Manuel Losada

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.

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

Abstract 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.

Ferredoxin-nitrite reductase from spinach

Summary 1. Nitrite reductase from a crude homogenate of spinach leaves has been purified about 500-fold and freed from NADP-reductase (EC 1.6.99.4) and nitrate reductase (EC 1.6.6.2). The enzyme, which does not seem to be a flavoprotein, catalyzes the reduction of nitrite to ammonia with a variety of enzyme systems as electron donors and requires ferredoxin as electron carrier. Flavin nucleotides and menadione are not capable of replacing ferredoxin, but the artificial electron carrier, methyl viologen, is also effective in the reaction. In the absence of spinach NADP-reductase, ferredoxin-nitrite reductase cannot use NADPH2 as electron donor. 2 A method based on the reduction of nitrite by ferredoxin (of methyl viologen) chemically reduced with hydrosulfite has been successfully applied to the aerobic assay of the enzyme. Nitrite reductase itself is inhibited by cyanide but not by p-chloromercuribenzoate or azide. The affinities of the various substrates and the offect of pH have also been investigated. 3. Isolated spinach chloroplasts contain nitrite reductase. On a protein basis, the activity of nitrite reductase in the chloroplasts is higher than in the rest of the cell as a whole. By breaking the chloroplasts, it has been shown that most of the enzyme is recovered in the chloroplast extract and only a small part remains bound to the grana.

Flavin nucleotide nitrate reductase from spinach

Abstract 1. 1. Nitrate reductase from spinach has been purified 130-fold by a procedure which includes, as the main steps, adsorption on calcium phosphate gel and chromatography on hydroxylapatite column. By this procedure it has been shown that NADP reductase and nitrate reductase are different proteins. 2. 2. A method based on the reduction of NO3− to NO2− with flavin nucleotides reduced by S2O42− has been successfully applied to the assay of the enzyme. 3. 3. FMN and FAD are the natural cofactors which in their reduced form mediate the transfer of electrons to the nitrate-nitrate reductase system. Menadione and ferredoxin are not capable of replacing the flavin nucleotides, but benzyl and methyl viologen are affective electron carriers in the system. 4. 4. Nitrate reductase has a pH optinum of 7.6. The Michaelis constant for either FMN or FAD is roughly 0.02 mM and that for nitrate, 0.2 mM. 5. 5. Due to the fact that FMN and FAD are the effective electron donors for the reduction of NO3− in higher plants and that they can be reduced by a variety of enzyme systems, the enzyme until now known as NAD(P)H2: nitrate oxidoreductase (EC 1.6.6.2) has to be considered as a mixture of two different proteins, NADP reductase and nitrate reductase itself, and the latter will be systematically classified as FMNH2(FADH2): nitrate oxidoreductase.

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO2 fixation products antagonizing the inhibitory effect of ammonium derivatives.

Factors affecting the production of biomass by a nitrogen-fixing blue-green alga in outdoor culture

The effectiveness of an aeration-shaking (air-lift) system for outdoor biomass photoproduction by the N2-fixing filamentous blue-green alga Anabaena variabilis was evaluated and the influence of relevant factors on the productivity of the system was assessed. Air at a flow rate of 60 liters per liter of cell suspension per h was enough, by itself, to promote adequate turbulence and to supply the gaseous nutrients (CO2 and N2) needed for maximal productivity. The addition of either or both, CO2 and combined nitrogen (as KNO3 or NH4Cl), did not result in any increase in productivity. In summer and winter, optimal cell density for a suspension depth of 25 cm was 0·2–0·3 g (dry weight) liter−1 and 0·1–0·2 g (dry weight) liter−1 respectively. Reciprocally, optimal suspension depth for a cell density of 0·2 g (dry weight) liter−1 was 20–25 cm in summer and below 15 cm in winter. Optimal values for pH and temperature were 8·2–8·4 and 30–35°C, respectively. Under optimal conditions, mean productivity values were about 13 g (dry weight) m−2 day−1 in summer and 6 g (dry weight) m−2 day−1 in winter. Net protein content of A. variabilis cells was higher than 50%, and nitrogen accounted for 10% of the dry biomass. From the productivity and nitrogen content data, the N2 fixation rate in outdoor cultures of A. variabilis can be estimated to be higher than 1 g N m−2 day−1, i.e. more than 3 t N per hectare per year when values are extrapolated both in time and area.

Differential Regulation of Soluble and Membrane-Bound Inorganic Pyrophosphatases in the Photosynthetic Bacterium Rhodospirillum rubrum Provides Insights into Pyrophosphate-Based Stress Bioenergetics

Soluble and membrane-bound inorganic pyrophosphatases (sPPase and H(+)-PPase, respectively) of the purple nonsulfur bacterium Rhodospirillum rubrum are differentially regulated by environmental growth conditions. Both proteins and their transcripts were found in cells of anaerobic phototrophic batch cultures along all growth phases, although they displayed different time patterns. However, in aerobic cells that grow in the dark, which exhibited the highest growth rates, Northern and Western blot analyses as well as activity assays demonstrated high sPPase levels but no H(+)-PPase. It is noteworthy that H(+)-PPase is highly expressed in aerobic cells under acute salt stress (1 M NaCl). H(+)-PPase was also present in anaerobic cells growing at reduced rates in the dark under either fermentative or anaerobic respiratory conditions. Since H(+)-PPase was detected not only under all anaerobic growth conditions but also under salt stress in aerobiosis, the corresponding gene is not invariably repressed by oxygen. Primer extension analyses showed that, under all anaerobic conditions tested, the R. rubrum H(+)-PPase gene utilizes two activator-dependent tandem promoters, one with an FNR-like sequence motif and the other with a RegA motif, whereas in aerobiosis under salt stress, the H(+)-PPase gene is transcribed from two further tandem promoters involving other transcription factors. These results demonstrate a tight transcriptional regulation of the H(+)-PPase gene, which appears to be induced in response to a variety of environmental conditions, all of which constrain cell energetics.

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H+-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure–function relationships of this type of proton pumps.

Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene

A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non‐phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3‐phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde‐3‐phosphate dehydrogenases. According to the irreversible catabolic character of the non‐phosphorylating reaction, the GapN‐complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden‐Meyerhof pathway with no net energy yield is feasible.

A thermostable K+‐stimulated vacuolar‐type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima

Current evidence suggests the occurrence of two classes of vacuolar‐type H+‐translocating inorganic pyrophosphatases (V‐PPases): K+‐insensitive proteins, identified in eukaryotes, bacteria and archaea, and K+‐stimulated V‐PPases, identified to date only in eukaryotes. Here, we describe the functional characterization of a thermostable V‐PPase from the anaerobic hyperthermophilic bacterium Thermotoga maritima by heterologous expression in Saccharomyces cerevisiae. The activity of this 71‐kDa membrane‐embedded polypeptide has a near obligate requirement for K+, like the plant V‐PPase, and its thermostability depends on the binding of Mg2+. Phylogenetic analysis of protein sequences consistently assigned the T. maritima V‐PPase to the K+‐sensitive class of V‐PPases so far only known for eukaryotes. The finding of a K+‐stimulated V‐PPase also in a member of a primitive eubacterial lineage strongly supports an ancient evolutionary origin of this group of pyrophosphate‐energized proton pumps.