Nitrogen‐isotope fractionation in the nitrate respiration by the marine bacterium Serratia marinorubra

Abstract

The isotopic composition (δ¹⁵N) of nitrite produced during nitrate respiration by both growing and washed cells of the marine bacterium, Serratia marinorubra, was determined. In both the growing and washed cells, δ¹⁵N of the nitrite changed considerably with time. At least two reaction steps, producing different isotope effects (active transport of nitrate across membranes and reduction of nitrate to nitrite), appear to be involved. With washed cells, a fractionation factor as high as 1.039 was obtained—the highest ever reported for biologicalnitrate reduction. The physiological state of nitrate‐respiring bacteria in oxygen‐depleted subsurface waters of the sea is discussed from the viewpoint of isotope fractionation.     

Nitrogen fixation and transfer in open ocean diatom–cyanobacterial symbioses

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N₂-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N₂ fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N₂ fixation (1.15–71.5 fmol N per cell h⁻¹) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N₂ fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N₂ fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.     

The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.     

Amino acid δ<Superscript>15</Superscript>N indicates lack of N isotope fractionation during soil organic nitrogen decomposition

We explore the question of how long elements cycle in terrestrial ecosystems and show that to address this question, a broader conceptual framework is needed that specifies ages and transit times. We calculated age and transit time distributions of five elements in a forest and two grassland ecosystems. Moreover, we assessed how ages and transit times of elements change in various scenarios. Mean age and mean transit time of all elements were smaller in the two grassland ecosystems than in the forest ecosystem due to the smaller element stocks in the grasslands in relation to the inputs. Phosphorus (P) had the largest mean transit time and mean age of all elements in the forest ecosystem (450 and 469 years) as well as in the high elevation grassland (82 and 80 years). Mean ages and mean transit times changed linearly with the stock in one pool. Changes in the internal cycling of elements in the ecosystem that did not imply the introduction of another pool had no effect on age and transit time. However, the introduction of a stable P pool in the mineral soil led to a divergence of mean transit time and mean age of P. Taken together, based on the probabilistic approach proposed here, we were able to precisely calculate not only the mean times elements need to transit different ecosystems and the ages they reach while cycling the ecosystems, but also the probability distribution of ages and transit times.     

Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Nitrogen fixation and diversity of benthic cyanobacterial mats on coral reefs in Curaçao

Coral Reefs - Benthic cyanobacterial mats (BCMs) have increased in abundance on coral reefs worldwide. However, their species diversity and role in nitrogen fixation are poorly understood. We...     

δ15N as an indicator of N2-fixation by cyanobacterial mats in tropical marshes

Cyanobacterial mats (CBM) are important components of wetland ecosystems in limestone-based regions of the Caribbean. During two sampling periods (July 1999 and January 2000) we measured N₂-fixation in samples from 23 different marshes simultaneously with measurements of relevant environmental factors. Samples were evaluated for abundance of five groups of cyanobacteria: (1) Leptolyngbya, (2) Oscillatoria, (3) Chroococcales, (4) Nostoc-& Stigonematales, and (5) dead sheaths. Differences in nitrogen fixation, expressed as nitrogenase activity in nmol C₂H₄ cm^–2 h^–1, were best explained by the proportion of heterocyst-forming cyanobacteria. The samples were analyzed for the natural abundance of ¹⁵N. ¹⁵N values ranged from –1.99 to 11.44 and were strongly negatively correlated with N₂-fixation. With all data included, ¹⁵N was also strongly correlated with nitrates in water. With the samples from Little Belize (high nitrate content marshes) excluded, the effect of nitrate became insignificant. N₂-fixation predicted from ¹⁵N measured on an independent data set from September 2000 was moderately accurate (r² = 0.68, 0.52 and 0.54 for predictions based on July 1999, January 2000 and combined data sets, respectively). When individual sample sets were divided into two groups with ¹⁵N < 2 and ¹⁵N > 2, the two groups were always highly significantly different in terms of their N₂-fixation. The presented evidence suggests that ¹⁵N can be used as a reliable indicator of N₂-fixation by CBM.     

Essential metals for nitrogen fixation in a free-living N₂-fixing bacterium: chelation, homeostasis and high use efficiency

Biological nitrogen fixation, the main source of new nitrogen to the Earth's ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo‐nitrogenase, the V‐nitrogenase and the Fe‐only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal‐replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo‐nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N₂ fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal‐limited soil environments.     

Computational analysis of the oscillatory dynamics in the processes of CO₂ assimilation and photorespiration

The computational analysis of the model system consisting of the processes of CO₂ assimilation and photorespiration shows the appearance of sustained oscillations in the system which might reflect their presence in photosynthesizing cells. Concentrations of CO₂ and O₂ oscillate in opposite phases causing Rubisco switching continuously between the carboxylase (CO₂ assimilation) and the oxygenase (photorespiration) reactions. The results of modeling are consistent with carbon isotopic and other observed data. They show that the oscillation period varies from about 1 s to 3 s depending on the values of parameters taken. Too high concentrations of O₂ suppress the oscillations.     

Is birch a suitable reference in estimating dinitrogen fixation in alders by isotope dilution?

Seedlings ofAlnus incana (nodulated and non-nodulated) andBetula papyrifera were fertilized with varying amounts (0, 10, 25, 50, 100, 250 and 500 g N g^–1 soil) of labelled ammonium-N or nitrate-N ( 5.2 A% excess¹⁵N as ammonium sulphate or potassium nitrate). After 4 months in the greenhouse,¹⁵N excess in the plants were determined and an isotope dilution equation was applied to determine the percent of biomass N fixed by theA. incana/Frankia system. When ammonium was used as the sole N source and birch as the non-fixing reference, N-fixation accounted for 95%, 87% and 60% of the plant nitrogen yields with 10, 25 and 50 g N g^–1 rates, additions respectively. At the 100 g N g^–1 fertilization and above N-fixation accounted for less than 10% of the N yield. Similar results were obtained when non-nodulatedA. incana was used as non-fixing reference. With nitrate as the sole N source, N-fixation accounted for 98%, 97%, 97%, 86%, 56% and 12% of N yield with 10, 25, 50, 100, 250 and 500 g N g^–1 additions respectively. These values were similar for both types of reference plants. The direct isotope dilution method was compared to that of the total nitrogen difference method. There was good agreement between the two methods up to 50 g N g^–1 for ammonium and up to 100 g N g^–1 for nitrate. The difference method produced negative values at high concentrations of nitrogen fertilization. Again similar results were obtained by the two reference plants. The results indicate that birch can be used as a non-fixing control in isotope dilution studies but that care must be exercised in selecting the type and quantity of labelled nitrogen fertilizer.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Ionic balance,proton efflux,nitrate reductase activity and growth ofHippophaë rhamnoides L. ssp.rhamnoides as influenced by combined-N nutrition or N2 fixation

Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N₂ fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH ₄ ⁺ , a mixture of NH ₄ ⁺ and NO ₃ ^? producing the highest yields. Growth was severely reduced when solely dependent on N₂ fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N₂-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO ₃ ^? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO ₃ ^? and NH ₄ ⁺ , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH ₄ ⁺ , NO ₃ ^? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO ₃ ^? +NH ₄ ⁺ plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH ₄ ⁺ plants than in the NO ₃ ^? plants. Measured extrusions of H⁺ or OH^? (HCO ₃ ^? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N₂ fixation amounted to 0.45–0.55 meq H⁺. (mmol N_org)^?1. Without acidity control and in the presence of NH ₄ ⁺ , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N₂-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

Large 13C/12C and small 15N/14N isotope fractionation in an experimental detrital foodweb (litter–fungi–collembolans)

Correctly estimating the trophic fractionation factors (Δ¹⁵N and Δ¹³C) in controlled laboratory conditions is essential for the application of stable isotope analysis in studies on the trophic structure of soil communities. Laboratory experiments usually suggest large ¹⁵N/¹⁴N and small ¹³C/¹²C trophic fractionation, but in field studies litter-dwelling microarthropods and other invertebrates are consistently enriched in ¹³C relative to plant litter. In the present study, we report data from two laboratory experiments investigating both fungi–collembolans and litter–fungi–collembolans systems. In the fungi–collembolans system, Δ¹⁵N and Δ¹³C averaged 1.4 ± 0.1 and 1.0 ± 0.2 ‰, respectively. In microcosms with fungi-inoculated litter, the difference in δ¹⁵N between collembolans and plant litter averaged 1.5 ± 0.2 ‰, confirming the relatively small ¹⁵N/¹⁴N trophic fractionation at the basal level of detrital foodwebs reported in numerous field studies. In full agreement with field observations, the difference in δ¹³C between bulk litter and collembolans in laboratory microcosms averaged 3.6 ± 0.1 ‰ and only little depended on collembolan species identities or the presence of water-soluble compounds in the litter. We conclude that increased δ¹³C values typical of litter-dwelling decomposers are largely determined by an increased ¹³C content in saprotrophic microorganisms.     

Carbohydrate utilization by normal and γ-sterilized Dacus oleae

Non-sterilized adult olive fruit flies were able to survive and reproduce on mannose, glucose, fructose, sucrose, melibiose, trehalose, maltose, melezitose, and sorbitol, out of a series of 23 carbohydrates γ-sterilized flies were able to utilize the same carbohydrates with the exception of melezitose, indicating that sterilization with a 10 Krad dose did not affect their ability to utilize various carbohydrates. Since these carbohydrates are common constituents of the fly's natural food sources, the released sterile flies should be able to survive satisfactorily in the field. The possible presence of three carbohydrases is discussed.     

Forest productivity under elevated CO₂ and O₃: positive feedbacks to soil N cycling sustain decade-long net primary productivity enhancement by CO₂

The accumulation of anthropogenic CO? in the Earth's atmosphere, and hence the rate of climate warming, is sensitive to stimulation of plant growth by higher concentrations of atmospheric CO?. Here, we synthesise data from a field experiment in which three developing northern forest communities have been exposed to factorial combinations of elevated CO? and O?. Enhanced net primary productivity (NPP) (c. 26% increase) under elevated CO? was sustained by greater root exploration of soil for growth-limiting N, as well as more rapid rates of litter decomposition and microbial N release during decay. Despite initial declines in forest productivity under elevated O?, compensatory growth of O? -tolerant individuals resulted in equivalent NPP under ambient and elevated O?. After a decade, NPP has remained enhanced under elevated CO? and has recovered under elevated O? by mechanisms that remain un-calibrated or not considered in coupled climate-biogeochemical models simulating interactions between the global C cycle and climate warming.     

Why no feeding frenzy? Mechanisms of nutrient acquisition and utilization during infection by the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae is a devastating pathogen of rice and wheat. It is a hemibiotroph that exhibits symptomless biotrophic growth for the first 4 to 5 days of infection of susceptible cultivars before becoming necrotrophic. Here, we review recent advances in our understanding of how M. oryzae is able to grow, acquire nutrients, and interact with the plant cell during infection. In particular, we describe direct mechanisms (such as the integration of carbon and nitrogen metabolism by trehalose-6-phosphate synthase 1) and indirect mechanisms (such as the suppression of host responses) that allow M. oryzae to utilize available host nutrient. We contrast the ability of M. oryzae to voraciously metabolize a wide range of carbon and nitrogen sources in vitro with the carefully orchestrated development it displays during the biotrophic phase of in planta growth and ask how the two observations can be reconciled. We also look at how nutrient acquisition and effector biology might be linked in order to facilitate rapid colonization of the plant host.     

Impaired cardiac reserve and severely diminished skeletal muscle O₂ utilization mediate exercise intolerance in Barth syndrome

Barth syndrome (BTHS) is a mitochondrial myopathy characterized by reports of exercise intolerance. We sought to determine if 1) BTHS leads to abnormalities of skeletal muscle O(2) extraction/utilization and 2) exercise intolerance in BTHS is related to impaired O(2) extraction/utilization, impaired cardiac function, or both. Participants with BTHS (age: 17 ± 5 yr, n = 15) and control participants (age: 13 ± 4 yr, n = 9) underwent graded exercise testing on a cycle ergometer with continuous ECG and metabolic measurements. Echocardiography was performed at rest and at peak exercise. Near-infrared spectroscopy of the vastus lateralis muscle was continuously recorded for measurements of skeletal muscle O(2) extraction. Adjusting for age, peak O(2) consumption (16.5 ± 4.0 vs. 39.5 ± 12.3 ml·kg(-1)·min(-1), P < 0.001) and peak work rate (58 ± 19 vs. 166 ± 60 W, P < 0.001) were significantly lower in BTHS than control participants. The percent increase from rest to peak exercise in ejection fraction (BTHS: 3 ± 10 vs. control: 19 ± 4%, P < 0.01) was blunted in BTHS compared with control participants. The muscle tissue O(2) saturation change from rest to peak exercise was paradoxically opposite (BTHS: 8 ± 16 vs. control: -5 ± 9, P < 0.01), and the deoxyhemoglobin change was blunted (BTHS: 0 ± 12 vs. control: 10 ± 8, P < 0.09) in BTHS compared with control participants, indicating impaired skeletal muscle extraction in BTHS. In conclusion, severe exercise intolerance in BTHS is due to both cardiac and skeletal muscle impairments that are consistent with cardiac and skeletal mitochondrial myopathy. These findings provide further insight to the pathophysiology of BTHS.