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Biological nitrogen fixation in the terrestrial environment of a high Arctic ecosystem (Truelove Lowland, Devon Island, N.W.T.).

Arranged in descending order of nitrogen-fixing (acetylene-reducing) potential the sites examined were mesic meadow and peat polygon troughs (equal rank), transition zone between mesic meadow and gravel ridge, gravel ridge, polar dessert, and peat polygon tops. The dominant nitrogen-fixing microorganisms, as in other Arctic areas, were blue-green bacteria, especially those epiphytic on Arctic mosses. The epiphytic association exhibited an optimum temperature for fixation of 20 degrees C. Other bacteria potentially able to fix nitrogen were present in the soils examined but their activity was severely restricted by low soil temperatures and lack of readily utilizable energy sources. These bacteria included members of the genera Klebsiella (the most numerous), Bacillus, Clostridium, and Beijerinckia (scarce). Also present at many of the sites was an unidentified yellow-pigmented fixer which was not Mycobacterium flavum. All fixers were psychotrophic rather than psychrophilic, having an optimum temperature greater than 20 degrees C but capable of slow growth at 5 degrees C or lower. The rate of acetylene reduction by the epiphytic system increased with the number of successive exposures to acetylene, a phenomenon of some significance in any calculations designed to measure the amount of nitrogen fixed in certain ecosystems.

Acetylene↗

Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

Two Azotobacter vinelandii strains capable of growing on N2(Nif+) were isolated from two different mutant strains that lacked dinitrogenase activity (Nif-). Extracts of N2-grown cells of the two Nif+ strains lacked significant amounts of the "conventional" dinitrogenase protein subunits, as determined by two-dimensional gel electrophoresis. Instead, the extracts contained at least four new proteins that appeared to be ammonia-repressible (i.e., they were not detected in extracts of ammonia-grown cells). Based on the results of genetic backcrosses, the two Nif+ strains were shown to be pseudorevertants. Both Nif+ pseudorevertant strains were able to grow in N-free media lacking molybdenum but containing tungsten (conditions that prevented growth of the wild-type strain). The four new proteins were observed in extracts of N2-fixing cells of the Nif+ pseudorevertants regardless of whether the cells were grown in the presence of molybdenum-starved wild-type A. vinelandii cells grown under N2-fixing conditions. Under conditions of molybdenum deprivation, Nif- mutant strains of several different phenotypic classes underwent phenotypic reversal to Nif+, as shown by their ability to incorporate 15N2 and to grow in N-free media. These results provide evidence that A. vinelandii possesses an alternative N2-fixation system that is expressed during conditions of molybdenum deficiency.

Azotobacter↗

Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-beta-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii.

Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-beta-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation in ptsP leads to a failure in poly-beta-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

Amino Acid Sequence↗

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems.

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O2.- and H2O2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 micrograms/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N2 fixation in nodules.

Ascorbate Peroxidases↗

[Ability of obligate methylotrophs to perform nitrogen fixation].

The ability for nitrification was studied among mesophilic and thermophilic cultures of obligate methylotrophs Methylobacter ucrainicus, Methylomonas methanica, and Methylococcus thermophilus. The strains were almost incapable of nitrification under autotrophic conditions. In the presence of methane, however, they oxidized NH+4 TO NO-2: over 150 mg/litre NO-2 nitrogen was found in the cultural broth. Therefore, obligate methylotrophs are capable of heterotrophic nitrification. The level of nitrification suggests that it is not a side reaction but a necessary stage of the metabolism of obligate methylotrophs. The phenotype relation between obligate methylotrophs and nitrifying organisms is discussed.

Bacteria↗