Nitrogen flux into metabolites and microcystins changes in response to different nitrogen sources in Microcystis aeruginosa NIES-843

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N₂-fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing ¹⁵N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, ¹⁵N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply

The extent to which individual plants utilise nitrate and ammonium, the two principal nitrogen sources in the rhizosphere, is variable and many species require a balance between the two forms for optimal growth. The effects of nitrate and ammonium on gene expression, enzyme activity and metabolite composition have been documented extensively with the aim of understanding the way in which plant cells respond to the different forms of nitrogen, but ultimately the impact of these changes on the organisation and operation of the central metabolic network can only be addressed by analysing the fluxes supported by the network. Accordingly steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown in Murashige and Skoog and ammonium‐free media, treatments that influenced growth and biomass composition. Fluxes through the central metabolic network were deduced from the redistribution of label into metabolic intermediates and end products observed when cells were labelled with [1‐¹³C]‐, [2‐¹³C]‐ or [¹³C₆]glucose, in tandem with ¹⁴C‐measurements of the net accumulation of biomass. Analysis of the flux maps showed that: (i) flux through the oxidative pentose phosphate pathway varied independently of the reductant demand for biosynthesis, (ii) non‐plastidic processes made a significant and variable contribution to the provision of reducing power for the plastid, and (iii) the inclusion of ammonium in the growth medium increased cell maintenance costs, in agreement with the futile cycling model of ammonium toxicity. These conclusions highlight the complexity of the metabolic response to a change in nitrogen nutrition.     

Organic and inorganic nitrogen uptake in lichens

In order to learn more about nitrogen (N) acquisition in lichens, and to see whether different lichens differ in their affinity to various N sources, N uptake was measured in 14 various lichen associations (species). These species represented various morphologies (fruticose or foliose), contrasting microhabitat preferences (epiphytic or terricolous), and had green algal, cyanobacterial or both forms of photobionts. N was supplied under non-limiting conditions as an amino acid mixture, ammonium, or nitrate, using ¹⁵N to quantify uptake. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to separate active and passive uptake. Thallus N, amino acids, soluble polyol concentrations, and the biont-specific markers chlorophyll a and ergosterol were quantified, aiming to test if these metabolites or markers were correlated with N uptake capacity. Ammonium uptake was significantly greater and to a higher extent passive, relative to the other two N sources. Nitrate uptake differed among lichen photobiont groups, cyanobacterial lichens having a lower uptake rate. All lichens had the capacity to assimilate amino acids, in many species at rates equal to nitrate uptake or even higher, suggesting that organic N compounds could potentially have an important role in the N nutrition of these organisms. There were no clear correlations between N uptake rates and any of the measured metabolites or markers. The relative uptake rates of ammonium, nitrate and amino acids were not related to morphology or microhabitat.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - Chl Chlorophyll - N Nitrogen     

The influence of different nitrogen sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically on 9 different nitrogen sources and the production of volatile compounds determined by a gas chromatographic head-space technique. Excellent growth was supported by glutamine, aspartic acid, asparagine, (NH₄)₂-tartrate and NH₄H₂PO₄. Valine, leucine, and particularly isoleucine were utilized with a somewhat lower growth rate. Lysine was rapidly utilized after a prolonged lag phase.The highest production of volatile compounds was obtained from leucine and isoleucine. At least 20 volatile compounds were formed from each of them and many products were detected in high concentrations. Intermediate amounts of volatile compounds were produced from asparagine, the ammonium salts and valine, and low amounts from lysine, glutamine and aspartic acid.Ethyl acetate was a major product irrespective of the nitrogen source used. Regarding the pattern of volatile compounds produced, leucine, isoleucine and valine had much in common. Most of the volatile products formed from these amino acids contained a branched carbon chain and at least three high-boiling components eluted later than n-amyl acetate from the gas chromatographic column. The other six nitrogen sources could be grouped together. In general the same volatile compounds were formed from these sources, but the quantities of the individual compounds differed. Only one component eluted later than n-amyl acetate. No basic difference in production of volatile compounds was observed between the ammonium salts and -amino compounds like lysine and asparagine.     

Ammonium and methylammonium uptake in a fertilizer-degrading strain of Ochrobactrum anthropi

The transport of ammonium and methylammonium was studied in a strain of Ochrobactrum anthropi, a microorganism isolated from garden soil and able to degrade methyleneureas which are used as slow-release nitrogen fertilizer. The activity of both transport systems was determined using [¹⁴C]methylammonium. Differences between the two transport systems were observed with regard to their pH- and temperature dependence as well as their kinetic parameters and regulation during growth with various nitrogen sources. Ammonium transport was subject to repression by ammonium and to derepression in its absence, while the methylammonium carrier was induced in the presence of methylamine. The ammonium but not the methylammonium transport system was severely inhibited by ammonium, and metabolic poisons inhibited both uptake systems. The analysis of intracellular metabolites using thin-layer chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that methylammonium was rapidly metabolized to N-methylglutamate via -N-methylglutamine.     

Comparative utilization of inorganic and organic compounds as sole nitrogen sources by the submergent duckweed,Lemna trisulca L.

The capability of the submergentLemna trisulca L. to utilize various inorganic and organic sources of nitrogen was studied using both non-axenic and axenic cultures. When doubling time for frond production was measured, the nitrogen sources in order of effectiveness were urea, aspartic acid, nitrate, glutamic acid, arginine, ammonium and casein hydrolysate. Nitrite supported a relatively rapid growth rate after an initial lag of 7 days. Other parameters of growth such as fresh or dry weight per frond or ohlorophyll content did not oorrelate well with rate of frond production. Casein hydrolysate and urea were found to elicit a morphology different from that seen in cultures containing the other nitrogen sources. These preferences for source of nitrogen were different than those known for the emergent species ofLemnaceae. The unique value ofL. trisulca as a subject for plant physiological research is discussed. This study also provides a possible explanation for the existence of nutritional niches existing in aquatio ecosystems containing several different species ofLemnaceae.     

Molecular analysis of the diversity of nitrifying bacteria in the soils of the forest and steppe zones of European Russia

Nitrifying bacteria play a key role in the global nitrogen cycle due to their ability to convert reduced nitrogen compounds (ammonium) to oxidized ones (nitrite and nitrate). Recent investigations based on the methods of molecular ecology revealed that bacteria are responsible for nitrification in natural ecosystems. At the same time, data on the species composition of the nitrifiers in soil microbial communities are scarce. Soil samples collected in the forest and steppe areas of European Russia and the enrichment cultures of nitrifying bacteria isolated from these samples were used for molecular studies of the diversity of the amoA gene encoding the synthesis of the key enzyme of autotrophic ammonium oxidation. The nitrifying bacteria of the genera Nitrosospira and Nitrosovibrio were found in all the studied soils from natural biocenoses and agrocenoses.     

The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook

Analysis of soil solution from forest sites dominated by Eucalyptus grandis and Eucalyptus maculata indicates that soluble forms of organic nitrogen (amino acids and protein) are present in concentrations similar to those of mineral nitrogen (nitrate and ammonium). Experiments were conducted to determine the extent to which mycorrhizal associations might broaden nitrogen source utilization in Eucalyptus seedlings to include organic nitrogen. In isolation, species of ectomycorrhizal fungi from northern Australia show varying abilities to utilize mineral and organic forms of nitrogen as sole sources. Pisolithus sp. displayed strongest growth on NH₄⁺, glutamine and asparagine, but grew poorly on protein, while Amanita sp. grew well both on mineral sources and on a range of organic sources (e.g. arginine, asparagine, glutamine and protein). In sterile culture, non-mycorrhizal seedlings of Eucalyptus grandis and Eucalyptus maculata grew well on mineral sources of nitrogen, but showed no ability to grow on sources of organic nitrogen other than glutamine. In contrast, mycorrhizal seedlings grew well on a range of organic nitrogen sources. These observations indicate that mycorrhizal associations confer on species of Eucalyptus the ability to broaden their resource base substantially with respect to nitrogen. This ability to utilize organic nitrogen was not directly related to that of the fungal symbiont in isolation. Seedlings mycorrhizal with Pisolithus sp. were able to assimilate sources of nitrogen (in particular histidine and protein) on which the fungus in pure culture appeared to grow weakly. Experiments in which plants were fed ¹⁵N-labelled ammonium were undertaken in order to investigate the influence of mycorrhizal colonization on the pathway of nitrogen metabolism. In roots and shoots of all seedlings, ¹⁵N was incorporated into the amide group of glutamine, and label was also found in the amino groups of glutamine, glutamic acid, γ-aminobutyric acid and alanine. Mycorrhizal colonization appeared to have no effect on the assimilation pathway and metabolism of [¹⁵N]H₄⁺; labelling data were consistent with the operation of the glutamate synthase cycle in plants infected with either Pisolithus sp. (which in isolation assimilates via the glutamate synthase cycle) or Elaphomyces sp. (which assimilates via glutamate dehydrogenase). It is likely that the control of carbon supply to the mycorrhizal fungus from the host may have a profound effect on both the assimilatory pathway and the range of nitrogen sources that can be utilized by the association.     

Nitrate enhancement of CAM activity in two Kalanchoë species is associated with increased vacuolar proton transport capacity

Among species that perform CAM photosynthesis, members of the genus Kalanchoë have been studied frequently to investigate the effect of environmental factors on the magnitude of CAM activity. In particular, different nitrogen sources have been shown to influence the rate of nocturnal CO₂ fixation and organic‐acid accumulation in several species of Kalanchoë. However, there has been little investigation of the interrelationship between nitrogen source (nitrate versus ammonium), concentration and the activity of the vacuolar proton pumps responsible for driving nocturnal organic‐acid accumulation in these species. In the present study with Kalanchoë laxiflora and Kalanchoë delagoensis cultivated on different nitrogen sources, both species were found to show highest total nocturnal organic‐acid accumulation and highest rates of ATP‐ and PPi‐dependent vacuolar proton transport on 2.5 mM nitrate, whereas plants cultivated on 5.0 mM ammonium showed the lowest values. In both species malate was the principal organic‐acid accumulated during the night, but the second‐most accumulated organic‐acid was fumarate for K. laxiflora and citrate for K. delagoensis. Higher ATP‐ and PPi‐dependent vacuolar proton transport rates and greater nocturnal acid accumulation were observed in K. delagoensis compared with K. laxiflora. These results show that the effect of nitrogen source on CAM activity in Kalanchoë species is reflected in corresponding differences in activity of the tonoplast proton pumps responsible for driving sequestration of these acids in the vacuole of CAM‐performing cells.     

Mineral Nutrient Requirements of Pinus silvestris and Picea abies Seedlings

The mineral nutrient requirements of Pinus silvestris L. and Picea abies Karst. were studied according to previously published methods applied to a series of various plant species. The optimum nutrient proportions are similar to those of Vaccinium, with a lower relative potassium requirement than birch and other broad-leaf species. Various ratios between ammonium and nitrate nitrogen were about equally efficient except for a minor growth reduction with pure nitrate, which gave a comparatively low nitrogen content and a high cation/nitrogen ratio. The rate of ammonium uptake was much higher than that of nitrate when both sources were supplied. The required total concentration in the nutrient solution for maximum growth is lower in pine than in spruce, but both fall within the low salt range. Both species, especially pine, are sensitive to high salt concentrations. Although pine and spruce grow on the same type of soils as Vaccinium— leached soils with low base saturation – accumulation of calcium or other cations is not as pronounced as in Vaccinium, especially not in pine. The results are compared with results from similar experiments with a series of other conifers. All the conifers have more flexible cation uptake mechanisms than Vaccinium but the results indicate tendencies to accumulation of anions, nitrate and phosphate. Recommended fertilizer compositions for forest fertilization and nurseries are discussed.     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Ammonium and urea removal by Spirulina platensis

Different concentrations either of ammonium chloride or urea were used in batch and fed-batch cultivations of Spirulina platensis to evaluate the possibility of substituting nitrate by cheaper reduced nitrogen sources in wastewaters biotreatment. The maximum nitrogen concentration able to sustain the batch growth of this microalga without inhibition was 1.7 mM in both cases. Ammonium chloride was limiting for the growth at lower concentrations, whereas inhibition took place at higher levels. This inhibition effect was less marked with urea, likely because the enzymatic hydrolysis of this compound by urease controlled the ammonia transfer into the cell. Fed-batch experiments carried out by pulse-feeding either ammonium or urea proved that the use of these compounds as nitrogen sources can sustain the long term-cultivation of S. platensis, provided that the conditions for their feeding are accurately optimized.     

Assimilation of Inorganic Nitrogen by Marine Invertebrates and Their Chemoautotrophic and Methanotrophic Symbionts

Symbioses between marine invertebrates and their chemoautotrophic and methanotrophic symbionts are now known to exist in a variety of habitats where reduced chemical species are present. The utilization of chemical energy and reliance on C₁ compounds by these symbioses are well documented. Much less is known about their metabolism of nitrogen. Earlier work has shown that the tissues of organisms in these associations are depleted of ¹⁵N compared with those of other marine organisms, indicating that local sources of nitrogen are assimilated and that novel mechanisms of nitrogen metabolism may be involved. Although these symbioses have access to rich sources of ammonium (NH₄⁺ and NH₃) and/or nitrate, several investigators have proposed that N₂ fixation may account for some of these isotope values. Here we report that [¹⁵N]ammonium and, to a lesser degree, [¹⁵N]nitrate are assimilated into organic compounds by Solemya reidi, a gutless clam containing S-oxidizing bacteria, and seep mussel Ia, an undescribed mytilid containing methanotrophic bacteria. In contrast, Riftia pachyptila, the giant hydrothermal vent tube worm symbiotic with S-oxidizing bacteria, assimilated nitrate but not exogenous ammonium. The rates of assimilation of these sources are sufficient to at least partially support C₁ compound metabolism. N₂ assimilation was not exhibited by the symbionts tested.     

Prediction of proton exchange and bacterial growth on various substrates using constraint-based modeling approach

Proton exchange between cells and medium is one of the most important factors affecting culture pH, and hence its prediction is advantageous. In this research, proton exchange flux across the cell membrane was predicted using a genome-scale model. Calculated proton exchange flux was then exploited as a criterion to predict the trends and intensities of pH changes in cultures of Bacillus subtilis containing various C-sources, i.e. glucose, sucrose, glycerol, lactate, and citrate, as well as N-sources, i.e. ammonium chloride, sodium nitrate, urea, and histidine. The results showed that glucose, sucrose, and glycerol lowered culture pH as compared to citrate and lactate, which raised it. With regard to N-sources, the model predicted that ammonium chloride lowered culture pH while other sources raised pH. Furthermore, maximum theoretical biomass yield using the various C&N-sources was calculated, and sensitivity of microbial growth to proton exchange flux was investigated using robustness analysis to identify the effect of pH on growth of B. subtilis using different substrates. Finally, the effect of ammonium nitrate, a widely used nitrogen source, on growth of B. subtilis was studied. Experimental data obtained by cultivation of B. subtilis DSM 3256 on mineral salt media containing various C&N-sources were used to confirm model predictions. Model predictions were in good agreement with the experimental results for all of the examined C-sources as well as ammonium chloride and sodium nitrate as N-sources. However, the predictions for the N-sources urea and histidine showed deviations, possibly because these two compounds serve as both C&N-sources.     

Nitrogen acquisition from inorganic and organic sources by boreal forest plants in the field

A wide range of recent studies have indicated that organic nitrogen may be of great importance to plant nitrogen (N) nutrition. Most of these studies have, however, been conducted in laboratory settings, excluding important factors for actual plant uptake, such as competition, mycorrhizal associations and soil interactions. In order to accurately evaluate the importance of different N compounds to plant N nutrition, field studies are crucial. In this study, we investigated short- as well as long-term plant nitrogen uptake by Deschampsia flexuosa, Picea abies and Vaccinium myrtillus from ¹⁵NO₃^–, ¹⁵NH₄⁺ and (U-¹³C, ¹⁵N) arginine, glycine or peptides. Root N uptake was analysed after 6 h and 64 days following injections. Our results show that all three species, irrespective of their type of associated mycorrhiza (arbuscular, ecto- or ericoid, respectively) rapidly acquired similar amounts of N from the entire range of added N sources. After 64 days, P. abies and V. myrtillus had acquired similar amounts of N from all N sources, while for D. flexuosa, the uptake from all N sources except ammonium was significantly lower than that from nitrate. Furthermore, soil analyses indicate that glycine was rapidly decarboxylated after injections, while other organic compounds exhibited slower turnover. In all, these results suggest that a wide range of N compounds may be of importance for the N nutrition of these boreal forest plants, and that the type of mycorrhiza may be of great importance for N scavenging, but less important to the N uptake capacity of plants.     

Sequential Use of Nitrogen Compounds by Saccharomyces cerevisiae during Wine Fermentation: a Model Based on Kinetic and Regulation Characteristics of Nitrogen Permeases

The efficiency of nitrogen use is a key determinant of the completion of alcoholic fermentation. We analyzed the kinetics of consumption of 18 nitrogen compounds by 14 Saccharomyces cerevisiae strains of various origins in a synthetic medium that mimicked a grape must. The kinetic profiles of total nitrogen consumption were diverse, but the order of nitrogen source consumption was similar for all strains. The nitrogen compounds could be classified into three groups, according to their order of use: prematurely consumed (Lys), early consumed (Asp, Thr, Glu, Leu, His, Met, Ile, Ser, Gln, and Phe), and late consumed (ammonium, Val, Arg, Ala, Trp, and Tyr). The initial concentrations of these compounds did not alter the order in which they were consumed, except for arginine and ammonium. Early consumed amino acids are transported by specific permeases under Ssy1p-Ptr3p-Ssy5 (SPS)-mediated control that are expressed at the beginning of consumption. Most nitrogen compounds consumed late are transported by permeases under nitrogen catabolite repression (NCR), and others (Val, Trp, and Tyr) are transported by SPS-regulated low-affinity permeases. Therefore, the kinetic characteristics of transporters, as well as SPS and NCR, are likely key factors controlling the temporal sequence of consumption of nitrogen compounds and constitute a system highly conserved in S. cerevisiae species. This work sheds new light on the mechanistic basis of the sequential use of different nitrogen compounds in complex environments.     

Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum

The ectomycorrhizal (ECM) fungus Hebeloma cylindrosporum is an appropriate model to study the intraspecific functional diversity of ECM fungi in forest ecosystems. Numerous metabolic genes, specifically genes related to nitrogen assimilation, have been characterised for this species and the spatial and temporal structures of its natural populations have been extensively worked out. In this paper, we reveal the extent to which intraspecific variation exists within this fungus for the ability to use organic nitrogen, an important functional characteristic of ECM fungi. In addition to ammonium and nitrate, H. cylindrosporum can use at least 13 different amino acids out of 21 tested as sole nitrogen source, as well as urea and proteins. By screening 22 genetically different wild type haploid strains we identified obvious differences in use of six nitrogen sources: alanine, glycine, phenylalanine, serine, bovine serum albumin and gelatine. Of the 22 haploid strains, 11 could not use at least one of these six nitrogen sources. The inability of some haploid strains to use a nitrogen source was found to be a recessive character. Nevertheless, obvious differences in use of the four amino acids tested were also measured between wild type dikaryons colonising a common Pinus pinaster root system. This study constitutes the basis for future experiments that will address the consequences of the functional diversity of an ECM fungus on the functioning of the ECM symbiosis under natural conditions.