Shift in carbon flow and stimulation of amino-acid turnover induced by nitrate and ammonium assimilation in Anacystis nidulans

The influence of nitrate and ammonium assimilation on the flow of recently fixed carbon has been determined in intact Anacystis nidulans cells actively fixing CO₂. Assimilation of nitrate or ammonium resulted in substantial increases in the incorporation of carbon into acid-soluble metabolites, the magnitude of the effect being dependent on the irradiance. The radiolabel in sugar phosphate was virtually unaffected by nitrogen assimilation, whereas that in organic acids and, in particular, in amino acids was markedly increased. Enhancement of carbon incorporation into amino acids induced by nitrogen assimilation was not accompanied by parallel increases in the size of the amino acid pools. This resulted in an appreciable increase of the specific radioactivity of most amino acids under conditions of nitrogen assimilation. The data indicate that nitrate and ammonium assimilation induce an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to oxaloacetate and α-ketoglutarate, as well as a stimulation of amino-acid turnover. These effects were more pronounced at saturating irradiance. We thank the Dirección General de Investigación Científica y Técnica, Spain (research grant PB88-0019) and the Plan Andaluz de Investigación (grupo 3101) for financial support, and P. Pérez de León for excellent secretarial assistance.     

The intracellular localisation of malate dehydrogenase in Anacystis nidulans

Abstract Malate dehydrogenase has been reported to be active as a Krebs cycle enzyme in Anabaena cylindrica and Anacystis nidulans [1,2] and as an enzyme of the glycollate pathway in Anabaena cylindrica [1,3]. This enzyme was also reported in Oscillatoria spp. [4] and in Nostoc muscorum [5]. The isoenzyme of eukaryotic organisms was known to participate in various metabolic pathways and to be localized in different subcellular organelles [6–9]. Kovatcheva and Bergman [5] have purified the enzyme from the reddish-brown 20 000 × g × 20 min supernatant. We have determined the intracellular distribution of malate dehydrogenese of Anacystis nidulans and present evidence that it is largely associated with the thylakoids. The significance of this study is discussed in terms of the dual role of cyanobacterial thylakoids in photosynthesis and respiration.     

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.     

Nucleotide Sequence of a Putative Sensory Kinase Gene from the Cyanobacterium, Anacystis nidulans 6301

The nucleotide sequence of a 2,146 bp portion of the Anacystisnidulans (Synechococcus PCC6301) genome has been determined.This region contains an open reading frame (ORF) of 392 codons,whose predicted protein sequence shows partial homology to thoseof E. coli phoM and envZ. Hence ORF392 is suggested to be asensory kinase gene in cyanobacteria.     

Impermeant electron acceptors and donors to the plasma membrane-bound respiratory chain of intact cyanobacterium Anacystis nidulans

Membrane-impermeant redox compounds ferricyanide and horse heart ferrocytochrome c acted as electron acceptor and donor, respectively, for intact cells or spheroplasts of Anacystis nidulans (Synechococcus ATCC 27144) in the dark. The anaerobic reduction of ferricyanide was faster than aerobic reduction. KCN significantly enhanced the reaction under aerobic conditions. Light did not influence ferricyanide reduction. The oxidation of exogenous ferrocytochrome c was oxygen-dependent and inhibited by KCN. Either type of redox reaction was accompanied by vectorial proton translocation out of the cells. Arrhenius plots for the temperature dependence of both ferricyanide reduction and cytochrome c oxidation gave one distinct break point reflecting the lipid phase transition temperature of the plasma membrane. The results are presented as evidence for a respiratory chain in the plasma membrane of A. nidulans.     

Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans

Photoinhibition of photosynthesis and its reactivation was studied in the cyanobaterium A. nidulans in the presence of the respiratory inhibitor sodium azide, the uncouplers carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone (FCCP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) and the photosystem I elicitor phenazine methosulphate (PMS). Inhibition of dark respiration by azide increased the susceptibility of the cyanobacterium to photoinhibition. Both FCCP and CCCP also remarkably affected the process of photoinhibition in A. nidulans. The PMS at lower photoinhibitory light intensity partially protected A. nidulans from photoinhibition. The recovery from photoinhibition in the presence of azide or FCCP was slow and normal photosynthesis could not be resumed even after a longer period of incubation under suitable reactivating condition. Thus dark respiration has a key function in the process of photoinhibition of photosynthesis and its reactivation in the cyanobacterium A. nidulans.     

蓝细菌Anacystis nidulans R-2藻胆体中43 Kd蛋白细胞定位的初步研究

高盐浓度条件下分离了蓝细菌Anacystis nidulans R-2的藻胆体,藻胆体中存在一种43kD的蛋白。Western blotting分析表明,该蛋白能与蓝细菌Fd：NADP氧还酶中FNRE占构域的抗体发生反应,解聚的藻胆体具有FNR黄递酶的活性,初步证明该43kD蛋白就是Fd：NADP氧还酶。Triton X-114分相实验表明,这种43kD的蛋白不能进入Triton X-114相。对藻胆体的部分解聚合实验表明,富含外周杆的组分中不存在43kD的蛋白。     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Oxidation of exogenous c-type cytochromes by intact spheroplasts of Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize reduced c-type cytochromes derived from horse heart, tuna, Saccharomyces oviformis, Candida krusei, Rhodocyclus purpureus, Rhodopseudomonas plustris and Paracoccus denitrificans with characteristics similar to those observed with isolated membranes. Rates of cytochrome c oxidation by the spheroplasts were only 10% of those measured with isolated membranes in which thylakoid-bound cytochrome oxidase contributes to the overall rates. Small amounts of an endogenous c-type cytochrome were released upon lysozyme treatment of the cells. The results appear to indicate the presence of cytochrome oxidase in the cytoplasmic membrane of A. nidulans.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - cyt cytochrome(s)     

The assimilation of nucleic acid precursors by intact cells and protoplasts of the blue-green alga Anacystis nidulans

Summary The entry of both nucleotides and base precursors into whole cells and the nucleic acid fraction of the blue-green alga Anacystis nidulans has been examined. Very slow rates of uptake were observed with intact organisms but protoplast preparations assimilated the nucleic acid precursors much more readily.     

Distribution of reducing power between photosynthetic carbon and nitrogen assimilation in Scenedesmus

Fixation of CO₂ and N assimilation were studied in synchronous cultures of Scenedesmus obtusiusculus Chod. under saturating and limiting light. Within the photon-flux range studied, the cells maintained C to N assimilation ratios of 7–10 with either NO ₃ ^- , NO ₂ ⁺ or NH ₄ ⁺ as the N source. Competitive interactions between C and N assimilation were pronounced under light limitation and were proportional to the oxidation status of the N source. Fixation of CO₂ at saturating light was also slightly reduced by NO ₂ ^- and NH ₄ ⁺ . In the absence of CO₂, NO ₃ ^- uptake and reduction was light-saturated at a comparatively low photon flux, whereas NO ₂ ^- uptake and reduction was considerably faster in the absence of CO₂ than in its presence. The pools of reduced pyridine nucleotides (NADPH and NADH) were largely unaffected by the presence or absence of the different N sources. The regulatory influences of CO₂ fixation on N assimilation are discussed in terms of coupling between the rates of CO₂ fixation and NH ₄ ⁺ assimilation, as well as the existance of control mechanisms for NO ₃ ^- uptake and reduction.Abbreviations Chl chlorophyll - PF photon flux     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

Physiological performances of the blue-green alga Anacystis nidulans in continuous turbidostat fermenter culture

A sterile continuous turbidostat culture in a 2-1 fermenter was used to systematically measure the gas exchange rates of Anacystis nidulans in a highly turbulent system under strictly controlled environmental conditions. An extensive physiological characterization of Anacystis is given in terms of photosynthesis rates (CO₂ uptake and O₂ evolution) and dark respiration rates as function of different parameters such as stirrer speed, temperature, CO₂ and O₂ concentration, light intensity, culture density and pH. Steady state ATP levels and apparent photophosphorylation rates complete the performance data. The dependence of the photosynthetic quotient from the parameters enables a physiological characterization of the light dependent nitrate assimilation.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Bioenergetic characterization of transient state phosphate uptake by the cyanobacterium Anacystis nidulans

A force flow relationship based on nonequilibrium thermodynamics was derived to analyze the variable transient state phosphate uptake phenomena of cyanobacteria seen under different growth conditions and external phosphate concentrations. This relationship postulates the following basic properties of the uptake system: First, a threshold value exists, below which incorporation is energetically impossible. Second, threshold values are influenced by the activity of the phosphate uptake system, such that a decrease of the activity increases the threshold level. Third, near the thermodynamic equilibrium the uptake rate is linearly dependent on the free energy of polyphosphate formation and the pH-gradient at the thylakoid membrane. Experiments performed with Anacystis nidulans showed that phosphate uptake characteristics conformed to the properties predicted by the linear force-flow relationship. Linearity extented into regions far form thermodynamic equilibrium, e.g. to high phosphate concentrations, when algae were preconditioned to high phosphate levels. Under phosphate limited growth linearity was confined to a small concentration range, threshold values decreased below 10 nM, and the external concentration approached threshold. The data suggest that the uptake system responds to changes in the external phosphate concentration in the same way as sensory systems to input stimuli by amplifying signals and adapting to them.Abbreviations chl chlorophyll - H _e ⁺ , H _C ⁺ , H _T ⁺ protons in the external medium, the cytoplasmic and thylakoid space respectively - P_c phosphate in the cytoplasmic space - P_e phosphate in the external medium - P_n, P_n+1 polyphosphates - pH_T pH-gradient across the thylakoid membrane     

Changes in growth and activity of enzymes involved in nitrate reduction and ammonium assimilation in tomato seedlings in response to NaCl stress

BACKGROUND AND AIMS: In Tunisia, salt water is largely used for tomato irrigation. In this work, a study was made of the changes in the nitrate reduction and ammonium assimilation into amino acids in tomato seedlings under salinity in order to providee further insight into the salt effects on plant growth. Methods Ten-day-old tomatoes (Solanum lycopersicum) were subjected to 100 mm NaCl stress, and nitrogen metabolism in leaves and roots was studied. KEY RESULTS: The concentrations of Na+ and Cl- rapidly increased in the leaves and in the roots following exposure of tomato seedlings to NaCl stress. In contrast, the NO3- concentrations were lowered first in the roots and later in the leaves. From 5 to 10 d of treatment, salt ions provoked a decrease in the dry weight and an increase in the NH4+ concentrations in the leaves. Inhibition was observed in the leaves for the activities of nitrate reductase (NR, EC 1.6.6.1), ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and deaminating glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2). NaCl affected these enzyme activities less in the roots than in leaves. This was in accordance with the pronounced decrease of dry weight by salt in leaves compared with that in the roots. CONCLUSIONS: NaCl stress effects on growth, metabolite concentrations and enzyme activities depended on the duration of salt treatment and the plant tissue.     

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans,a cyanobacterium

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth.Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceeded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found.In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.     

The effect of nitrogen refeeding on the carbohydrate content into nitrogen-starved cells of Platymonas striata Butcher

Studies on the mean cellular carbohydrate contents of Platymonas striata Butcher under conditions of nitrogen-starvation, and after refeeding these starved cultures with either nitrate or ammonium ions (growing under continuous illumination or with an alternating light/dark regime) have shown that nitrogen-starved cells accumulated abnormal amounts of cellular carbohydrate and that nitrogen refeeding produced a marked drop in the cellular carbohydrate. Cells grown in a light/dark regime accumulated less carbohydrates than those grown in continuous light. The mean cellular carbohydrate levels 16 h after nitrogen refeeding were still much in excess of those of cells grown with normal nutrition. It was therefore suggested that the differences in nitrogen uptakes in this period — when comparing either the uptake of cells grown in continuous light with that of cells grown in a light/dark regime; or when comparing the uptakes of cells presented with either nitrate or ammonium ions and grown in a light/dark regime —cannot be directly due to shortages of carbohydrate for the provision of carbon skeletons for nitrogen assimilation.     

The DNA,RNA and protein composition of the cyanobacterium Anacystis nidulans grown in light- and carbon dioxide-limited chemostats

The DNA, RNA and protein content of the cyanobacterium Anacystis nidulans was determined in light-limited and carbon dioxide-limited chemostat cultures over the dilution rate range, D=0.02 h^-1 to 0.19 h^-1. The macromolecular contents as a percentage of the dry weight and on a per cell basis varied significantly as a function of organism growth rate and the nature of the growth conditions. For both limitations the RNA content per cell increased [20–55 fg RNA (cell)^-1] with increasing dilution rate and also showed an increase as a percentage of the dry weight. The DNA content as a percentage of the dry weight showed a 2-fold decrease with increasing dilution rate over the range examined. On a per cell basis DNA reached a peak at D=0.1 h^-1 [4.5 fg DNA (cell)^-1] for light-limited organisms and at D=0.08 h^-1 [8.0 fg DNA (cell)^-1] for carbon dioxide-limited organisms. The q _RNA increased with increasing dilution rates over the complete growth rate range examined whilst q _DNA reached a maximum at D=0.09 to 0.10 h^-1. The protein content as a percentage of the dry weight was greater in CO₂-limited organisms than light-limited organisms but in both cultures declined as the dilution rate was increased above D=0.10 h^-1.