Catalina Lara

Involvement of the C-terminal Domain of an ATP-binding Subunit in the Regulation of the ABC-type Nitrate/Nitrite Transporter of the Cyanobacterium Synechococcus sp. Strain PCC 7942

In Synechococcus sp. strain PCC 7942, an ATP-binding cassette transporter encoded by the genes nrtA, nrtB, nrtC, and nrtD mediates active transport of nitrate and nitrite, which is inhibited by ammonium, a preferred source of nitrogen for the cyanobacterium. One of the ATP-binding subunits of the transporter, NrtC, has a distinct C-terminal domain of 380 amino acid residues. A mutant NC2, constructed by removal of this domain using genetic engineering techniques, assimilated low concentrations of nitrate and nitrite and accumulated nitrate intracellularly, showing that the domain is not essential for the transporter activities. Assimilation of low concentrations of nitrite was only partially inhibited by ammonium in NC2 but was completely inhibited in the wild-type cells. Cells of NC2 and its derivative (nitrate reductase-less strain NC4) carrying the truncated NrtC but not the cells with the wild-type NrtC accumulated nitrate intracellularly in the presence of ammonium in medium. These findings indicated that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the nitrate/nitrite transporter. In the presence of ammonium, NC2 could not assimilate nitrate despite its ability to accumulate nitrate intracellularly, which suggested that reduction of intracellular nitrate by nitrate reductase is also subject to inhibition by ammonium.

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (Pi). Addition of Ca2+ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO2 fixation, indicating that NR activation follows saturation kinetics with respect to CO2 fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO2 fixation.

The ferredoxin/thioredoxin system of enzyme regulation in a cyanobacterium

Cell-free preparations of the cyanobacterium (bluegreen alga) Nostoc muscorum were assayed for thioredoxins and enzymes catalyzing the ferredoxin and NADP-linked reduction of thioredoxin. Nostoc was found to have two different thioredoxins: one of approximate molecular weight 16,000 (designated Nostoc thioredoxin f) that selectively activated chloroplast fructose 1,6-bisphosphatase, and another of approximate molecular weight 9,000 (designated Nostoc thioredoxin m) that selcetively activated chloroplast NADP-malate dehydrogenase. The two thioredoxins could be reduced either chemically with dithiothreitol or photochemically with ferredoxin and ferredoxin-thioredoxin reductase which, like the recently found regulatory iron-sulfur protein ferralterin, was present in Nostoc cells. Nostoc ferredoxin-thioredoxin reductase appeared to be similar to its chloroplast counterpart in enzyme specificity, molecular weight, and spectral properties. The Nostoc and spinach chloroplast ferredoxin-thioredoxin reductases as well as their thioredoxins, ferredoxins, and chlorophyll containing membranes were interchangeable in activating chloroplast fructose 1,6-bisphosphatase and NADP-malate dehydrogenase. There was no evidence for an NADP-linked thioredoxin reductase such as that of E. coli. The results are in accord with the conclusion that the cyanobacteria resemble higher plants in having a functional ferredoxin/thioredoxin system rather than an NADP/thioredoxin system typical of other bacteria.

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.

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans.

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.

Physiochemical properties of ferralterin. A regulatory iron-sulfur protein functional in oxygenic photosynthesis.

Abstract Ferralterin, an iron-sulfur protein identified earlier in chloroplasts and cyanobacteria, was purified to homogeneity from spinach leaves and Nostoc muscorum cells. When isolated from both sources, ferralterin showed a molecular weight of about 28,000 and was comprised of three subunits: one of molecular weight 12,000 and two, apparently identical, of molecular weight 7000. Based on the Lowry method of protein estimation, ferralterin contained approximately 3 g atoms each of nonheme iron and acid-labile sulfide per mole. The iron-sulfur cluster of ferralterin showed unusual redox and electron paramagnetic resonance (EPR) properties. Ferralterin was EPR silent as isolated and did not show an EPR signal on addition of reductants such as sodium dithionite or on exposure to illuminated chloroplast membranes. These reducing conditions also had no significant effect on the absorption spectrum of isolated ferralterin. The ferralterin iron-sulfur cluster was oxidized selectively by ferricyanide and showed a midpoint redox potential of +410 mV. Ferricyanide-oxidized ferralterin was characterized by a low-temperature EPR signal with g values of 2.10, 2.05, and 2.00 (spinach) and 2.09, 2.04, and 1.98 ( Nostoc ). When oxidized by ferricyanide, the iron-sulfur cluster could be reduced by a variety of reductants, including illuminated chloroplast membranes. The results are consistent with the conclusion that, like several other iron-sulfur enzymes (aconitase, glutamine phospho-ribosylpyrophosphate amidotransferase, hydrogenase), ferralterin achieves its catalytic effect via an active group independently of a redox change in the iron-sulfur chromophore.

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.

SODIUM-DEPENDENT NITRATE TRANSPORT AND ENERGETICS OF CYANOBACTERIA

Nitrate is a major source of nitrogen for organisms in aquatic environments. The first and least understood step in nitrate assimilation is the transport of this anion into the cell. Among the microalgae, cyanobacteria represent a convenient model system to study the transport of nitrate. Early studies on nitrate transport in the unicellular cyanobacterium Anacjstis (Sjnechococcus) showed endergonic accumulation of nitrate into the cells and, hence, the operation of an active nitrate transport system (Lara et al. 1987). Nitrate transport in Anacjstis has proved to have some unusual and interesting features. The nitrate transport activity was shown to be sensitive to the regulation exerted by products of both ammonium and CO, assimilation, thus providing evidence that photosynthetic nitrate assimilation in cyanobacteria is regulated at the level of substrate supply to the cell (Lara et al. 1987). The expression of the nitrate transport system was also shown to be under nitrogen control, being repressed when ammonium is used as the nitrogen source and derepressed upon transfer of the cells to media containing nitrate or no nitrogen source (Sivak et al. 1989). A 47-kDa polypeptide, which is a major plasma membrane component in nitrate-grown cells but is virtually absent from ammonium-grown cells, was identified as an essential component of the nitrate transporter (Omata et al. 1989, Sivak et al. 1989). More recent studies have provided evidence of the Na+ dependence of active nitrate transport, A,GNa+ appearing to be the driving force for a sodiumnitrate symport system (Rodriguez et al. 1992). In this article, the operation of such a sodiumdependent nitrate transport system is discussed in relation to the bioenergetics of cyanobacteria. A personal view, rather than a comprehensive review, on these matters is presented.

Ferralterin: An iron-sulfur protein functional in enzyme regulation in photosynthesis

The chloroplast new protein factor that was recently shown to link light to the activation of fructose 1,6-bisphosphatase was identified as a previously unrecognized iron-sulfur protein. This protein, given the name “ferralterin,” was purified to homogeneity from spinach leaves and from the blue-green alga (cyanobacterium) Nostoc muscorum. Ferralterin from both sources showed a visible absorption peak at 410nm, a molecular weight of about 30,000 and (provisionally) 4 g-atoms per mole each of nonheme iron and acid labile sulfide. The homogeneous ferralterin preparations catalyzed a light-dependent activation of chloroplast fructose 1,6-bisphosphatase that was dependent only on chlorophyll-containing membranes.

A new protein factor functional in the ferredoxin-independent light activation of chloroplast fructose 1,6-bisphosphatase.

Abstract A protein purified from chloroplasts (the “new protein factor”) activated Fru-P 2 ase in a photochemical reaction that depended only on chloroplast membranes. The results suggest that chloroplasts utilize the newly found mechanism for the photoregulation of Fru-P 2 ase in addition to the recently described ferredoxin/thioredoxin system.