Modulation of nitrate uptake in Anacystis nidulans by the balance between ammonium assimilation and CO2 fixation

Gradual inhibition of ammonium assimilation in Anacystis nidulans cells by increasing concentrations of 5-hydroxylysine resulted in a progressive enhancement of nitrate uptake. For 5-hydroxylysine-treated cells, the magnitude of the inhibition of nitrate uptake promoted by added ammonium was dependent on the ammonium assimilation capacity. In cells with a moderate ammonium assimilation activity, acceleration of CO2 fixation induced by bicarbonate addition antagonized the negative effect of ammonium, allowing full nitrate uptake activity. The results support the contention that nitrate utilization is under the feed-back control exerted by products of its own assimilation via ammonium, the inhibitory effect being potentiated by ammonium addition and alleviated by enhanced CO2 fixation. Results of amino acid analysis in cells exhibiting different capacities to utilize nitrate speak against these compounds as direct effectors of nitrate uptake.     

Dependence of nitrate utilization upon active CO2 fixation in Anacystis nidulans: a regulatory aspect of the interaction between photosynthetic carbon and nitrogen metabolism

Specific inhibition of photosynthetic CO2 fixation in Anacystis nidulans cells by D,L-glyceraldehyde resulted in the simultaneous inhibition of nitrate utilization, indicating a dependence of the latter process upon the provision of CO2-fixation products. This dependence was lost in cells treated with L-methionine-D,L-sulfoximine or azaserine, effective inhibitors of ammonium assimilation. In these cells, nitrate uptake could proceed at rates similar to those in control cells even if CO2 fixation was severely inhibited by D,L-glyceraldehyde. The results support the contention that CO2-fixation products participate in the control of nitrate uptake in A. nidulans by preventing the accumulation of certain ammonium derivatives which are negative effectors of nitrate uptake.     

Inhibition of nitrate consumption by nitrite in entrapped Chlamydomonas reinhardtii cells.

Whereas in freely suspended cell cultures growing photoautotrophically under non-limiting carbon conditions nitrite and nitrate were simultaneously consumed after ammonium consumption was complete, in alginate-entrapped cell cultures a sequential consumption of nitrite (first) and nitrate was observed after ammonium had almost been fully removed. In this paper results are reported that show inhibition of nitrate consumption by nitrite in immobilized cells. However no inhibition of nitrate active transport was observed. The sequential consumption of ammonium, nitrite and nitrate by Ca-alginate immobilized cells is explained on the basis of local ammonium accumulation due to its photoproduction by photorespiration, that could be caused by the increase of the O2/CO2 ratio around the entrapped cells. Measurements of light-dependent oxygen production (LDOP) and activity levels of nitrogen assimilation enzymes, including nitrite reductase (NiR) and glutamine synthetase (GS) in immobilized cells, determined under photorespiration stimulating conditions, are shown that support this explanation.     

The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport

A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO(2) conditions.     

Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation

Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO₂ evolved O₂ at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO₂ fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO₂ fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO₂ fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO₂ fixation are discussed.     

Independent induction of two blue light-dependent monovalent anion transport systems in the plasma membrane of Monoraphidium braunii

In the plasma membrane of the green alga Monoraphidium braunii there are at least two monovalent anion transport systems. One of them is specific for bicarbonate. This transport system is activated by blue light and its induction is triggered by a decrease in the external CO2 concentration. The second transport system is responsible for nitrate uptake at least. This transport system is also activated by blue light and its induction occurs when there is no ammonium in the external medium. Both transport systems are synthesized independently. Hence, when M. braunii cells grow with nitrate as the only nitrogen source under high CO2, they have a nitrate transport system but lack a bicarbonate transporter. Conversely, cells grown with ammonium under low CO2, have a bicarbonate transport system but lack a nitrate transporter. Both transport systems are induced in cells irradiated with white light in the absence of a carbon source, suggesting that there may be precursors in the plasma membrane that only need the synthesis and assembly of some component(s) to become fully active. The induction of nitrate and nitrite reductases, however, only takes place when a carbon source is supplied to the cells.     

Distinctive Light and CO(2)-Fixation Requirements of Nitrate and Ammonium Utilization by the Cyanobacterium Anacystis nidulans

The effect of light intensity on the rates of ammonium and nitrate uptake and of CO₂ fixation has been determined in intact Anacystis nidulans cells. Ammonium uptake became saturated at photon flux values of about 60 microeinsteins per square meter per second, whereas both nitrate uptake and CO₂ fixation reached saturation at about 250 microeinsteins per square meter per second, the rates of the two latter processes being tightly correlated at any light intensity assayed. Inhibition of ammonium assimilation resulted in the loss of correlation between CO₂ fixation and nitrate uptake, the latter process exhibiting then a reduced light requirement. The results establish a clear distinction between ammonium utilization and nitrate utilization with regard to their light requirement and to the nature of their dependence upon CO₂ fixation.     

Short-term nitrate (nitrite) inhibition of nitrogen fixation in Azotobacter chroococcum.

Nitrate-grown Azotobacter chroococcum ATCC 4412 cells lack the ability to fix N2. Nitrogenase activity developed after the cells were suspended in a combined nitrogen-free medium and was paralleled by a concomitant decrease in nitrate assimilation capacity. In such treated cells exhibiting transitory nitrate assimilation and N2-fixation capacity, nitrate or nitrite caused a short-term inhibitory effect on nitrogenase activity which ceased once the anion was exhausted from the medium. The analog L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, prevented inhibition of nitrogenase activity by nitrate or nitrite without affecting the uptake of these antions, which were reduced and stoichiometrically released into the external medium as ammonium. Inhibition of nitrogenase by nitrate (nitrite) did not take place in A. chroococcum MCD1, which is unable to assimilate either. We conclude that the short-term inhibitory effect of nitrate (nitrite) on nitrogenase activity is due to some organic product(s) formed during the assimilation of the ammonium resulting from nitrate (nitrite) reduction.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Nitrate transport in the cyanobacterium Anacystis nidulans

A significant progress in the knowledge of different aspects of nitrate transport in the unicellular cyanobacterium Anacystis (Synechococcus ) has been achieved in the last few years. The main contributions of our group are summarized in this article and discussed in relation to other information available. Endergonic accumulation of nitrate into the cells, indicative of the operation of an active nitrate transport system, has been experimentally substantiated and methods established to evaluate and analyze the activity of the system. Nitrate transport activity is sensitive to regulation exerted by products of both ammonium and CO₂ assimilation, thus providing evidence that photosynthetic nitrate assimilation in cyanobacteria is primarily controlled at the level of substrate supply to the cell. The expression of nitrate transport was also shown to be under nitrogen control, being repressed when ammonium is used as the nitrogen source. A 47-kDa polypeptide, which is a major plasma membrane component in nitrate-grown cells but is virtually absent in ammonium-grown cells, was identified as an essential component of the nitrate transporter. More recently, evidence of a strict Na'-dependence of active nitrate transport has been obtained, Δμ(Na⁺) appearing as the driving force of a sodium-nitrate symport system. Kinetic studies indicate also that the nitrate transporter may transport nitrite into the cell.     

A mutant of Chlamydomonas reinhardtii altered in the transport of ammonium and methylammonium

Summary A methylammonium-resistant mutant, named hereafter strain 2170 (ma-1), was isolated for the first time from a eukaryotic phototrophic organism. Mutant 2170 from Chlamydomonas reinhardtii carries a single mendelian mutation which results in a decreased rate of uptake of both ammonium and methylammonium without being affected either in uptake of nitrate or nitrite or any of the tested enzyme activities related to ammonium assimilation. Mutant cells could not use methylammonium as nitrogen source nor excrete ammonium into the medium but they had derepressed nitrate and nitrite reductases when growing in the presence of ammonium. Mutant 2170 also exhibited a diminished methylammonium transport rate in comparison with the wild-type cells. We conclude that mutant 2170 is affected in a transport system responsible for the entrance of both ammonium and methylammonium into the cells.Abbreviations CHES 2-(N-Cyclohexylamino)ethanesulphonic acid - MOPS 3(N-morpholine)propanesulphonic acid     

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l-methionine dl-sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO₂, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l-methionine dl-sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO₂-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.     

Inorganic Carbon-Stimulated O2 Photoreduction Is Suppressed by NO2- Assimilation in Air-Grown Cells of Synechococcus UTEX 625

The effect of NO2- assimilation on O2 exchange and CO2 fixation of the cyanobacterium, Synechococcus UTEX 625, was studied mass spectrometrically. Upon addition of 1 mM inorganic carbon to the medium, inorganic carbon pools developed and accelerated O2 photoreduction 5-fold when CO2 fixation was inhibited. During steady-state photosynthesis at saturating light, O2 uptake represented 32% of O2 evolution and balanced that portion of O2 evolution that could not be accounted for by CO2 fixation. Under these conditions, NO2- assimilation reduced O2 uptake by 59% but had no influence on CO2 fixation. NO2- assimilation decreased both CO2 fixation and O2 photoreduction at low light and and increased net O2 evolution at all light intensities. The increase in net O2 evolution observed during simultaneous assimilation of carbon and nitrogen over carbon alone was due to a suppression of O2 photoreduction by NO2- assimilation. When CO2 fixation was precluded, NO2- assimilation inhibited O2 photoreduction and stimulated O2 evolution. When the electron supply was limiting (low light), competition among O2, CO2, and NO2- for electrons could be observed, but when the electron supply was not limiting (saturating light), O2 photoreduction and/or NO2- reduction caused electron transport that was additive to that for maximum CO2 fixation.     

Nitrate reduction and assimilation in Chlorella

1. Nitrate reduction and assimilation have been studied in Chlorella pyrenoidosa under growth conditions by observing effects on the CO(2)/O(2) gas exchange quotient. 2. During assimilation of glucose in the dark, nitrate reduction is noted as an increase in the R.Q. to about 1.6 caused by an increased rate of carbon dioxide production. 3. During photosynthesis at low light intensity nitrate reduction is evidenced by a reduction in the CO(2)O(2) quotient to about 0.7 caused by a decreased rate of carbon dioxide uptake. 4. Chlorella will assimilate nitrogen from either nitrate or ammonia. When both sources are supplied, only ammonia is utilized and no nitrate reduction occurs. It is inferred that under the usual conditions of growth nitrate is reduced only at a rate required for subsequent cellular syntheses. The effect of nitrate reduction on the CO(2)O(2) quotient therefore provides a measure of the relative rate of nitrogen assimilation. 5. Over-all photosynthetic metabolism may be described from elementary analysis of the cells since excretory products are negligible. The gas exchange predicted in this way is in good agreement with the observed CO(2)/O(2) quotients.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.