Absence Of Ammonium Research Articles

Histidine Utilization by the Unicellular Alga Dunaliella tertiolecta

The unicellular alga Dunaliella tertiolecta was cultivated axenically with histidine, or ammonium, or histidine and ammonium ions as nitrogen sources. In the presence of histidine as the sole source of nitrogen, cell growth is comparable to that observed with the same concentration of nitrogen in ammonium form. In the presence of both histidine and ammonium, histidine degradation is observed only when the concentration of ammonium falls below 4 mM. Under these conditions, the first two enzymes of histidine degradation pathway, histidase and urocanase, as well as histidine permease, are produced. Linear relationships are observed between histidase and urocanase and histidase and histidine permease, respectively, showing that, in D. tertiolecta enzyme, systems are coordinately regulated. Histidase activity is also controlled by succinate, glutamate and acetate as carbon sources. D. tertiolecta is unable to grow on a medium containing acetate and histidine together in the absence of ammonium.

Dynamics of artificially immobilizedNitrosomonas europaea: Effect of biomass decay

The dynamics of growth and death of immobilized Nitrosomonas europaea were studied. For this, the death rate of suspended cells was determined in the absence of ammonium or oxygen by following the loss of respiration activity and by fluorescein-diacetate (FDA)/lissamine-green staining techniques. The death rates obtained (1.06 x 10(-6) s(-1) or 4.97 x 10(-6) s(-1) in the absence of oxygen or ammonium, respectively) were incorporated in a dynamic growth model and the effects on the performance of the immobilized-cell process illustrated by model simulations.These model simulations and experimental validation show that if decay of biomass occurs the biomass concentration in the center of the bead decreases. As a result, the systems react slower to changes in substrate concentrations than if all cells remain viable.To show that cells in the center of the bead died, the FDA and lissamine-green staining techniques were adapted for immobilized cells. It was shown that biomass decay occurred, especially in the center of the bead; the amount of cells decreased there, and the remaining cells were all stained with lissamine green indicating cell death. After the substrate availability was decreased, also cells near the surface of the bead lost their viability. The number of viable cells increased again after increasing the substrate concentration as the result of cell multiplication. At low substrate concentrations and low hydraulic retention times, as for example in the treatment of domestic wastewater, the death rate of cells is thus an important parameter for the performance of the immobilized-cell system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 630-641, 1997.

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.

Accumulation of poly(3-hydroxybutyric acid) and overproduction of exopolysaccharides in a mutant of a methylotrophic bacterium

The pink-pigmented facultatively methylotrophic bacterium Methylobacterium rhodesianum MB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.

Effect of growth conditions on the activities of methylotrophic enzymes inMethylophilusW3A1

The specific activities of four methylotrophic enzymes were measured in batch and chemostat cultures of Methylophilus W3A1. Methanol dehydrogenase was a constitutive enzyme. Methylamine dehydrogenase was regulated by an induction/repression mechanism. Trimethylamine dehydrogenase and dimethylamine monooxygenase were induced co-ordinately, probably by dimethylamine. Methanol completely prevented synthesis of the methyl-amine oxidising enzymes when ammonium was also present; the absence of ammonium relieved this repression. The significance of the results for the evolution of methylotrophic pathways in restricted facultative methylotrophs is discussed.

Molecular mechanism for the operation of nitrogen control in cyanobacteria.

In cyanobacteria, ammonium exerts a negative regulation of the expression of proteins involved in the assimilation of nitrogen sources alternative to ammonium. In Synechococcus, mRNA levels of genes encoding proteins for nitrate and ammonium assimilation were observed to be negatively regulated by ammonium, and ammonium-regulated transcription start points were identified for those genes. The NtcA protein is a positive regulator of genes subjected to nitrogen control by ammonium. Mutants lacking NtcA exhibited only basal mRNA levels of the regulated genes, even in the absence of ammonium, indicating that NtcA exerts its regulatory action by positively influencing mRNA levels of the nitrogen-regulated genes. NtcA was observed to bind directly to the promoters of nitrogen-regulated genes, and the palindromic DNA sequence GTAN8TAC was identified as a sequence signature for NtcA-target sites. The structure of the nitrogen-, NtcA-regulated promoters of Synechococcus was determined to be constituted by a -10, Pribnow-like box in the form TAN3T, and an NtcA-binding site that substituted for the canonical -35 box.

Arrest of cell division but not protein synthesis in sodium-deficient cells of the marine diatom Phaeodactylum tricornutum Bohlin

Cell division in the marine diatom Phaeodactylum tricornutum was prevented when cultures were maintained in the absence of sodium, regardless of the nitrogen status of the cells or medium. Addition of 10 mM ammonium and 50 mM sodium to cultures preconditioned in nitrogen and sodium-deficient medium for 5 d led to a recovery in cell division and chlorophyll a, and net protein synthesis. Sodium added in the absence of ammonium led to a recovery in cell division, but not net protein synthesis. Ammonium added in the absence of sodium was partially assimilated (as NH3) and resulted in a small amount of protein synthesis, but without cell division. This effect was enhanced if the cells had lower protein quotas prior to ammonium addition, with total consumption of the added 1 mM ammonium and appreciable net protein synthesis. Respiration was enhanced by 1 or 10 mM ammonium or 10 mM methylammonium addition to nitrogen-deficient cultures maintained in the presence or absence of sodium. In contrast to respiration, photosynthesis was inhibited by these additions in sodium-replete cultures, but was enhanced in sodium-deficient cultures.

Ammonium selectively inhibits the regulated pathway of protein secretion in two endocrine cell lines

The effect of ammonium on protein processing and secretion from mouse insulinoma βTC3 cells and recombinant mouse pituitary AtT-20 cells was investigated. Protein stored in granules of the regulated secretion pathway was discharged from cells with secretagogues, and the addition of new protein was encouraged by recharging cells in serum-containing medium with or without added ammonium. Ammonium at 6 m m inhibits the addition of insulin-related peptides to intracellular stores in both cell lines; it is not clear whether this effect is dose dependent for concentrations between 0 and 6 m m. There is a slight increase in insulin-related proteins secreted during recharging of cells in the presence of ammonium. Using reverse-phase high-performance liquid chromatography to separate proinsulin from insulin, we found that extracts from βTC3 cells recharged in the presence of 6 m m ammonium contain significantly less insulin than control cells recharged in the absence of ammonium.

Production of protease with Bacillus licheniformis mutants insensitive to repression of exoenzyme biosynthesis

Two mutant strains of Bacillus licheniformis insensitive to catabolite repression were selected by classical mutagenesis in connection with the development of a fed-batch procedure for protease production. B. licheniformis 4a produced up to 20 U (Anson-Units) subtilisin Carlsberg/ml in fed-batch experiments in the presence of up to 1.5 m glycerol, but was inhibited by excess ammonium. Formation of spores, excretion of α-amylase and the biosynthesis of citrate synthase and isocitrate dehydrogenase were likewise not repressed by glycerol. The strain was characterized by unusually low activity of the α-oxoglutarate dehydrogenase complex and increased biosynthesis of polyglutamic acid in the presence and excretion of α-oxoglutarate in the absence of ammonium, respectively. The results are discussed in view of a possible connection between the defect in the α-oxoglutarate dehydrogenase complex and insensitivity to catabolite repression. The second strain B. licheniformis 114 was able to synthesize 11.5 U protease/ml independently of the glycerol and ammonium concentration in the medium.

Short-term interaction between nitrate and ammonium uptake inThalassiosira pseudonana: Effect of preconditioning nitrogen source and growth rate

The effect of preconditioning nitrogen source and growth rate on the interaction between nitrate and ammonium uptake was determined inThalassiosira pseudonana (Clone 3H). A new method, using cells on a filter (Parslow et al. 1985), allowed continuous measurement of uptake from 0.5 to 9 min after the addition of nitrate, ammonium, or both, with no variation in concentration during the course of the experiment. For each preconditioning N source and growth rate, a series of uptake experiments was conducted, including controls with only nitrate or only ammonium, and others with different combinations of concentrations of nitrate and ammonium. For the first time, preference for ammonium was separated from inhibition of nitrate uptake by ammonium. Ammonium was the preferred N source, i.e. if nitrate and ammonium were presented separately, ammonium uptake rates exceeded nitrate uptake rates. Preference for ammonium varied with both preconditioning N source and growth rate. Inhibition of nitrate uptake by ammonium, determined by comparing nitrate uptake in the presence and absence of ammonium, was observed at ammonium concentrations > 1µM, but was rarely complete. Inhibition of nitrate uptake by ammonium was less in the ammonium-limited culture than in the cultures growing on nitrate, but invariant with growth rate in the nitrate-grown cultures. Below 1µM ammonium, nitrate uptake was often stimulated and rates exceeded those in the controls without ammonium. Ammonium uptake was not inhibited by the presence of nitrate.T. pseudonana fits the classical view of the interaction between nitrate and ammonium uptake in some respects, such as preference for ammonium, and inhibition of nitrate uptake by ammonium concentrations > 1µM. However, at ammonium concentrations typical of most marine environments, nitrate uptake occurs at rapid rates. In other respects, N uptake inT. pseudonana deviates from the classical view in the following ways: (1) stimulation of nitrate uptake by low concentrations of ammonium; (2) lack of inhibition of nitrate uptake by ammonium at low nitrate concentrations; and (3) variation in preference and inhibition with preconditioning, which is markedly different for other species. Because of the apparent enormous species variation in the interaction between nitrate and ammonium uptake and the lack of detailed information for a variety of species, it is difficult to generalize about the effect of ammonium on nitrate uptake, especially in the field, where prior N availability and species composition are not usually addressed.

Nitrate Uptake and Partitioning by Corn Root Systems

The relative effects of ammonium on nitrate uptake and partitioning during induction were compared among decapitated seedlings of three corn (Zea mays L.) genotypes at two developmental stages. This study tested the hypothesis that root systems efficient at translocating products of ammonium assimilation away from sites of nitrate uptake or reduction would exhibit less inhibition of nitrate uptake by ammonium compared to root systems with inefficient N translocation efficiency. Inhibition of nitrate uptake by ammonium was relatively slight at day 5 ranging from 0% to 20% among the three genotypes, as compared to greater inhibition, from 20% to 37%, at day 8. Five-day-old roots exhibited negligible xylem translocation capacity in comparison with those grown for 8 days. Thus, although the capability to translocate ammonium assimilates out of the root increased between days 5 and 8, inhibitory effects of ammonium also increased. In the absence of ammonium, nitrate uptake per unit root mass increased between days 5 and 8. This increased activity of the uptake system was proportionally more sensitive to ammonium.Partitioning of entering nitrate into the reduction process was positively correlated with lateral root development of the inbred root systems at 5 and 8 days. This is supportive of a localization of a major portion of nitrate reduction occurring in root apical regions. Nitrate reduction was the partitioning process most severely inhibited by ammonium in all cases, ranging from 39% to 55% inhibition. In contrast, ammonium-inhibition of nitrate accumulation in the root tissue and translocation via xylem vessels varied with genotype and root age.Two mechanisms of ammonium-inhibition of nitrate are implicated, one which directly affects nitrate reduction and the uptake system associated with it, and another which may involve potassium as an intermediate regulator of nitrate accumulation in the root tissue and nitrate translocation out of the root tissue.

Effects of various parameters on carbon monoxide oxidation by ammonium oxidizers

The effects of temperature, pH, nitrogen source, cell concentration, and the interactions between ammonium and carbon monoxide were examined with respect to the oxidation of carbon monoxide by several chemolithotrophic ammonium-oxidizing bacteria. The ammonium oxidizers Nitrosomonas europaea, Nitrosomonas sp. 4W30, a marine isolate, and Nitrosococcus oceanus were examined. All of the organisms were able to oxidize significant amounts of CO over wide ranges of temperature and pH. Ammonium at concentrations as low as 1 mg/L [Formula: see text] N initially inhibited CO oxidation in all three organisms; however, after 48 h, the presence of ammonium stimulated the CO-oxidizing ability of Nitrosomonas europaea and Nitrosomonas sp. 4W30, while Nitrosococcus oceanus remained inhibited. None of the other nitrogen sources examined had a significant effect on CO oxidation. No carbon from CO was incorporated into cellular material in the absence of ammonium, even in the presence of alternate nitrogen sources. Cells incubated in the presence of ammonium at concentrations as low as 1.0 mg/L [Formula: see text] N were able to incorporate CO carbon into cellular material. Increasing [Formula: see text] N concentrations to 50 mg/L stimulated the incorporation of CO carbon by Nitrosococcus oceanus. Concentrations of 10 mg/L gave the highest incorporation levels for Nitrosomonas europaea and Nitrosomonas sp. 4W30 and 50 mg/L inhibited the incorporation by these two organisms. The presence of CO inhibited the oxidation of ammonium by all of the organisms tested.

Carbon monoxide oxidation by chemolithotrophic ammonium oxidizers

Chemolithotrophic nitrifying bacteria were examined with respect to their ability to oxidize carbon monoxide in the presence and absence of ammonium. All of the ammonium oxidizers tested, including Nitrosomonas europaea and Nitrosococcus oceanus, were able to oxidize CO to CO2 in the presence and absence of ammonium. None of the organisms tested incorporated any of the carbon from CO into cellular components in the absence of ammonium. All were able to oxidize CO at trace concentrations of <0.5 nM. None of the nitrite oxidizers examined were capable of CO oxidation. CO oxidation by Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosomonas sp. 4S30, a marine ammonium oxidizer, were examined with respect to CO concentration and time course of CO oxidation, both in the presence and absence of 10 mg/L NH4 N. The CO oxidation rates at a CO concentration of 300 nL/L in the absence of ammonium ranged from 0.048 nmol/h per 106 cells for Nitrosomonas sp. 4W30 to 0.0025 nmol/h per 106 cells for N. europaea.

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

Root or secondary leaf segments from maize (Zea mays L. cv. Ganga safed‐2) seedlings were incubated with 9‐amino acids and two amides separately, each at 5 mM for 24 h, to study their effects on glutamate dehydrogenase (GDH) activity. Most of the compounds tested inhibited the specific activity of NADH‐GDH and increased that of NAD+‐GDH in the roots in the presence as well as in the absence of ammonium. In the leaves, such effects were recorded only with a few amino acids. Total soluble protein in the root and leaf tissues increased with the supply of most of the amino compounds. The effect of glutamate on enzyme activity and protein was concentration dependent in both tissues. When the enzyme extracts from root or leaf tissues were incubated with some of the amino acids, NADH‐GDH declined while NAD+‐GDH increased in most cases. The inhibition of NADH‐GDH increased with increasing concentration of cysteine from 1 to 5 mM. The experiments demonstrate that most of the amino acids regulated GDH activity, possibly through some physicochemical modulation of the enzyme molecule.

Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea

Chemolithotrophic ammonium-oxidizing and nitrite-oxidizing bacteria including Nitrosomonas europaea, Nitrosococcus oceanus, Nitrobacter sp., Nitiospina gracilis, and Nitrococcus mobilis were examined as to their ability to oxidize methane in the absence of ammonium or nitrite. All ammonium oxidizers tested had the ability to oxidize significant amounts of methane to CO(2) and incorporate various amounts into cellular components. None of the nitrite-oxidizing bacteria were capable of methane oxidation. The methane-oxidizing capabilities of Nitrosococcus oceanus and Nitrosomonas europaea were examined with respect to ammonium and methane concentrations, nitrogen source, and pH. The addition of ammonium stimulated both CO(2) production and cellular incorporation of methane-carbon by both organisms. Less than 0.1 mM CH(4) in solution inhibited the oxidation of ammonium by Nitrosococcus oceanus by 87%. Methane concentrations up to 1.0 mM had no inhibitory effects on ammonium oxidation by Nitrosomonas europaea. In the absence of NH(4)-N, Nitrosococcus oceanus achieved a maximum methane oxidation rate of 2.20 x 10 mumol of CH(4) h mg (dry weight) of cells, which remained constant as the methane concentration was increased. In the presence of NH(4)-N (10 ppm [10 mug/ml]), its maximum rate was 26.4 x 10 mumol of CH(4) h mg (dry weight) of cells at a methane concentration of 1.19 x 10 mM. Increasing the methane concentration above this level decreased CO(2) production, whereas cellular incorporation of methane-carbon continued to increase. Nitrosomonas europaea showed a linear response throughout the test range, with an activity of 196.0 x 10 mumol of CH(4) h mg (dry weight) of cells at a methane concentration of 1.38 x 10 mM. Both nitrite and nitrate stimulated the oxidation of methane. The pH range was similar to that for ammonium oxidation, but the points of maximum activity were at lower values for the oxidation of methane.

Nitrate assimilation by decapitated corn root systems: effects of ammonium during induction

Development of the accelerated phase of nitrate uptake by darkgrown decapitated corn seedlings upon first exposure to nitrate (1.0 mM, pH 6, 30°C) was restricted by the presence of ambient ammonium (0.5 mM (NH 4) 2SO 4). Upon transfer to ammonium-free solutions, the increase in the nitrate uptake rate paralleled the increase in seedlings not exposed to ammonium, but did not, within 6 h, completely recover to yield the rate occuring in the absence of ammonium throughout. In contrast, the restriction in potassium uptake was completely eliminated within 2 h following transfer. Presence of ammonium decreased reduction of [ 15N]nitrate and in vitro nitrate reductase activity (NRA) of the root tissue, but the decrease in [ 15N]nitrate reduction was not sufficient to account for the decrease in uptake. The data are interpreted as indicating that ammonium exerted a detrimental effect on the formation of the nitrate uptake system.

Influence of Nitrogen Form and Nitrogen Absence on Utilization of Assimilates for Growth and Maintenance in Tops and Roots of Lolium multiflorum

AbstractUtilization of assimilates for growth and maintenance of tops and roots of Lolium multiflorum was determined for plants supplied with either nitrate or ammonium. Carbon dioxide exchange rates were measured continuously for tops and roots separately. Three‐day periods were applied for two irradiation levels. On the last day of each three‐day period no nitrogen was supplied to the two treatments. In the nitrate treatment, the coefficient of utilization for converting assimilates into constructive growth (YG) remained unaffected in absence of nitrate. However, in absence of nitrate the maintenance respiration (M) for both tops and roots was only one third of that in presence of nitrate. In the treatment with ammonium the maintenance respiration of the plants was not influenced by the absence of ammonium. However, especially for the tops YG increased in absence of ammonium. In both the treatments, growth respiration of the roots was inefficient compared to that of the tops. Only in the case of absence of nitrate, maintenance respiration of the roots was similar to that of the tops.

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.

Nitrogen assimilation and regulation of nitrate and nitrite reductases in cultured Ipomoea cells

Ipomoea cells grown in a medium containing ammonium and nitrate preferentially used ammonium during the initial stages of growth but in the later stages assimilated nitrate rapidly regardless of the presence or absence of ammonium. Cells grown on nitrate and maintained at pH 4.8 released ammonia into the medium, whereas when they were maintained at pH 6.5 they secreted nitrite. The enzymes of nitrate assimilation were inducible by nitrate and the activities changed considerably in response to nitrogen source. The addition of ammonium 3 days after inoculation to cells highly induced for the reductases did not result in the suppression of further synthesis of the enzymes. The levels of nitrate (EC 1.6.6.1) and nitrite (EC 1.6.6.4) reductases detected in cells grown on ammonium as the nitrogen source were about 25% and 66%, respectively, of the fully induced level. The addition of nitrate to ammonium-assimilating cells resulted in a low level of induction of both reductases. Addition of nitrite had no effect. With both ammonium and nitrate initially present in the medium, the ammonium was utilized quickly but no induction of the reductases was observed for 24 h. the time interval when the assimilation of nitrate was low. This was followed by the induction of the enzymes to a higher level than the activity in cultures of similar age that had been grown continually in nitrate, supplied at the same concentration. Thus, for nitrate and nitrite reductases, repression-like effects were produced by ammonium.

Effect of ammonium on growth of plant cells (Ipomoea sp.) in suspension cultures

Maintenance of the ammonium level in batch cultures of a plant cell line initiated from Ipomoea root tissue resulted in cells with a higher nitrogen content and a greater total accumulation of nitrogen in the cells. Utilization of nitrate was not suppressed. The increased supply of ammonium did not, by itself, increase the yield of dry cells, but it did permit a twofold or greater increase in cell yield when sucrose was added subsequently.Supplementary feeding of ammonium and sucrose to a batch culture that had exhausted the initial supply of ammonium rapidly returned the cells to a state of high nitrogen content, favored further utilization of nitrate, and greatly increased cell yield.These results are discussed in terms of growth phases. The presence of ammonium in the medium appeared to induce and maintain a growth phase characterized by a high nitrogen level in the cells. After the ammonium supply was depleted a second phase occurred, characterized by a rapid increase in dry weight without proportional accumulation of nitrogen. This second phase was not caused by a limiting rate of nitrate utilization, nor was it directly related to observed pH changes. We conclude that the two phases relate to the presence and absence of ammonium in the medium.