Utilization of Nitrite as a Nitrogen Source by Botryococcus Braunii

Nitrite at 2 mM did not affect the growth of Botryococcus braunii and served as the sole nitrogen source giving a minimum biomass doubling time of 4.2 d, which was equal to that of the culture using 4 mM nitrate as nitrogen source. With nitrite at 4 mM, after a lag phase of about 10 d, the alga grew quickly, reaching 1.1 g l(-1) at the end. Respective nitrite removals were 100% and 99.7%. There were few differences in the hydrocarbons produced using different nitrogen sources.     

Metabolism of Glycollate by Lemna minor L. Grown on Nitrate or Ammonium as Nitrogen Source

Marques, I. A., Oberholzer, M. J. and Erismann, K. H. 1985.Metabolism of glycollate by Lemna minor L. grown on nitrateor ammonium as nitrogen source.—J. exp. Bot. 36: 1685–1697. Duckweed, Lemna minor L., grown on inorganic nutrient solutionscontaining either NH₄⁺ or NO₃^– as nitrogen source wasallowed to assimilate [1-¹⁴C]- or [2-¹⁴C]glycollate during a20 min period in darkness or in light. The incorporation ofradioactivity into water-soluble metabolites, the insolublefraction, and into the CO₂ released was measured. In additionthe extractable activity of phosphoenolpyruvate carboxylasewas determined. During the metabolism of [2-¹⁴C]glycollate in darkness, as wellas in the light, NH₄⁺ grown plants evolved more ¹⁴CO₂ than NO₃^–grown plants. Formate was labelled only from [2-¹⁴C]glycollateand in NH₄⁺ grown plants it was significantly less labelledin light than in darkness. In NO₃^– grown plants formateshowed similar radioactivity after dark and light labelling.The radioactivity in glycine was little influenced by the nitrogensource. Amounts of radioactivity in serine implied that thefurther metabolism of serine was reduced in darkness comparedwith its metabolism in the light under both nitrogen regimes.In illuminated NH₄⁺ plants, serine was labelled through a pathwaystarting from phosphoglycerate. After [1-¹⁴C]glycollate feedingNH₄⁺ grown plants contained markedly more radioactive aspartateand malate than NO₃^– plants indicating a stimulated phosphoenolpyruvatecarboxylation in plants grown on NH₄⁺. Key words: Photorespiration, glycollate, nitrogen, Lemna     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Resistance to Phosphinothricin (Glufosinate) and Its Utilization as a Nitrogen Source by Chlamydomonas reinhardtii

Wild-type strain 21gr of the green alga Chlamydomonas reinhardtii was resistant to the ammonium salt of l-phosphinothricin (PPT, also called glufosinate), an irreversible inhibitor of glutamine synthetase activity and the main active component of the herbicide BASTA (AgrEvo, Frankfurt am Main, Germany). Under the same conditions, however, this strain was highly sensitive to l-methionine-S-sulfoximine, a structural analog of PPT which has been reported to be 5 to 10 times less effective than PPT as an inhibitor in plants. Moreover, this alga was able to grow with PPT as the sole nitrogen source when this compound was provided at low concentrations. This utilization of PPT was dependent upon the addition of acetate and light and did not take place in the presence of ammonium. Resistance was due neither to the presence of N-acetyltransferase or transaminase activity nor to the presence of glutamine synthetase isoforms resistant to PPT. By using l-[methyl-(sup14)C]PPT, we demonstrated that resistance is due to lack of PPT transport into the cells. This strongly suggests that PPT and l-methionine-S-sulfoximine enter the cells through different systems. Growth with PPT is supported by its deamination by an l-amino acid oxidase activity which has been previously described to be located at the periplasm.     

Nodule Nitrate Reductase as a Source of Reduced Nitrogen in Soybean, Glycine max

Hydroponically grown soybeans were fed ¹⁵N-enriched NaNO₃ at nine reproductive stages of development. The stem exudates contained excess ¹⁵N in the fully reduced nitrogen fraction. The soybean nodules had high nitrate reductase activity, whereas the roots had no detectable nitrate reductase activity. Based on these results, we concluded that the nodule nitrate reductase system has the potential of contributing significantly to the nitrogen economy of the plant.     

Utilization of Previously Accumulated and Concurrently Absorbed Nitrogen during Reproductive Growth in Maize : Influence of Prolificacy and Nitrogen Source

A prolific maize (Zea mays L.) genotype was grown to physiological maturity under greenhouse conditions to examine the effects of reproductive sink demand on (a) the remobilization of N accumulated during vegetative growth, and (b) the partitioning of N accumulated concurrent with ear development. One- and two-eared plants were treated with either a NO₃⁻ or NH₄⁺ source of ¹⁵N-labeled N during reproductive growth. Plants with two ears enhanced grain production, N remobilization from the stalk and roots, and N translocation to the grain from concurrently assimilated N. But, remobilization of leaf-N was unaffected by ear number. In addition, N uptake and total dry matter accumulation during the reproductive period were also unaffected, although P uptake was greater in the two-eared plants. Less than 15% of the total K⁺ uptake was accumulated after silking while during this time more than 40% of the total N and more than 50% of the total P were absorbed. The data also indicate that with NO₃⁻ nutrition, internal recirculation of K⁺ between shoots and roots may play a prominent role in the transport of nitrogenous solutes during grain development. N source had no effect on dry matter production and N uptake of both one- and two-eared plants. However, slightly greater partitioning of labeled-N from the NH₄⁺ source to the grain was observed in the two-eared plants.     

Utilization of Nitrogen Sources by Immature Soybean Cotyledons in Culture

Immature Glycine max (L.) Merrill cotyledons were cultured ina defined medium containing different nitrogen sources. Glutaminewas the most efficient source in terms of protein accumulationin the cotyledons. Asparagine was less efficient (about 70 percent that of glutamine) while allantoin was a poor source ofnitrogen. This was also true for older cotyledons where asparaginaseand allantoinase activities were maximal. The utilization ofboth asparagine and allantoin (but not glutamine) was totallyinhibited by methionine sulfoximine suggesting that their metabolisminvolves ammonia assimilation via glutamine synthetase. Apparently,neither exogenous or endogenously-generated ammonia had mucheffect on glutamine utilization, but ammonia did have a smallinhibitory effect on asparagine, which may in part account forthe lower efficiency observed with this amide. Glycine max, soybean, cotyledon culture, nitrogen metabolism     

Solute Accumulation In Plant Cells IV. Effects of Ammonium lons on Growth and Solute Content

Procedures previously described were used to study growth andsolute content of aseptically cultured carrot explants as affectedby supplementary salts in the medium. The salts chosen (KC1,KNO₃, NH₄,Cl, and NH⁴,NO₃) contrasted, with appropriate controls,the effects due to nitrate and ammonium. Growth was measuredin terms of fresh weight, the number and average size of cells:solute concentrations were recorded for total solutes, sugars,soluble nitrogen compounds, and the electrolytes K⁺, Na⁺, C1^–,NO^–₃, and organic acids. The time-response curves of thecultures were traced at a fixed concentration of the added saltsand the effects due to the concentration of the supplementarysalts were tested after a fixed time period, For the same nitrogensource the concentrations of metabolites and solutes in cellswere very similar despite some clonal differences in their growth.When cells in a nitrate medium were small and dividing, thecultures had a low osmotic value, contained K⁺ as the principalcation balanced by organic acid, had relatively low sugar content,and their enriched total nitrogen content emphasized proteinrather than soluble nitrogen compounds. Later, as the cellsbecame older and larger, salts (K⁺, organic anions, Cl^–)contributed substantially to their increased osmotic value butthey accumulated sugar as their main, osmotically active solute,and the ratio of soluble to protein nitrogen declined as proteinsynthesis progressed. The extra nitrogen supplied by the additionalpotassium nitrate contributed more to protein and caused potassium,organic acids, and sugars to accumulate to higher levela. Supplementaryammonium salts required that more sugar be metabolized to organicnitrogen compounds (e.g. glutamine), contributed more to solublethan to protein nitrogen, and sharply reduced. both the osmoticvalue of the cells and the potassium linked to organic anions.The selectivity of the growing cells for K⁺ over Na⁺ and theirdiscrimination. between alkali cations (Ka⁺+Na⁺) and halides(C1^–) were relaxed in the presence of ammonia. Attentionis drawn to the implications of these results for the accumulationof solutes, organic and inorganic, by dividing and enlargingcells.     

Aerobic Growth on Nitroglycerin as the Sole Carbon, Nitrogen, and Energy Source by a Mixed Bacterial Culture

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1,2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 ± 0.005 h⁻¹ (mean ± standard deviation) was estimated for the mixed culture at 25°C. Evidence of GTN toxicity was observed at GTN concentrations above 0.3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Accumulation of Soybean Lectin-Binding Polysaccharide During Growth of Rhizobium japonicum as Determined by Hemagglutination Inhibition Assay

A hemagglutination inhibition assay was used to estimate the presence of soybean lectin-binding polysaccharide in whole culture, culture supernatant, and isolated exopolysaccharide of Rhizobium japonicum USDA 138. The occurrence of 0.1 to 0.2 μg of lectin-binding polysaccharide could be detected within 2 h with a 0.5-ml total sample. Lectin-binding polysaccharide was detected in all preparations during both exponential and stationary growth phases. The formation of lectin-binding polysaccharide was not, whereas that of total exopolysaccharide was, markedly affected by culture conditions.     

Growth of Bacteria on 3-Nitropropionic Acid as a Sole Source of Carbon,Nitrogen, and Energy

3-Nitropropionic acid (3NPA) is a widespread nitroaliphatic toxin found in a variety of legumes and fungi. Several enzymes have been reported that can transform the compound, but none led to the mineralization of 3NPA. We report here the isolation of bacteria that grow on 3NPA and its anion, propionate-3-nitronate (P3N), as the sole source of carbon, nitrogen, and energy. Experiments with resting cells, cell extracts, and purified enzymes indicate that the pathway involves conversion of 3NPA to P3N, which upon denitration yields malonic semialdehyde, nitrate, nitrite, and traces of H₂O₂. Malonic semialdehyde is decarboxylated to acetyl coenzyme A. The gene that encodes the enzyme responsible for the denitration of P3N was cloned and expressed, and the enzyme was purified. Stoichiometry of the reaction indicates that the enzyme is a monooxygenase. The gene sequence is related to a large group of genes annotated as 2-nitropropane dioxygenases, but the P3N monooxygenase and closely related enzymes form a cluster within COG2070 that differs from previously characterized 2-nitropropane dioxygenases by their substrate specificities and reaction products. The results suggest that the P3N monooxygenases enable bacteria to exploit 3NPA in natural habitats as a growth substrate.Large-scale release of synthetic nitroaromatic compounds to the biosphere followed the invention of nitrobenzene around 1830. In less than 200 years, microorganisms adapted to the presence of nitroaromatic compounds in the environment by developing catalytic pathways to exploit them as growth substrates. Such rapid development suggests that the pathways did not develop de novo but evolved from preexisting degradation pathways such as might be found in microorganisms that degrade naturally occurring compounds.3-Nitropropionic acid (3NPA) is a widespread naturally occurring nitroaliphatic compound. It is a principal toxic component of Astragalus locoweeds and has been found in hundreds of species of legumes (20, 39) and a variety of fungi (6). The compound causes irreversible inhibition of succinate dehydrogenase, which makes it deadly to eukaryotes (1). Plants that make 3NPA also contain an enzyme, 3NPA oxidase (NPAO) (19, 20), which converts the compound to malonic semialdehyde (MSA) to protect the plant against the toxic effects of the compound (20). Given its widespread occurrence, we hypothesized that there must be bacteria in soil that degrade 3NPA and play a major role in determining the flux of the compound.Although bacteria that degrade 3NPA have previously been sought, the focus has been on organisms that ingest 3NPA-containing plant matter. Rumen microorganisms reduce 3NPA to β-alanine (4), and in the grasshopper gut, 3NPA is bound to glycine to form inert conjugates which are then eliminated (24). The plant enzyme NPAO converts 3NPA and O₂ to MSA, nitrate, nitrite, and hydrogen peroxide (19). It is similar to propionate-3-nitronate (P3N) oxidase (P3NO; EC 1.7.3.5) from Penicillium atrovenetum that converts the P3N form of the compound to MSA (36). The enzymes mentioned above are “orphan enzymes” (28), which means that the gene(s) has not been identified. None of the previously studied microorganisms can use 3NPA as a growth substrate, and the physiological roles of the enzymes have not been established.MSA appears to be a central intermediate in the eukaryotic transformation of 3NPA and its analogs. However, the transformations involve distinctly different types of reactions and metabolites released. P3N and 3NPA release nitrate and nitrite in a 2:1 ratio when attacked by the fungal or plant oxidases. When 3NPA is reduced to β-alanine by rumen microorganisms, β-alanine is further metabolized (4), possibly by deamination to MSA (18).We report here the isolation from soil of aerobic bacteria that grow on 3NPA as the sole source of carbon, nitrogen, and energy. The genes that encode the initial enzymes of the degradation pathway were cloned, and recombinant proteins were purified and partially characterized to allow determination of the initial steps in the catabolic pathway.(Preliminary reports of this work have been presented previously at the 106th General Meeting of the American Society for Microbiology [32a] and the 108th General Meeting of the American Society for Microbiology [32b].)     

Utilization of PPi as an Energy Source by a Clostridium sp

The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PP_i was stimulated by the presence of PP_i. The doubling time decreased and cell yield increased proportionately to PP_i concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PP_i has been reported. The PP_i-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PP_i, but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PP_i serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PP_i. The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen.     

Effects of Nitrogen Deficiency on the Absorption of Nitrate and Ammonium by Barley Plants

When young barley plants which had been supplied with nitratewere deprived of this source of N, an enhanced capacity forabsorption of either nitrate or ammonium ions developed, reachinga maximum in about 3 d under the particular experimental conditionsused. The net uptake rate of either nutrient was then approximatelythree times that in plants which had received nitrate throughout.Likewise, withholding external N from plants previously growingwith ammonium caused a 2.4-fold increase in their subsequentcapacity to absorb that ion, compared with control plants grownwith an uninterrupted ammonium supply. Accelerated nitrate uptakein N-starved plants was not accompanied by additional phosphateor sulphate absorption, but the plants had the capacity to absorbmore potassium, whether or not ammonium was also present inthe solution. Indirect evidence from analyses of root tissuesuggests that these responses to mild N-stress may depend onsome property of an N fraction which does not include nitrateor ammonium. Hordeum vulgare, barley, nitrogen, ammonium, nitrate, N-deficiency, absorption     

Growth and Metabolism of Soybean as Affected by Paclobutrazol

Paclobutrazol, an experimental growth retardant, was soil-appliedat the rate of 125 or 250 µg active ingredient per 10cm pot to 19 day-old soybean plants. This compound considerablyreduced plant height, leaf area, and stem dry weight. In addition,paclobutrazol-treated plants had numerous thickened lateralroots at the soil surface and had increased chlorophyll andsoluble protein contents compared to controls. During the first14 days after treatment, paclobutrazol increased the activitiesof NAD- and NADP-glyceraldehyde-3-phosphate dehydrogenase andaminotransferases but decreased the activity of nitrate reductase.Net photosynthesis (P_n) of the first and second trifoliatesof treated plants remained fairly constant throughout the studywhile control P_n declined during the latter portion of the experimentalperiod presumably due to leaf senescence. This decline of P_nin controls was accompanied by a decrease in the activitiesof NAD- and NADP-glyceraldehyde-3-phosphate dehydrogenase, nitratereductase, aminotransferases, and NAD malate dehydrogenase.Activities of these enzymes also tended to decline in paclobutrazol-treatedplants, but were still considerably higher than in controlsat the end of the experiment. The activities of RNase, protease,and glutamic dehydrogenase were higher in controls than in treatedplants. Our results suggest that paclobutrazol not only modifiesthe activity of a number of soybean enzymes but also delaysthe onset of senescence, thereby prolonging the period of normalmetabolic activity in a given leaf. ¹ Permanent address: Department of Botany, University of Jodhpur,India.     

氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响

共生根瘤的固氮效率受外界氮素的严格调控。除固氮酶活性外,豆血红蛋白(Lb)浓度亦是反应固氮能力的重要指标。为明确氮水平对生物固氮作用的影响,以大豆(Glycine max)为材料,在低氮(0.53 mmol·L–1)条件下接种根瘤菌,30天后再进行高氮(5.3、10、20、30和40 mmol·L–1)处理7天,分析L...     

Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain

Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.     

Deep-Sea Bacteria: Growth and Utilization of Hydrocarbons at Ambient and In Situ Pressure

Microorganisms present in Atlantic Ocean sediment samples collected at a depth of 4,940 m were found to be capable of utilizing hydrocarbons under both ambient and in situ pressures. The rate of utilization under in situ pressure (500 atm) and ambient temperature (20 C) was found to be significantly less compared with hydrocarbon utilization examined under conditions of ambient temperature (20 C) and pressure (1 atm).