Nitrogen fixation and nitrogen metabolism in heliobacteria

Three species of anoxygenic phototrophic heliobacteria, Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis, were studied for comparative nitrogen-fixing abilities and regulation of nitrogenase. Significant nitrogenase activity (acetylene reduction) was detected in all species grown photoheterotrophically on N₂, although cells of H. mobilis consistently had higher nitrogenase activity than did cells of either H. chlorum or H. gestii. Nitrogen-fixing cultures of all three species of heliobacteria were subject to switch-off of nitrogenase activity by ammonia; glutamine also served to switch-off nitrogenase activity but only in cells of H. mobilis and H. gestii. Placing photosynthetically grown heliobacterial cultures in darkness also served to switch-off nitrogenase activity. Dark-mediated switch-off was complete in lactate-grown heliobacteria but in pyruvate-grown cells substantial rates of nitrogenase activity continued in darkness. In all heliobacteria examined ammonia was assimilated primarily through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway although significant levels of alanine dehydrogenase were present in extracts of cells of H. gestii, but not in the other species. The results suggest that heliobacteria, like phototrophic purple bacteria, are active N₂-fixing bacteria and that despite their gram-positive phylogenetic roots, heliobacteria retain the capacity to control nitrogenase activity by a switch-off type of mechanism. Because of their ability to fix N₂ both photosynthetically and in darkness, it is possible that heliobacteria are significant contributors of fixed nitrogen in their paddy soil habitat.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

磁场对大豆共生固氮的效应

恒定磁场处理慢生大豆根瘤菌“005”和接种后的大豆植株,发现磁场可以提高根瘤的固氮活性。在一定的磁场强度(70—100mT)下,固氮活性平均可以提高4—5倍,植株的结瘤数和根瘤重量平均提高2—3倍。从这样的根瘤中所分离出的根瘤菌,由慢生型转变成快生型,在100植株中有17株的根瘤分离出快生菌。生长世代时间和培养溶液中的pH值与慢生型不同,而与快生型相同。     

The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti

Summary The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wildtype symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix⁺. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon; rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria

Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The ¹³C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (¹³C between-25.3 and-27.8) were significantly less depleted in ¹³C than needles (¹³C between-27.3 and-29.1), and ¹³C in twigs increased consistently with age. The ¹⁵N values of needles ranged between-2.5 and-4.1 and varied according to stand and age. In young needles ¹⁵N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in ¹⁵N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. ¹⁵N values in twigs were more negative than in needles (-3.5 to-5.2) and showed age- and stand-dependent trends that were similar to the needles. ¹⁵N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4 in the mineral soil. The ¹⁵N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed.     

Leaf lifespan as a determinant of leaf structure and function among 23 amazonian tree species

Summary The relationships between resource availability, plant succession, and species' life history traits are often considered key to understanding variation among species and communities. Leaf lifespan is one trait important in this regard. We observed that leaf lifespan varies 30-fold among 23 species from natural and disturbed communities within a 1-km radius in the northern Amazon basin, near San Carlos de Rio Negro, Venezuela. Moreover, leaf lifespan was highly correlated with a number of important leaf structural and functional characterisues. Stomatal conductance to water vapor (g) and both mass and area-based net photosynthesis decreased with increasing leaf lifespan (r²=0.74, 0.91 and 0.75, respectively). Specific leaf area (SLA) also decreased with increasing leaf lifespan (r²=0.78), while leaf toughness increased (r²=0.62). Correlations between leaf lifespan and leaf nitrogen and phosphorus concentrations were moderate on a weight basis and not significant on an area basis. On an absolute basis, changes in SLA, net photosynthesis and leaf chemistry were large as leaf lifespan varied from 1.5 to 12 months, but such changes were small as leaf lifespan increased from 1 to 5 years. Mass-based net photosynthesis (A/mass) was highly correlated with SLA (r²=0.90) and mass-based leaf nitrogen (N/mass) (r²=0.85), but area-based net photosynthesis (A/area) was not well correlated with any index of leaf structure or chemistry including N/area. Overall, these results indicate that species allocate resources towards a high photosynthetic assimilation rate for a brief time, or provide resistant physical structure that results in a lower rate of carbon assimilation over a longer time, but not both.     

The influence of salinity on the kinetics of NH inf4 sup+ uptake in Spartina alterniflora

Summary The effects of short- and long-term exposure to a range in concentration of sea salts on the kinetics of NH _inf4 ^sup+ uptake by Spartina alterniflora were examined in a laboratory culture experiment. Long-term exposure to increasing salinity up to 50 g/L resulted in a progressive increase in the apparent K_m but did not significantly affect V_max (mean V_max=4.23±1.97 mole·g^–1·h^–1). The apparent K_m increased in a nonlinear fashion from a mean of 2.66±1.10 mole/L at a salinity of 5 g/L to a mean of 17.56±4.10 mole/L at a salinity of 50 g/L. These results suggest that the long-term effect of exposure to total salt concentrations within the range 5–50 g/L was a competitive inhibition of NH _inf4 ^sup+ uptake in S. alterniflora. No significant NH _inf4 ^sup+ uptake was observed in S. alterniflora exposed to 65 g/L sea salts. Short-term exposure to rapid changes in salinity significantly affected both V_max and K_m. Reduction of solution salinity from 35 to 5 g/L did not change V_max but reduced K_m by 71%. However, exposing plants grown at 5 g/L salinity to 35 resulted in an decrease in V_max of approximately 50%. Exposure of plants grown at 35 g/L to a total sea salt concentration of 50 g/L for 48h completely inhibited uptake of NH _inf4 ^sup+ . For both experiments, increasing salinity led to an increase in the apparent K_m similar to that found in response to long-term exposure. Our data are consistent with a conceptual model of changes in the productivity of S. alterniflora in the salt marsh as a function of environmental modification of NH _inf4 ^sup+ uptake kinetics.     

Nitrate nutrition ofDeschampsia flexuosa (L.) Trin. in relation to nitrogen deposition in Sweden

Summary Current and maximally induced nitrate reductase activity (NRA), total-N, nitrate, K, P, Ca, Mg, Mo and sucrose in leaves ofDeschampsia flexuosa was measured three times during the vegetation period in forests along a deposition gradient (150 km) in south Sweden, in north Sweden where the nitrogen deposition is considerably lower, and at heavily N-fertilized plots. In addition, the interaction between nitrogen nutrition and light was studied along transects from clearings into forest in both south and north Sweden. Plants from sites with high nitrogen deposition had elevated current NRA compared to plants from less polluted sites, indicating high levels of available soil nitrate at the former. Current NRA and total N concentration in grass from sites with high deposition resembled those found at heavily N-fertilized plots. Under such circumstances, the ratio current NRA: maximally induced NRA as well as the concentration of nitrate was high, while the concentration of sucrose was low. This suggests that the grass at these sites was already utilizing a large portion of its capacity to assimilate nitrate. Light was found to play an important role in the assimilation of nitrate; leaf concentration of sucrose was found to be negatively correlated with both nitrate and total N. Consequently, grass growing under dense canopies in south Sweden is not able to dilute N by increasing growth. The diminished capacity of the grass to assimilate nitrate will increase leaching losses of N from forests approaching N saturation.     

The utilization of nitrogen from insect capture by different growth forms of Drosera from Southwest Australia

Summary Plants of Drosera species, neighbouring noncarnivorous plants, and arthropods on or near each Drosera sp. were collected at 11 contrasting habitat locations in SW Australia. At three of the sites clones of the rare glandless mutant form of D. erythrorhiza were collected alongside fully glandular counterparts. The ¹⁵N value (¹⁵N/¹⁴N natural isotope composition) of insect-free leaf and stem fractions was measured, and the data then used to estimate proportional dependence on insect N (%N_dI) for the respective species and growth forms of Drosera. The data indicated lower %N_dI values for rosette than for self-supporting erect or for climbing vine species. The latter two groups showed an average %N_dI value close to 50%. The %N_dI increased with length and biomass of climbing but not erect forms of Drosera. ¹⁵N values of stems were positively correlated with corresponding values for leaves of Drosera. Leaf material was on average significantly more ¹⁵N enriched than stems, possibly due to delayed transport of recent insect-derived N, or to discrimination against ¹⁵N in transfer from leaf to the rest of the plant. The comparison of ¹⁵N values of insects and arthropod prey, glandless and glandular plants of D. erythrorhiza indicated %N_dI values of 14.3, 12.2 and 32.2 at the respective sites, while matching comparisons based on ¹⁵N of insect, reference plants and glandular plants proved less definitive, with only one site recording a positive %N_dI (value of 10.4%) despite evidence at all sites of feeding on insects by the glandular plants. The use of the ¹⁵N technique for studying nutrition of carnivorous species and the ecological significance of insect feeding of different growth forms of Drosera growing in a large range of habitats is discussed.