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PubMed1976

Effect of protein additives on acetylene reduction (nitrogen fixation) by Rhizobium in the presence and absence of soybean cells.

The effect of protein additives on acetylene reduction (N(2) fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Merr.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/ml supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of alpha-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autoclaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction. The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32H1-soybean cell associations on media containing alpha-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N(2) under field conditions.

Journal Article↗
PubMed1984

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum.

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO(2) on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO(2) fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO(2)-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.

Journal Article↗
PubMed1999

Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon methanosarcina barkeri 227

The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases. Na+ and K+ had equivalent effects, whereas Mg2+ was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl- was more inhibitory than acetate was. Because M. barkeri 227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N2 than with NH4+. Cells grown with N2 and cells grown with NH4+ produced equal concentrations of alpha-glutamate at low salt concentrations and equal concentrations of Nepsilon-acetyl-beta-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.

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PubMed1995

Diversity of heterotrophic nitrogen fixation genes in a marine cyanobacterial mat.

The diversity of nitrogenase genes in a marine cyanobacterial mat was investigated through amplification of a fragment of nifH, which encodes the Fe protein of the nitrogenase complex. The amplified nifH products were characterized by DNA sequencing and were compared with the sequences of nitrogenase genes from cultivated organisms. Phylogenetic analysis showed that similar organisms clustered together, with the exception that anaerobic bacteria clustered together, even though they represented firmicutes, (delta)-proteobacteria, and (gamma)-proteobacteria. Mat nifH sequences were most closely related to those of the anaerobes, with a few being most closely related to the cluster of (gamma)-proteobacteria containing Klebsiella and Azotobacter species. No cyanobacterial nifH sequences were found from the mat collected in November when Microcoleus chthonoplastes was the dominant cyanobacterium, but sequences closely related to the cyanobacterium Lyngbya lagerheimeii were found during summer, when a Lyngbya strain was dominant. The results indicate that there is a high diversity of heterotrophic nitrogen-fixing organisms in marine cyanobacterial mats.

Journal Article↗
PubMed1994

Utilization of light for nitrogen fixation by a new synechocystis strain is extended by its low photosynthetic efficiency.

Performance of photosynthesis and nitrogenase activity in a novel cyanobacterium, Synechocystis sp. strain BO 8402, isolated from Lake Constance, located at the northern fringe of the Alps in central Europe, and of a stable derivative, strain BO 9201, were examined. Strain BO 8402 is characterized by an extraordinarily high level of autofluorescence originating from paracrystalline phycobiliprotein-linker complexes located in inclusion bodies (W. Reuter, M. Westermann, S. Brass, A. Ernst, P. Böger, and W. Wehrmeyer, J. Bacteriol. 176:896-904, 1994). Energy transfer between paracrystalline phycobiliproteins and the photosystems is inefficient, resulting in a high oxygen compensation point and a decreased growth rate. The derivative strain BO 9201 exhibits hemidiscoidal phycobilisomes that support a high growth rate, even under low light intensities. Because of the differences in photosynthetic performance, anaerobic light-stimulated nitrogenase activity is maintained at higher light intensity in the original strain BO 8402 than in the derivative strain BO 9201. The results indicate that the formation of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 represents a hitherto-unknown means for a unicellular cyanobacterium to extend its capacity to fix nitrogen in the light.

Journal Article↗
PubMed1990

Nitrogen fixation of nodulation mutants of soybean as affected by nitrate.

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C(2)H(2) reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C(2)H(2) reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C(2)H(2) reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.

Journal Article↗
PubMed1979

Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest.

Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity.Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants.No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed.

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PubMed2001

RpoN of Rhizobium leguminosarum bv. viciae strain VF39SM plays a central role in FnrN-dependent microaerobic regulation of genes involved in nitrogen fixation.

The rpoN gene, which codes for the alternative transcription factor sigma54, was cloned and sequenced from Rhizobium leguminosarum strain VF39SM. Construction of a rpoN mutant allowed analysis of the role of RpoN as a transcriptional regulator of genes carrying lacZ reporter fusions. Analysis of a rpoN::lacZ transcriptional fusion in the rpoN background revealed that this gene was negatively autoregulated. Site-directed mutagenesis was used to demonstrate that this autoregulation was dependent on a reverse complement RpoN binding site located upstream of the rpoN gene. rpoN was shown to be required for full microaerobic expression of both copies of fixGHIS, as well as of fixNOQP, despite the absence of apparent rpoN binding sites upstream of fixG. Moreover, rpoN was found to be required for full microaerobic expression of fnrN, which in turn is absolutely required for microaerobic induction of fixGHIS. This suggests that the reduced fixG::lacZ expression seen in the rpoN background is due to the dependence of fnrN expression on RpoN.

Bacterial Proteins↗
PubMed1988

The effect of crude oil and oil spill chemicals on nitrogen fixation in the cyanobacteria Nostoc sp.

The effects of crude oil and three oil spill dispersants (Corexit 9600, 9550 and 7664) on nitrogenase activity in the cyanobacteria Nostoc sp. were examined. The addition of oil to Nostoc sp. cultures resulted in a catastrophic decline in nitrogenase activity with activity ceasing 7 h after treatment. The addition of a dispersant with the oil did not ameliorate this effect. Cultures exposed to high concentrations of dispersants showed lower rates on nitrogenase activity than untreated cultures. However, it is unlikely that dispersant concentrations of this magnitude would occur in the field. At the lowest concentration tested, which approximates the manufacturer's recommended application rate, the effects of the dispersant appear to be negligible.

Journal Article↗
PubMed2003

The first example of a seven-coordinate vanadium(III) thiolate complex containing the hydrazine molecule, an intermediate of nitrogen fixation.

The first example of a seven-coordinate vanadium(III) thiolate complex, [V(PS3' ')(N(2)H(4))(3)] (1), where PS3' ' = [P(C(6)H(3)-3-Me(3)Si-2-S)(3)](3)(-), has been synthesized and characterized. Compound 1 contains a tetradentate ligand (PS3' ') and three hydrazine molecules, forming a capped octahedral geometry. A five-coordinate vanadium(III) complex, [V(PS3)(1-Me-Im)] (2), where PS3 = [P(C(6)H(4)-2-S)(3)](3)(-) and 1-Me-Im = 1-methyl-imidazole, was also obtained. Compound 2 adopts a trigonal bipyramidal geometry, in which the vanadium is ligated by the title ligand, PS3, and one 1-Me-Im molecule.

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