Dark Anaerobic Conditions Research Articles

Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, Mr=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.

Denitrifying and photoheterotrophic growth of Rhodobacter sphaeroides S under anaerobic-dark and -light conditions

Denitrifying growth characteristics of Rhodobacter shaeroides S were examined in batch and continuous cultures under anaerobic-dark and -light conditions. Growth yield based on the electron equivalent, Y eq., in anaerobic-dark conditions was 2–3 times lower than that in aerobic-dark conditions (aerobic-heterotropic growth). Under anaerobic-light conditions, denitrifying growth was observed with photosynthetic growth, but decreased with the increase of light intensity. The value of Y eq. in anaerobic-light conditions increased with the increase of light intensity. This phenomenon could be explained as the change of the fraction of cell mass grown by denitrification and photosynthesis in the total cell mass from the energetic analysis.

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c2 from mutant H41 demonstrated at the same time the participation of the c2-polypeptide in the regulation of cytochrome c oxidase.

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70 degrees C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65 degrees C site, with naturally high hydrogen levels, and a 55 degrees C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65 degrees C mat.

Protein Synthesis and Degradation during the Differentiation Cycle of Rhodomicrobium Vannielii Swarmer Cells

SUMMARY: Rhodomicrobium vannielii swarmer cells, when synchronized by selective filtration, differentiate synchronously with the loss of motility and synthesis of a prostheca, and subsequently form a daughter cell. Synchronized swarmer cells synthesized an 11·5 kDa protein under dark anaerobic conditions which disappeared from the soluble fraction when the culture was subsequently incubated in the light and the differentiation cycle thus initiated. The protein did not accumulate under dark aerobic conditions and it may play a role in the inhibition of differentiation in the light limited and thus energy limited swarmer cell.

Hydrogen production by a mixed culture of a green alga, Chlamydomonas reinhardtii and a photosynthetic bacterium, Rhodospirillum rubrum.

An about, 4-fold H2 evolution rate and a 5-fold H2 molar yield (mol H2/mol glucose) were obtained with a mixed culture of Chlamy domonas reinhardtii and Rhodosprillum rubrum, compared with in the case of an algal culture of C. reinhardtii alone. This increasing effect was due to the consumption of formate formed by C. reinhardtii; R. rubrum evolved hydrogen from formate via the formate hydrogen-lyase system under dark anaerobic (N2) conditions. Maximum H2 evolution by the mixed culture was observed with a ratio of 8 :2 (alga: bacterium) at a total cell concentration of above 0.6 mg dry wt/ml. Sustained H2 production with an alternating light/dark cycle in a membrane reactor, in which this alga and bacterium were cultured in separate compartments, was performed for one week.

Untersuchungen zum Atmungsstoffwechsel, ATP-Gehalt and Kornzuwachs bei Winterweizen: Investigations of Metabolism of Respiration, ATP Content and Grain Growth of Winter Wheat

In the grain filling period the dynamic of dry weight and total nitrogen increment of winter wheat grains was closely paralleled that of ATP level, mitochondria) respiration, succinate dehydrogenase activity and dark respiration. ATP content of grains was lower by about 12 % in the plants analyzed after 5 h light than in the plants analyzed after 15 h darkness. Anaerobic conditions in dark for 15 h decreased the ATP pool by more than 50 %. Darkness for 4 d didn't affect the protein synthesis, but the dry matter production was lower after darkness for more than 2 d due to limited energy and sucrose supply. 2,4-Dinitrophenol inhibited both accumulation of dry matter and of protein. It is concluded that the energy production by the dark respiration is more limiting than the supply of assimilates by the source organs under field conditions.

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.

Transmembrane Proton Electrochemical Gradients in Dark Aerobic and Anaerobic Cells of the Cyanobacterium (Blue-Green Alga) Anacystis nidulans

The transmembrane proton electrochemical potential gradient Deltamu(H+) in whole cells of Anacystis nidulans was measured in aerobic and anaerobic dark conditions using the distribution, between external medium and cell interior, of radioactively labeled weak acids (acetylsalicyclic acid, 5,5-dimethyloxazolidine-2,4-dione) or bases (imidazole, methylamine), and permeant ions (tetraphenylphosphonium cation, thiocyanate anion), as determined by flow dialysis. Alternatively, the movements across the plasma membrane of DeltapH-indicating atebrin or 9-aminoacridine, and of DeltaPsi-indicating 8-anilino-l-naphthalenesulfonate were qualitatively followed by fluorescence measurements. Attempts were made to discriminate between the individual chemiosmotic gradients across the cytoplasmic (plasmalemma) and the intracytoplasmic (thylakoid) membranes. By use of the ionophores nigericin, monensin, and valinomycin, the components of the proton motive force, namely the proton concentration gradient DeltapH and the electric membrane potential DeltaPsi were shown to be mutually exchangeable within the range of external pH values tested (3.2-11.0). Both components were depressed by the uncoupler carbonylcyanide m-chlorophenylhydrazone, though inhibition of DeltapH was much more pronounced than that of DeltaPsi, notably in the alkaline pH(0) range. The total proton electrochemical gradient across the plasma membrane was significantly higher in aerobic than in anaerobic cells and increased markedly (i.e. became more negative) towards lower pH(0) values. This increase was paralleled by a similar increase in the rate of endogenous respiration of the cells. At the same time the ATPase inhibitor dicyclohexylcarbodiimide only slightly affected the proton motive force across the plasma membrane of aerobic cells. The results will be discussed in terms of a respiratorily competent plasma membrane in Anacystis nidulans.

Endogenous ammonia production by Anacystis nidulans R-2 induced by methionine sulfoximine

Anacystis nidulans R-2 produced ammonia from endogenous sources for at least 6 h when illuminated without external nitrogen source but with CO2 in the presence of 50 μM methionine sulfoximine. The onset of ammonia release coinciding with complete inhibition of glutamine synthetase. The total quantity of ammonia which could be released exceeded the nitrogen content of small molecule pools, and suggested protein degradation as the most likely source of the nitrogen. Ammonia release was not accompanied by leakage of carbon compounds from the cells. Methionine sulfoximine-induced ammonia release was energy requiring, and was barely detectable under dark anaerobic conditions, or in the presence of 10 μM carbonyl cyanide m-chlorophenylhydrazone in light. Phenyl methyl sulfonylfluoride, an inhibitor of serine proteases, eliminated ammonia release, and the rate of release was reduced to one-third of control values, after a lag, in the presence of 50–75 μg/ml chloramphenicol. The rate of NH+4release was maximal (1.4 nmol·min-1·mg-1 protein) if suspensions were bubbled with 100% O2, but could not be reduced below 0.6 nmol·min-1·mg-1 protein in air: CO2, suggesting that release was at most only partly due to photorespiration.

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

1. The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source. 2. Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR+ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH 4 + , and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH 4 + . Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl. 3. Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR+. The nitrate reductase activity in cells of AD2 and N22DNAR+ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR+ has identical roles. These roles are suggested to include: (i) The first step in the assimilation of nitrate. (ii). Provision of an alternative electron acceptor to oxygen for generating a membrane potential. (iii). A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth. This type of nitrate reductase, especially in AD2 and N22DNAR+, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli. 4. In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

Regulation of nitrate assimilation in plants in light and dark

Assimilation of nitrate and nitrite accumulated in leaves was much more rapid in the light than in the dark. Under light aerobic and dark anaerobic conditions the extent of nitrate reduction was almost equal. Dark assimilation of nitrite was drastically inhibited by 2,4-dinitrophenol (2,4-DNP). Although the uncoupler did not show any significant effect on oxidation of NADH and succinate in isolated leaf mitochondria, it inhibited 14CO 2 evolved from endogenously labelled sugars, probably by blocking the formation of glucose 6-phosphate (G 6-P). It is suggested that for dark assimilation of nitrite, NADPH generated in the oxidative pentose phosphate pathway is the source of reductant.

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose‐6‐phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses‐phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo‐synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.

Dark activation of maize leaf NADP-malate dehydrogenase and pyruvate, orthophosphate dikinase in vivo under anaerobic conditions

Previous reports have shown that two enzymes of the C 4 pathway of photosynthesis, pyruvate, orthophosphate dikinase (PPDK) and NADP-malate dehydrogenase (NADP-MDH), are light-activated and dark-inactivated in vivo. In the present study with the C 4 plant maize, we found the two enzymes were activated in the dark under nitrogen. With NADP-MDH at temperatures of 17°, 25° and 32°C, near maximum activation was reached after 30–60 min of dark-N 2 treatment. With decreasing temperature the maximum level of activation increased. The level of activation under dark-anaerobic conditions at 17° and 25°C was up to 2-fold higher than under air in light at 25°C. After 60–90 min of treatment the level of activation decreased. With PPDK at temperatures of 17°, 25° and 32°C, the maximum level of dark-anaerobic activation occurred within 45–90 min, after which there was a decreased level of activation. In contrast to NADP-MDH, as temperature increased the degree of activation of PPDK increased. Carbon dioxide (0.03% CO 2) inhibited the dark activation of both NADP-MDH and PPDK under anaerobic conditions. The results are discussed in relation to known differences in the mechanisms of activation/inactivation of these two enzymes.

Assimilation of [15N]Nitrate and [15N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [(15)N]nitrate and [(15)N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [(15)N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [(15)N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.In dark anaerobic conditions, the assimilation of [(15)N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [(15)N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [(15)N]nitrite assimilation.

Active sodium extrusion reduces net efficiencies of oxidative phosphorylation in the strictly photoautotrophic cyanobacterium Anacystis nidulans

Efficiencies of oxidative phosphorylation ( P O ratios), intracellular high-energy phosphate pools (ATP and ADP) under aerobic and anaerobic dark conditions, and photosynthetic oxygen evolution measured with intact cells of Anacystis nidulans were found to be specifically depressed by NaCl, but not by KCl. A scheme is proposed which explains the deleterious effect of sodium on the energy metabolism of A. nidulans by competition for protons between ATP synthesis and active sodium extrusion.

Regulation of nitrate reduction in wheat leaves

Wheat leaves exposed to 710 nm monochromatic light, when only photosystem 1 operates, reduced small but significant amount of nitrate to nitrite. This could be due to partial inhibition of mitochondrial oxidation of NADH, brought about by cyclic photo-phosphorylation. Under dark aerobic conditions, citric acid cycle intermediates only slightly stimulated nitrate reduction. Under dark anaerobic conditions, when maximum reduction of nitrate occurred, the time course showed a 1:1 stoichiometry between nitrite and CO2. It is suggested that for maximum reduction of nitrate under physiological conditions, CO2 fixation and export of ATP via triose phosphate shuttle is essential.

In vivo and in vitro studies on ATP and electron donors to nitrogenase in Rhodospirillum rubrum

The photosynthetic bacterium, Rhodospirillum rubrum (ATCC 11170), was tested for its ability to fix nitrogen (acetylene reduction) under aerobic and dark-anaerobic conditions. Whole cells reduced acetylene under darkanaerobic conditions if pyruvate was supplied. Reactions of the cells were inhibited less by oxygen in the dark than in the light, and the cells were capable of acetylene reduction in the presence of low levels of oxygen (0.6%) in the dark. Crude extracts of R. rubrum reduced acetylene if pyruvate and Coenzyme A were added; ferredoxin from R. rubrum greatly increased the pyruvate-driven activity in crude extracts. It was not possible to demonstrate light-driven acetylene reduction in crude extracts unless a reductant (dithionite) was added.

ATP-dependent uptake of nitrate in Nostoc muscorum and inhibition by ammonium ions

A high rate of nitrate uptake was observed in Nostoc muscorum when cells were grown on elemental nitrogen as compared to that when they were grown on nitrate or ammonium. The uptake of nitrate was light dependent. However, supplementation with ATP (50 μM) stimulated nitrate uptake both in light and darkness. ADP, under similar conditions had no effect. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2- n-heptyl-4-hydroxyquinoline, (HOQNO) and KCN inhibitied nitrate uptake in light which could be partially reversed by adition of ATP. Inhibitiion by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler of photophosphorylation, was complete and could not be restored by the addition of ATP. N,N′-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of ATPase, blocked nitrate uptake in the presence or absence of externally added ATP. Although no nitrate uptake was observed under anaerobic conditions in dark, addition of ATP resulted in uptake of nitrate, which was similar in magnitude to that observed under aerobic condition in the light, and was inhibited by DCCD. Ammonium ions inhibited the uptake of nitrate in the absence of ATP but in its presence there was simultaneous uptake of nitrate and ammonium ions. However, uptake of ammonium ions alone was not affected by presence or absence of ATP in the external medium. It was concluded that nitrate ion uptake was energy dependent and may be linked with a proton gradient which can be formed either by photophosphorylation or ATP hydrolysis.

ORIGIN OF REDUCTANT FOR REDUCTION OF NITRATE AND NITRITE IN RICE AND WHEAT LEAVES IN VIVO

SUMMARYMalonate inhibited completely anaerobic dark reduction of nitrate in rice leaves and the inhibition was reversed by fumarate. Under dark anaerobic conditions l4CO2 evolution from endogenously labelled substrates was dependent on nitrate reduction. When nitrate reduction was inhibited by tungstate or cyanide, 14CO2 evolution was inhibited and conversely, when 14CO2 evolution via the citric acid cycle was inhibited by malonate, nitrate reduction was inhibited. These results suggest a mitochondrial origin of NADH for nitrate reduction and this suggestion was confirmed by following the evolution of 14CO2 from endogenous substrates, mostly organic acids, labelled in darkness. In comparison with the reduction of nitrite in light, the dark aerobic reduction of accumulated nitrite was very slow and commenced only after a time lag of 20 min. This could be a detoxification mechanism for the removal of nitrite which does not normally accumulate in the leaves under natural conditions. NADH acted as a physiological reductant for dark aerobic nitrite reduction.