Negative Effect Of Ammonium Research Articles

N fertilization in a Mediterranean ecosystem alters N and P turnover in soil, roots and the ectomycorrhizal community

Increased anthropogenic nitrogen (N) deposition is a major contributor to alteration of soil nutrient cycles particularly in nutrient poor ecosystems, such as the Mediterranean basin, where co-limitation of N and phosphorus (P) occurs and N addition might thus lead to an exacerbation of P limitation. Here, we measured the effect of medium term (6 years) N fertilization in different forms and doses (40 kg N ha−1 yr−1 as 1:1 NH4Cl and (NH4)2SO4; 40 and 80 kg N ha−1 yr−1 as NH4NO3) on nutrient stoichiometry, potential turnover rates and abundance of roots, ectomycorrhizal (ECM) root tips and adjacent soil in Cistus ladanifer L. In order to assess the impact of N addition at its most extreme point, we sampled roots and topsoil (10 cm) with and without plant influence in the summer months. We analysed N and P concentrations in soil and roots and determined the abundance of the most dominant mycorrhizal root tip morphotypes. We also assessed nutrient turnover in soil, roots and mycorrhizal root tips by measuring their N and C related enzyme activities (EAs) as well as acid phosphatase (AP) activity. Resultsshowed decreased soil Pinorg and increased soil N:Pinorg in the treatment plots. Also, a decline in Cenococcum geophilum in N addition plots was found and a general reduction in ECM colonization in the treatment receiving ammonium without nitrate. We also detected a decrease of C. geophilum absolute EA and AP, as well as N related EA in the whole soil compartment. Furthermore, we observed lower root AP activity and found a loss of correlation between N related EA and AP activity in all treatments, while a high correlation between N related EA and C related EA persisted in all plots. EA was also generally negatively related with root P/soil P, which we used as a measure for plant P status.The negative effect of ammonium on the ECM community of C. ladanifer and a putative loss of short distance exploration morphotypes, such as C. geophilum, together with decreased AP activity in the plant roots, might be connected with low Pinorg availability in soil with plant influence, thus being in line with the hypothesis of P depletion due to N addition. Furthermore, the decrease of N related EA in the soil compartment, as well as the decoupling of N and P cycles, might be signs of altered soil microbial communities. This decoupling, together with the strong dependence of EA on plant P status, could point to a shift from N and P scavenging ECM communities to more copiotrophic saprophytic fungi that rely on C and N acquisition from soil organic matter rather than plant C inputs. We posit that a decline in root colonization by ECM fungi and changes in N:P cycling could be detrimental to ecosystem development, as C. ladanifer is a common ECM species in early successional stages, providing a host for ECM fungi that also colonize late-successional plants.

Effects of elevated ammonium on glycosylation gene expression in CHO cells

The negative effects of ammonium on recombinant protein productivity and glycosylation have been well documented, but the interaction of ammonium on glycosylation genes has not been completely elucidated. In this study, the effects of elevated ammonium on 12 glycosylation related genes in Chinese hamster ovary cells were evaluated by quantitative real time reverse transcriptase polymerase chain reaction. Numerous cytosol and endoplasmic reticulum (ER) localized genes associated with early glycosylation steps were insensitive to the ammonium condition. The initial expression of uridine diphosphate (UDP)-galactose transporter was higher for the ammonium-treated culture, while the initial expressions of cytosine monophosphate (CMP)-sialic acid transporter, β(1,4)-galactosyltransferase, and UDP-glucose pyrophosphorylase were higher for the control culture. α(2,3)-sialyltransferase was observed to have lower expression level under the elevated ammonium condition compared to the control culture. This study indicates that galactosylation and sialylation inhibition is mainly due to decreased gene expression of galactosyltransferase, sialyltransferase, and CMP-sialic acid transporter and not due to sialidase. These unbalanced initial glycosylation and branching steps can explain the higher molecular heterogeneity under ammonium stress. Moreover, this study indicates that elevated ammonium has limited effects on the glycosylation genes associated with the ER and cytosol compared to the genes associated with the Golgi.

The importance of nitrate in ameliorating the effects of ammonium and urea nutrition on plant development: the relationships with free polyamines and plant proline contents.

In order to investigate the possible involvement of free polyamines and proline in the mechanism underlying the action of nitrate in correcting the negative effects associated with ammonium and urea nutrition in certain plant species, we studied plant contents of free polyamines and proline associated with nitrogen nutrition involving different nitrogen forms (nitrate, ammonium, urea) in two plant species, wheat and pepper. The results showed that ammonium nutrition and, to a lesser extent, urea nutrition were associated with significant increases in plant putrescine content that were well correlated with reductions in plant growth. These negative effects of ammonium and urea nutrition were corrected by the presence of nitrate in the nutrient solution; the presence of nitrate was also related to a significant decrease in the plant putrescine content. These results are compatible with a specific effect of nitrate reducing ammonium accumulation through the improvement of ammonium assimilation. As for the plant proline content, in pepper a slight increase in this parameter was associated with ammonium and urea nutrition, but it was also decreased by the presence of nitrate in the nutrient solution. These changes, however, were not so clearly related to the variations in plant growth as in the case of putrescine content. These results are compatible with the hypothesis that putrescine biosynthesis might be related to proline degradation by a specific pathway related to ammonium detoxification.

Ecological aspects of ntcA gene expression and its use as an indicator of the nitrogen status of marine Synechococcus spp.

Nitrogen nutrition in cyanobacteria is regulated by NtcA, a transcriptional activator that is subject to negative control by ammonium. Using Synechococcus sp. strain WH7803 as a model organism, we show that ntcA expression was induced when cells were exposed to nitrogen stress but not when they were subjected to phosphorus or iron deprivation. Transcript levels accumulated in cells grown on a variety of inorganic and organic nitrogen sources, with the sole exception of ammonium. ntcA transcription was induced when ammonium levels dropped below 1 microM and reached maximal levels within 2 h. Furthermore, the addition of more than 1 microM ammonium led to a rapid decline in ntcA mRNA. The negative effect of ammonium was prevented by the addition of L-methionine-D,L-sulfoximine (MSX) and azaserine, inhibitors of ammonium assimilation. Thus, basal ntcA transcript levels are indicative of ammonium utilization. Conversely, the highest ntcA transcript levels were found in cells lacking a nitrogen source capable of supporting growth. Therefore, maximal ntcA expression would indicate nitrogen deprivation. This state of nitrogen deprivation was induced by a 1-h incubation with MSX. The rapid response of ntcA gene expression to the addition of ammonium and MSX was used to design a protocol for assessing relative ntcA transcript levels in field populations of cyanobacteria, from which their nitrogen status can be inferred. ntcA was basally expressed in Synechococcus at a nutrient-enriched site at the northern tip of the Gulf of Aqaba, Red Sea. Therefore, these cyanobacteria were not nitrogen stressed, and their nitrogen requirements were met by regenerated nitrogen in the form of ammonium.

Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the expression of the Nia1 gene encoding NAD(P)H nitrate reductase is controlled at the transcriptional level, positively by light and negatively by ammonium. Previous work has shown that the region -279 to +269 with respect to the start site of transcription was sufficient to confer regulated expression of a promoterless arylsulfatase (Ars) reporter gene. To understand the mechanisms underlying this regulation, the -279 to +2 sequence was analysed for the presence of ammonium-responsive elements using either pJD54 (promoterless Ars gene) or pJD100 (minimal beta-tubulin promoter-driven Ars gene). The region lying between -195 and -120 was shown to be dispensable. Essential responsive elements were found in four distinct regions between -231 and -219, -120 and -100, -76 and -65 and -33 and -8. Each of these sequences is required for maximal expression in the absence of ammonium and a conserved GGA/TAGGGT motif is present in two of these regions. Several deletions within the region -33 to -77 were shown to partially relieve the transformants from the negative effect of ammonium. These experiments demonstrate that Nia1 expression is promoted by at least four elements between -231 and -8 and suggest that part of the repression by ammonium takes place through a proximal element located in the -51 to -33 sequence.

Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells. As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized. Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated. To reduce the negative effects of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expression. In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium. However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium. GPI was induced to a factor of two under ammonium-containing medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium.

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.

Ammonium ions repress δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase in Streptomyces clavuligerus NRRL 3585

Production of cephems (predominantly cephamycin C) by Streptomyces clavuligerus grown in chemically defined medium supplemented with 120 mM NH4Cl was sharply reduced. This concentration of ammonium ions in the medium repressed δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase formation by about 75%. Of the other cephalosporin synthases, cyclase was repressed by 70%, expandase by 50%, and epimerase only to a very small extent. Inhibition of the action of ACV synthetase was only slight in the presence of 100 mM NH4Cl. Repression of ACV synthetase, cyclase, and expandase appears to be the major factors contributing to the negative effect of ammonium on S. clavuligerus NRRL 3585. ACV synthetase is probably the rate-limiting step of cephalosporin biosynthesis in this strain.Key words: β-lactam biosynthesis, antibiotic biosynthesis, cephalosporins, Streptomyces clavuligerus, ACV synthetase.

The Assimilatory Nitrate Uptake in Azotobacter chroococcum. Induction by Nitrate and by Cyanate

Summary Induction of the assimilatory nitrate uptake in the strictly aerobic, nitrogen-fixing chemi-autrophic bacterium Azotobacter chroococcum is strictly dependent on the presence of nitrate or nitrite in the extracellular medium; induction requires an energy source and is inhibited by chloramphenicol. Cyanate was found to act as a gratuitous inducer of the assimilatory nitrate uptake and as an inhibitor of nitrate transport into the bacterial cells. Ammonium inhibited the development of assimilatory nitrate uptake. Blockage of ammonium assimilation by L-methionine-D,L-sulfoximine prevented the negative effect of ammonium on the assimilatory nitrate uptake induction, thus indicating that some organic products) derived from ammonium metabolism rather than ammonium itself is the actual regulator of the assimilatory nitrate uptake.