Stimulated Nitrogenase Activity Research Articles

Effects of metals on nitrogen fixation and denitrification in slurries of anoxic salt-marsh sediment

The effect of metals on nitrogen fixation, as measured by acetylene reduction, and on denitrification, as determined by nitrous oxide accumulation in the presence of acetylene (acetylene blockage), was examined in short-term experiments with saltmarsh sediments. At concentrations of 1000 ppm (weight metal : weight dry sediment), HgCI,, PbCl,, CdC12, ZnSO,, CuCl,, K,Cr207, K,CrO,, and Na,MoO, decreased acetylene reduction throughout the experiments by more than 30 %. FeC13 decreased it to a lesser extent, while NiC12 greatly stimulated nitrogenase activity. Initial rate of nitrous oxide production was inhibited by HgCl,, PbCl,.. NiCI,, K2Cr207, K,CrO,. ZnSO,, CuC1,. FeCl,, and CdCI,, but maximum production was stimulated substantially by PbCl,, K2Cr20,, and K,CrO,, and somewhat by Na2Mo0,, ZnSO,, and CuCl,. In contrast, NiCl, depressed both initial and maximum nitrous oxide production. Lower levels of Ni and Hg (10 and 100 ppm) caused effects that were intermediate between 1000 ppm and controls. We conclude that metal pollution could considerably alter nitrogen dynamics in marine sediments, at least in the short term; and could have repercussions on water-column productivity.

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. α-ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.

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 N(2)-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 KNO(3) 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 KNO(3) 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 KNO(3) or KNO(2) 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.

Field measurements of nitrogenase activity in soils amended with wheat straw

Nitrogenase activity and decomposition of straw were examined in situ in two areas (Gunnedah and Cowra) representative of large areas of the New South Wales wheat belt. Measurements of nitrogenase activity were made by adapting the acetylene reduction assay for use in the field. Evolution of CO2 was monitored as an indirect measure of decomposition of straw. The addition of straw to soil stimulated nitrogenase activity which was related to the amount of straw added and the rate of straw decomposition. There were significant levels of activity provided the soil was moist and warm. Nitrogenase activity increased with mean daily soil temperature (up to at least 30�C) and decreased as the soil dried from field capacity. It is concluded that nitrogen fixation by free-living nitrogen-fixing microorganisms in soils amended with straw may contribute to the nitrogen status of the soil and thus reduce the need for nitrogen fertilizers.

Stimulation of Nitrogenase Activity in in vitro Associations of Petunia Plantlets with Rhizobium 32H1 by Plant Growth Substances

Summary Non-leguminous tissues and even plants like petunias and wheat are able to induce nitrogenase activity in Rhizobium. The effect of 6-benzyladenine, α-naphthylacetic acid, and gibberellic acid on the induction of nitrogenase activity in associations of Petunia hybrida with Rhizobium 32H1 was studied. All the three growth regulators stimulated nitrogenase activity. The highest value, an 11-fold increase compared with Petunia-Rhizobium associations without hormones, was found with a 6-benzyladenine concentration which was superoptimal for growth and development of the petunias. Under the conditions used, neither petunias nor rhizobia alone showed nitrogenase activity. These data gained with the model plant Petunia are basic for similar investigations using plants of nutritional value like wheat.

Nitrogenase regulation in Anabaena cylindrica

The regulation of nitrogenase. biosynthesis in the heterocystous blue-green alga, Anabaena cylindrica was studied in vivo. The action of ammonia on nitrogenase activity during heterocyst induction and in fully differentiated cultures was compared with that of selective inhibitors of transcription and translation. The process of heterocyst differentiation is known to become refractory to inhibition by ammonia at some intermediate stage. We now report that nitrogenase induction is also inhibited only in the early stages of heterocyst development. Later in the differentiation process, nitrogenase activity continued to increase in the presence of ammonia. The addition of ammonia to logarithmically growing cultures had little effect on nitrogenase activity on a volume basis; however, no new heterocysts were formed and specific activity declined as the cultures grew. Nitrogenase under these conditions is not stable since inhibition of protein synthesis resulted in rapid loss of enzyme activity. Thus nitrogenase biosynthesis does not appear to be inhibited in heterocysts. Dramatic losses of nitrogenase activity were found after prolonged ammonia treatment of linear phase cultures. H2 stimulated nitrogenase activity to nearly control levels and sensitivity to DCMU (in the presence of O2) increased while dark activities were lost. These findings suggest indirect effects of ammonia or its metabolites (e.g. glutamine) on heterocyst differentiation, reductant supply, and oxygen protection, rather than a direct effect on nitrogenase biosynthesis.

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 α-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.

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir-) were also capable of reducing nitrous oxide to N2.

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cylindrica.

Exogenous pyruvate added to cultures of the blue-green alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO2 (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with, or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O2, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O2. Malate, citrate, alpha-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of 14C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.

Nitrogenase activity and photosynthesis in Plectonema boryanum.

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.