A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

Achromobacter denitrificans YD35 is an NO₂⁻-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO₂⁻. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10⁻³ min⁻¹, which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S]²⁺ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO₂⁻ levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10⁻³ min⁻¹, which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO₂⁻, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO₂⁻ tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO₂⁻ milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO₂⁻ tolerance mechanism in this strain.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Isolation of Lightning-Competent Soil Bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl₂) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two “lightning-competent” soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc^r, Sp^r, Sm^r). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10⁻³ to 10⁻⁴ by electroporation and 10⁻⁴ to 10⁻⁵ by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Isolation and Antifungal and Antioomycete Activities of Aerugine Produced by Pseudomonas fluorescens Strain MM-B16

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C₁₀H₁₁NO₂S (M⁺, m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 μg ml⁻¹). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 μg ml⁻¹. Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO₃⁻ and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO₃⁻, either by amplifying the effect of low endogenous NO₃⁻ levels, or by making NO₃⁻ available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO₃⁻ and benzyladenine, additive responses were obtained. The effects of NO₃⁻ and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO₃⁻, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO₃⁻ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO₃⁻ and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO₃⁻. At the time of the second exposure to benzyladenine, no NO₃⁻ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO₃⁻.     

Role of Nitrate and Nitrite in the Induction of Nitrite Reductase in Leaves of Barley Seedlings

The role of NO₃⁻ and NO₂⁻ in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO₂⁻/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO₂⁻ and NO₃⁻ induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO₂⁻ and NO₃⁻ over a 24 hour period). In NO₃⁻-supplied leaves, NiR induction occurred at an ambient NO₃⁻ concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 millimolar. Nitrate accumulated in NO₂⁻-fed leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NiR as did equivalent amounts of NO₃⁻ accumulating in NO₃⁻-fed leaves. Induction of NiR in NO₂⁻-fed leaves was not seen until NO₃⁻ was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO₃⁻, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO₃⁻ to NO₂⁻ was inhibited by WO₄²⁻, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO₃⁻ directly, i.e. without being reduced to NO₂⁻, and that absorbed NO₂⁻ induces the enzyme activity indirectly after being oxidized to NO₃⁻ within the leaf.     

Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations

Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO₃⁻, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO₃⁻in vivo, −NO₃⁻in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO₃⁻ to the in vivo NR assay medium increased activity (over that of the −NO₃⁻in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO₃⁻ was rate-limiting. The stimulation of in vivo NR activity by NO₃⁻ was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO₃⁻ was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO₂⁻ [g fresh weight, hr]⁻¹) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.     

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Ecology of Thioploca spp.: Nitrate and Sulfur Storage in Relation to Chemical Microgradients and Influence of Thioploca spp. on the Sedimentary Nitrogen Cycle

Microsensors, including a recently developed NO₃⁻ biosensor, were applied to measure O₂ and NO₃⁻ profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of Thioploca spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O₂, high NO₃⁻, and bottom water current. On addition of NO₃⁻ and NO₂⁻, Thioploca spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with Thioploca, the penetration depth of NO₃⁻ was only 0.5 mm and a sharp maximum of NO₃⁻ uptake was observed 0.5 mm above the sediment surface. In sediments with only few Thioploca spp., NO₃⁻ was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N₂O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces intracellular NO₃⁻ to NH₄⁺. Measurements of the intracellular NO₃⁻ and S⁰ pools in Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO₃⁻ were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of Thioploca spp. and nitrate transport into the sediment.     

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Most studies of bacterial denitrification have used nitrate (NO₃⁻) as the first electron acceptor, whereas relatively less is understood about nitrite (NO₂⁻) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO₂⁻ (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO₂⁻, as well as NO₃⁻. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO₂⁻ reductase containing copper that was involved in bacterial NO₂⁻ reduction. The strain denitrified up to 40 mM NO₂⁻ to dinitrogen under anaerobic conditions in which other denitrifiers or NO₃⁻ reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO₂⁻. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188^T proliferated even in the presence of higher levels of NO₂⁻ (100 mM), and both were considerably more resistant to acidic NO₂⁻ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO₃⁻) or nitrite (NO₂⁻) is reduced to gaseous nitrogen forms such as N₂ and nitrous oxide (N₂O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO₃⁻ reductase (Nar), NO₂⁻ reductase (Nir), nitric oxide (NO) reductase (Nor), and N₂O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO₃⁻ to generate NO₂⁻ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO₂⁻ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds (11), organophosphorus pesticides (45), and other hydrocarbons (38) and remove heavy metal ions such as chromium and cadmium (24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence (7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level (33). Some O. anthropi strains have been identified as denitrifiers (21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO₂⁻ (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO₂⁻ under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS.     

Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sediment slurries at in situ NO₂⁻ concentrations in the presence and absence of NO₃⁻. With single enrichment above 10 μM ¹⁴NO₂⁻ or ¹⁴NO₃⁻ and ¹⁵NH₄⁺, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO₂⁻ or NO₃⁻, including an elevated response at lower concentrations. With 100 μM NO₃⁻, no significant transient accumulation of NO₂⁻ could be measured, and the starting concentration of NO₂⁻ could therefore be regulated. With dual enrichment (1 to 20 μM NO₂⁻ plus 100 μM NO₃⁻), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO₂⁻, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO₂⁻ added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO₂⁻ added at 5 μM for these two months, respectively. Decreasing the concentration of NO₃⁻ but holding NO₂⁻ at 5 μM decreased the significance of anammox as a sink for NO₂⁻. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO₃⁻ and NO₂⁻ and the relative size or activity of the anammox population.     

Studies of the Regulation of Nitrate Influx by Barley Seedlings Using NO(3)

Using ¹³NO₃⁻, effects of various NO₃⁻ pretreatments upon NO₃⁻ influx were studied in intact roots of barley (Hordeum vulgare L. cv Klondike). Prior exposure of roots to NO₃⁻ increased NO₃⁻ influx and net NO₃⁻ uptake. This `induction' of NO<sub>3</sub><sup>−</sup> uptake was dependent both on time and external NO<sub>3</sub><sup>−</sup> concentration ([NO<sub>3</sub><sup>−</sup>]). During induction influx was positively correlated with root [NO<sub>3</sub><sup>−</sup>]. In the postinduction period, however, NO<sub>3</sub><sup>−</sup> influx declined as root [NO<sub>3</sub><sup>−</sup>] increased. It is suggested that induction and negative feedback regulation are independent processes: Induction appears to depend upon some critical cytoplasmic [NO<sub>3</sub><sup>−</sup>]; removal of external NO<sub>3</sub><sup>−</sup> caused a reduction of <sup>13</sup>NO<sub>3</sub><sup>−</sup> influx even though mean root [NO<sub>3</sub><sup>−</sup>] remained high. It is proposed that cytoplasmic [NO<sub>3</sub><sup>−</sup>] is depleted rapidly under these conditions resulting in `deinduction' of the NO₃⁻ transport system. Beyond 50 micromoles per gram [NO₃⁻], ¹³NO₃⁻ influx was negatively correlated with root [NO₃⁻]. However, it is unclear whether root [NO₃⁻] per se or some product(s) of NO₃⁻ assimilation are responsible for the negative feedback effects.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Comparative Induction of Nitrate Reductase by Nitrate and Nitrite in Barley Leaves

The comparative induction of nitrate reductase (NR) by ambient NO₃⁻ and NO₂⁻ as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO₃⁻ influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO₃⁻. As the ambient concentration of NO₃⁻ increased, the relative influences imposed by influx and reduction on NO₃⁻ accumulation changed with influx becoming a more predominant regulant. Significant levels of NO₃⁻ accumulated in NO₂⁻-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO₂⁻, about 60% more NO₃⁻ accumulated in the leaves than in the absence of the inhibitors. In NO₃⁻-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mm. No NR induction occurred in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 mm. In fact, NR induction from NO₂⁻ solutions was not seen until NO₃⁻ was detected in the leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NR as did equivalent amounts of NO₃⁻ accumulating from NO₃⁻-fed leaves. In all cases the internal concentration of NO₃⁻, but not NO₂⁻, was highly correlated with the amount of NR induced. The evidence indicated that NO₃⁻ was a more likely inducer of NR than was NO₂⁻.     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.