Methylamine uptake in Pseudomonas species strain MA: utilization of methylamine as the sole nitrogen source.

The uptake of methylamine as the sole nitrogen, but not carbon, source by Pseudomonas sp. strain MA was investigated. Under these growth conditions, a high-affinity, low-capacity uptake system was present having a Km of 16 microM and Vmax of 2 nmol.min-.mg (dry weight) of cells that was competitively inhibited by ammonium chloride. The transport system was induced by growth on succinate with methylamine as the sole nitrogen source.     

Transport of methylamine by Pseudomonas sp. MA.

Pseudomonas sp. MA grows on methylamines as a sole source of carbon, nitrogen, and energy. The transport of methylamine into the organism was investigated. It was found that this organism possesses an inducible transport system for methylamine having the following physical parameters: pH optimum, 7.2; temperature optimum, 30 to 35 degrees C; Km, 1 to 30 mM; Vmax, 90 to 120 nmol/min per mg (dry weight) of cells. Methylamine uptake was curtailed by azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone; osmotic shock treatment reduced the uptake by 50%. The uptake was not effectively inhibited by ammonium ion, amino acids, or amides, but was competitively inhibited by short-chain alkylamines. Cells grown on succinate-ammonium chloride did not possess the transport system, but it could be induced in such cells by methylamine in 20 h. Cells grown with methylamine as a sole nitrogen, but not carbon, source transported methylamine at a reduced rate.     

Interactions between nitrate and ammonium during uptake by carob seedlings and the effect of the form of earlier nitrogen nutrition

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were used in two sets of experiments in order to evaluate; (1) the reciprocal effects of each nitrogen form on net uptake of nitrate and ammonium, and (2) the effect of earlier nitrogen nutrition on ammonium versus nitrate uptake. In the former group of experiments we studied the kinetics of nitrate and ammonium uptake as well as the interference of each of the two forms with net uptake of ammonium and nitrate by both nitrogen depleted and nitrogen fed carob seedlings. On the whole, nitrogen depletion led to increase in both affinity and V_max of the system for both forms of nitrogen, at the same time as the effects of nitrate on uptake of ammonium and vice versa were concentration dependent. In the second group of experiments the effects of earlier nitrogen nutrition on nitrate and ammonium uptake were characterized, and in this case we observed that: (a) if only one form of N was supplied, ammonium was taken up in greater amounts than nitrate; (b) the presence of ammonium enhanced nitrate uptake; (c) ammonium uptake was inhibited by nitrate; (d) there was a significant effect of the earlier nitrogen nutrition on the response of the plants to a different nitrogen source. The latter was evident mainly as regards ammonium uptake by plants grown in ammonium nitrate. The interactions between nitrate and ammonium uptake systems are discussed on the basis of the adaptation to the nitrogen source during early growth.     

Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [(14)C]methylamine (MA), had an apparent EC(50) of 1.6 mm and a maximum uptake rate (V(max)) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na(+) gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H(+) gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC(50) = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H(+)-coupled counter-transport mechanism.     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Mineralization of 3-chloro-4-methylaniline via an ortho-cleavage pathway by Pseudomonas cepacia strain CMA1

Pseudomonas cepacia strain CMA1, which was isolated from soil, utilized 3-chloro-4-methylaniline (3C4MA) in concentrations up to 1.4 mm (0.2 g·l^–1) as the sole source of carbon, nitrogen, and energy. In addition, 3-chloroaniline, 4-chloroaniline and phenol, but not aniline or methylanilines, were degraded by strain CMA1. Biodegradation of the anilines was coupled to the liberation of ammonium and chloride. The broad specificities of the aniline- and catechol-oxidizing enzymes were demonstrated in oxygen uptake experiments, which in addition showed higher activities for ring-cleaving than for aniline-oxidizing enzymes. Two ring-cleaving catechol 1,2-dioxygenases, which were induced selectively after growth on 3C4MA (pyrocatechase type II) and phenol (pyrocatechase type I), respectively, were discerned after partial purification by DEAE-cellulose chromatography. Correspondence to: F. Streichsbier     

The simultaneous assimilation of ammonium and l-arginine by the marine diatom Phaeodactylum tricornutum Bohlin

Nitrogen-replete cells of Phaeodactylum tricomutum Bohlin assimilated ammonium and the amino acid l-arginine simultaneously. Arginine was taken up at rates expected to supply at least 30% of the cells' requirement for nitrogen; arginme-carbon mainly entered protein but, when uptake was in darkness, ≈40% was respired. Cells grown in a 12:12 h light:dark cycle with ammonium as the sole nitrogen source took up ammonium throughout the growth cycle, whereas cells grown with the addition of arginine took up little ammonium during the dark phase. The uptake of ammonium over the course of the cycle was reduced by 30% when arginine was present. Cells grown with arginine as the sole nitrogen source took up the amino acid at the rate required for growth. In contrast, cells grown on ammonium, while growing at the same rate as those on arginine, assimilated nitrogen at twice the rate. Cells grown with both sources of nitrogen present, took up arginine at the same rate as before, but more of the arginine-carbon was respired (60% as compared with 40% when ammonium was absent). The uptake of ammonium was reduced by 30%, but the total nitrogen assimilation again exceeded immediate requirements. A high uptake rate of arginine was indicative of cells assimilating ammonium only; a low uptake rate of ammonium during the dark phase of growth was indicative of cells assimilating arginine. It is not known whether the findings with P. tricomutum are applicable to other marine phytoplankton. If they are, arginine may be of greater significance as a natural source of nitrogen for phytoplankton than is generally thought.     

Constitutive choline transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent K_m value of 53 μM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.     

DIFFERENCES IN GROWTH AND PHYSIOLOGY OF MARINE SYNECHOCOCCUS (CYANOBACTERIA) ON NITRATE VERSUS AMMONIUM ARE NOT DETERMINED SOLELY BY NITROGEN SOURCE REDOX STATE1

The preference of phytoplankton for ammonium over nitrate has traditionally been explained by the greater metabolic cost of reducing oxidized forms of nitrogen. This “metabolic cost hypothesis” implies that there should be a growth disadvantage on nitrate compared to ammonium or other forms of reduced nitrogen such as urea, especially when light limits growth, but in a variety of phytoplankton taxa, this predicted difference has not been observed. Our experiments with three strains of marine Synechococcus (WH7803, WH7805, and WH8112) did not reveal consistently faster growth (cell division) on ammonium or urea as compared to nitrate. Urease and glutamine synthetase (GS) activities varied with nitrogen source in a manner consistent with regulation by cellular nitrogen status via NtcA (rather than by external availability of nitrogen) in all three strains and indicated that each strain experienced some degree of nitrogen insufficiency during growth on nitrate. At light intensities that strongly limited growth, the composition (carbon, nitrogen, and pigment quotas) of WH7805 cells using nitrate was indistinguishable from that of cells using ammonium, but at saturating light intensities, cellular carbon, nitrogen, and pigment quotas were significantly lower in cells using nitrate than ammonium. These and similar results from other phytoplankton taxa suggest that a limitation in some step of nitrate uptake or assimilation, rather than the extra cost of reducing nitrate per se, may be the cause of differences in growth and physiology between cells using nitrate and ammonium.     

Ammonium (methylammonium) uptake by Alcaligenes eutrophus H16

The uptake of the radioactive ammoniumanalogue ¹⁴C-methylammonium¹ was measured in heterotrophically grown cells of Alcaligenes eutrophus H16 in order to study the mechanism of NH ₄ ⁺ uptake. MA gradients of up to 200 were built up by a substrate-specific and energy-dependent system which showed a K _m of 35–111 M and a V _max of 0.4–1.8 nmol MA/min per mg protein. The involved carrier exhibited a higher affinity towards NH ₄ ⁺ than towards CH₃NH ₃ ⁺ indicating that ammonium rather than MA was its natural substrate. Cold shock with hypotonic but not with hypertonic solutions caused the efflux of almost the entire accumulated MA. Osmotic shock did not affect the uptake reaction, suggesting that no periplasmic binding proteins were involved. Indirect observations indicate the membrane potential as driving force for MA uptake. High rates of uptake were observed in cells grown under nitrogen deficiency or with nitrate as nitrogen source. The uptake rate decreased during growth at high ammonium concentrations indicating that biosynthesis of nitrogenous compounds was supported by passive diffusion of NH₃. The data suggest that the formation of the carrier is subject to nitrogen control.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphe-nylhydazone - MA methylammonium - pCMB para-chlormercuribenzoate     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

冬小麦等4种作物对铵,硝态氮的吸收能力

采用水培试验探讨了冬小麦、大豆、油菜和莴笋４种作物对硝、铵态氮的相对吸收能力以及这两种氮源对它们生长发育的影响。试验表明：（１）不同氮源对供试作物的生长发育影响极大。供给硝态氮,这些作物生长发育良好,供给等量的ＮＯ＾－３和ＮＨ＾－４（１：１）时,蔬菜作物莴笋生长量下降幅度最大;供给铵态氨,莴笋和大豆极为敏感,供给ＮＯ＾－３时莴笋吸氮量显著高于供给等氮量ＮＯ＾－３和ＮＨ＾＋４,莴上麦供给等量ＮＯ＾－     

Interactions in the uptake of amino acids, ammonium and nitrate ions in the Arctic salt-marsh grass, Puccinellia phryganodes

The uptake of amino acids and inorganic nitrogen by roots of Puccinellia phryganodes was examined to assess the potential contribution of soluble organic nitrogen to plant nitrogen uptake in Arctic coastal marshes, where free amino acids constitute a substantial fraction of the soil‐soluble N pool. Short‐term excised root uptake experiments were performed using tillers grown hydroponically under controlled conditions in the field. The percentage reductions in ammonium uptake at moderate salinity (150 mm NaCl) compared with uptake at low salinity (50 mm NaCl) were double those of glycine, but glycine uptake was more adversely affected than ammonium uptake by low temperatures. Glycine uptake was higher at pH 5·7 than at pH 7·0 or 8·2. The glycine uptake was up‐regulated in response to glycine, whereas ammonium uptake was up‐regulated in response to ammonium starvation. Nitrate uptake was strongly down‐regulated when tillers were grown on either ammonium or glycine. In contrast to N‐starved roots, which absorbed ammonium ions more rapidly than glycine, the roots grown on glycine, ammonium and nitrate and not N‐starved prior to uptake absorbed glycine as rapidly as ammonium and nitrate ions combined. Overall, the results indicate that amino acids are probably an important source of nitrogen for P. phryganodes in Arctic coastal marshes.     

Uptake of ammonium and nitrate by carob (Ceratonia siliqua) as affected by root temperature and inhibitors

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown in nutrient solution culture for 5 weeks, with or without nitrogen at different root temperatures (10, 16, 22, 30, 35 or 40deg;C) and with the air temperature kept between 20 and 24°C. The nitrogen was given as either ammonium or nitrate. At all root temperatures studied, nitrogen-depleted plants developed higher net uptake rates for nitrogen than plants grown in the presence of nitrogen. Temperature affected the kinetic parameters of nitrate uptake more than those of ammonium uptake. With increasing root temperature, the K_m of ammonium uptake decreased, but to a lesser extent than the K_m for nitrate. The increase in V_max of ammonium uptake with temperature was also less noticeable than that for nitrate uptake. Ammonium and nitrate uptakes were inhibited in a similar way by respiratory or protein synthesis inhibitors. It may be noted that ammonium uptake in the presence of inhibitors at 40°C was higher than uptake at 10°C without inhibitors. Some similarities between the transport mechanisms for nitrate and ammonium are underlined in the present work. Components of both transport systems displayed saturation kinetics and depended on protein synthesis and energy. The following components of nitrate uptake were distinguished: (a) a passive net influx into the apparent free space; (b) a constitutive active uptake and (c) active uptake dependent on protein synthesis. We may similarly define three ammonium uptake systems: (a) a passive influx into the apparent free space; (b) passive diffusion uptake at high temperature and (c) active uptake dependent on protein synthesis. The possible role of the ratio between mechanism (c) and mechanism (b) as determinant of ammonium sensitivity is discussed.     

Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.     

Solid-state NMR studies of regulation of N-(phosphonomethyl)glycine and glycine metabolism in Pseudomonas sp. strain PG2982

Lyophilized samples of Pseudomonas sp. PG2982 grown on 13C- and 15N-labeled glyphosate have been analyzed by single and double cross-polarization 13C NMR. Both the carbon and nitrogen metabolism of glyphosate are significantly influenced by the nitrogen source used for the growth of the organism. When ammonium sulfate is the source of nitrogen, the glycyl moiety of glyphosate is utilized intact for the biosynthesis of purines and proteins. But when the organism is grown on glycine as the source of nitrogen, the carbons and nitrogen of glyphosate are scrambled, consistent with incorporation into serine and pyruvate, and hence participation in general metabolism. When both ammonium and glycine are present in the growth medium, regulation of the metabolic fluxes along each of the two major pathways appears to be determined by the intracellular glycine concentration.     

Factors affecting ammonium uptake in streams – an inter-biome perspective

1. The Lotic Intersite Nitrogen eXperiment (LINX) was a coordinated study of the relationships between North American biomes and factors governing ammonium uptake in streams. Our objective was to relate inter‐biome variability of ammonium uptake to physical, chemical and biological processes. 2. Data were collected from 11 streams ranging from arctic to tropical and from desert to rainforest. Measurements at each site included physical, hydraulic and chemical characteristics, biological parameters, whole‐stream metabolism and ammonium uptake. Ammonium uptake was measured by injection of ¹⁵N‐ammonium and downstream measurements of ¹⁵N‐ammonium concentration. 3. We found no general, statistically significant relationships that explained the variability in ammonium uptake among sites. However, this approach does not account for the multiple mechanisms of ammonium uptake in streams. When we estimated biological demand for inorganic nitrogen based on our measurements of in‐stream metabolism, we found good correspondence between calculated nitrogen demand and measured assimilative nitrogen uptake. 4. Nitrogen uptake varied little among sites, reflecting metabolic compensation in streams in a variety of distinctly different biomes (autotrophic production is high where allochthonous inputs are relatively low and vice versa). 5. Both autotrophic and heterotrophic metabolism require nitrogen and these biotic processes dominate inorganic nitrogen retention in streams. Factors that affect the relative balance of autotrophic and heterotrophic metabolism indirectly control inorganic nitrogen uptake.     

Functional and physiological evidence for a rhesus-type ammonia transporter in Nitrosomonas europaea

Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The K(m) value for the MA uptake of N. europaea was 1.8+/-0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [K(i)(NH(4) (+)) 0.3+/-0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH(4) (+) trap.