitrogen was studied. The roots of rice plants exposed to low levels of combined nitrogen (20--40 kg N/ha) harboured Spirillum sp. possessing higher nitrogen-fixing efficiency as compared to the cultures from plants receiving 60--100 kg N/ha. Moreover, the nitrogen-fixing efficiency of these Spirillum spp. varied with age of the plant, irrespective of the dosage of combined nitrogen.

Fertilizers

Effects of some inorganic elements on nitrogen-fixation in blue-green algae and some ecological aspects of pollution.

Nitrogen-fixation by two species of Nostoc, one of them a lichen phycobiont, was generally stimulated by low concentrations of arsenic, cadmium, nickel, lead, palladium, and zinc. Higher concentrations (0.025 to 0.125 ppm) of arsenic, nickel, and palladium were also stimulatory; however, higher concentrations of cadmium, lead, and zinc tended to inhibit fixation. With the exception of palladium and zinc at low concentrations these six tested elements tended to inhibit nitrogen-fixation in Chlorogloea fritschii and Westiellopsis sp.

Arsenic