Synthesis Of Nitrate Reductase Research Articles

Effects of temperature on photosynthetic performance and nitrate reductase activity in vivo assay in Gracilariopsis lemaneiformis (Rhodophyta)

Gracilariopsis lemaneiformis is an economically-valued species and widely cultured in China at present. After being acclimated to different growth temperatures (15, 20, 25, and 30°C) for 7 days, the relative growth rate (RGR), nitrate reductase activity, soluble protein content and chlorophyll a fluorescence of G. lemaneiformis were examined. Results show that RGR was markedly affected by temperature especially at 20°C at which G. lemaneiformis exhibited the highest effective quantum yield of PSII [Y(II)] and light-saturated electron transport rate (ETRmax), but the lowest non-photochemical quenching. Irrespective of growth temperature, the nitrate reductase activity increased with the incubation temperature from 15 to 30°C. In addition, the greatest nitrate reductase activity was found in the thalli grown at 20°C. The value of temperature coefficient Q10 of alga cultured in 15°C was the greatest among those of other temperatures tested. Results indicate that the optimum temperature for nitrate reductase synthesis was relatively lower than that for nitrate reductase activity, and the relationship among growth, photosynthesis, and nitrate reductase activity showed that the optimum temperature for activity of nitrate reductase in vivo assay should be the same to the optimal growth temperature.

Nitrate bioreduction dynamics in hyporheic zone sediments under cyclic changes of chemical compositions

Hyporheic zone (HZ) is the hotspot of contaminant biotransformation in ecosystems that affects water quality in both groundwater and river water. HZ is characterized by its dynamic changes in water flow direction that lead to the cyclic changes in aqueous chemical compositions such as dissolved oxygen (DO), nitrate, dissolved organic carbon, and their concentrations. Here we report a study on the effects of cyclic changes in the chemical conditions on the rates of nitrate bioreduction in sediments from a hyporheic zone, and the corresponding dynamics of the synthesis and decay of nitrate reductases. The results indicated that the rate of nitrate bioreduction in the second and third cycles was faster than that in the first one because the nitrate reductases produced in the first cycle still partially maintained their reactivity. The presence of DO significantly decreased the rates of nitrate reduction and nitrate reductase synthesis, but such an inhibition effect diminished in the later cycle. The coupled changes in nitrate concentrations and functional enzymes and genes abundances were generally consistent with an enzyme-based biogeochemical kinetic model. Overall the results indicated that enzyme growth dynamics and functions were complexly affected by the cyclic changes of environmental conditions, and implied that HZ sediments have a memory-type system behavior in reducing nitrate that would be affected by the frequency and magnitude of groundwater and river water exchanges, and chemical compositions in the groundwater and river water.

Nitric oxide is involved in nano-titanium dioxide-induced activation of antioxidant defense system and accumulation of osmolytes under water-deficit stress in Vicia faba L.

Nano-titanium dioxide (nTiO2) has been reported to improve tolerance of plants against different environmental stresses by modulating various physiological and biochemical processes. Nitric oxide (NO) has been shown to act as an important stress signaling molecule during plant responses to abiotic stresses. The present work was planned to investigate the involvement of endogenous NO in nTiO2-induced activation of defense system of fava bean (Vicia faba L.) plants under water-deficit stress (WDS) conditions. Water-suffered plants showed increased concentration of hydrogen peroxide (H2O2) and superoxide (O2-) content coupled with increased electrolyte leakage and lipid peroxidation which adversely affected nitrate reductase (NR) activity, chlorophyll content and growth of the plants. However, application of 15mgL-1 nTiO2 to stressed plants significantly induced NR activity and synthesis of NO which elevated enzymatic and non-enzymatic defense system of the stressed plants and suppressed the generation of H2O2 and O2- content, leakage of electrolytes, and lipid peroxidation. Application of nTiO2, in association with NO, also enhanced the accumulation of osmolytes (proline and glycine betaine) that assisted the stressed plants in osmotic adjustment as witnessed by improved hydration level of the plants. Involvement of NO in nTiO2-induced activation of defense system was confirmed with NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] which caused recurrence of WDS.

Hypoxia-induced synthesis of respiratory nitrate reductase 2 of Streptomyces coelicolor A3(2) depends on the histidine kinase OsdK in mycelium but not in spores.

The three nitrate reductases (Nar) of the saprophytic aerobic actinobacterium Streptomyces coelicolor A3(2) contribute to survival when oxygen becomes limiting. In the current study, we focused on synthesis of the Nar2 enzyme, which is the main Nar enzyme present and active in exponentially growing mycelium. Synthesis of Nar2 can, however, also be induced in spores after extended periods of anoxic incubation. The osdRK genes (oxygen stress and development) were recently identified to encode a two-component system important for expression of the nar2 operon in mycelium. OsdK is a predicted histidine kinase and we show here that an osdK mutant completely lacks Nar2 enzyme activity in mycelium. Recovery of Nar2 enzyme activity was achieved by re-introduction of the osdRK genes into the mutant on an integrative plasmid. In anoxically incubated spores, however, the osdK mutant retained the ability to synthesize NarG2, the catalytic subunit of Nar2. We could also demonstrate that synthesis of NarG2 in spores occurred only under hypoxic conditions; anoxia, as well as O2 concentrations significantly higher than 1 % in the gas-phase, failed to result in induction of NarG2 synthesis. Together, these findings indicate that, although Nar2 synthesis in both mycelium and spores is induced by oxygen limitation, different mechanisms control these processes and only Nar2 synthesis in mycelium is under the control of the OsdKR two-component system.

Is Ulva sp. able to be an efficient biofilter for mariculture effluents?

Nitrogenous compounds such as ammonia, nitrate, and dissolved organic nitrogen (DON) are the main waste components of marine fish pond effluents. These compounds are also regarded as the primary nitrogen sources for seaweed. Aiming at designing an efficient and cost-effective extractive biofilter for fishpond effluents, Ulva lactuca performance and the dynamics of dissolved inorganic (DIN) and DON uptake by this alga grown in the effluents of a land-based integrated multi-trophic aquaculture (IMTA) system were studied. Stocking densities of 1 and 3 kg m−2 were found to be optimal for yield along with specific growth rate (SGR) and protein content, respectively. The presence of ammonia inhibited nitrate uptake by U. lactuca and carbon limitation reduced SGR and yield. However, protein levels of U. lactuca tissue in a carbon-limited situation were higher than when unlimited carbon was made available. When compared with 3 kg m−2 stocking densities, the high C/N ratio in U. lactuca tissue cultured at 1 kg m−2 likely indicated carbon limitation. Ammonia assimilation rate was density dependent. At 1 kg m−2, ammonia uptake was relatively fast, at 4.31 μmole N h−1, and nitrate uptake started only 24 h after ammonia depletion, suggesting this period to be the time required for nitrate reductase (NR) synthesis in the algae tissue. At 2 kg m−2, ammonia uptake was 2.51 μmole N h−1 and nitrate uptake started 24 h after that observed in 1 kg m−2, suggesting that the lower ammonia threshold for uptake by the U. lactuca is around 1.4 μmole L−1. Contrary to 1 and 2 kg m−2 stocking densities, in the 3 kg m−2 stocking density, ammonia uptake was as low as 1.51 μmole N h−1 and no uptake of nitrate appeared to have taken place due to the presence of ammonia in the water. The additional ammonia in the water was found to be due to DON-ammonifying bacteria on the surface of U. lactuca thalli. In the 1ow stocking density, the additional ammonia was relatively low compared to that measured at the high algae density. In the light of the better understanding of the system dynamics achieved in our study, we hypothesize that a set of similar bioreactors using U. lactuca can intensify the system purification efficiency manifold.

Interactive effect of nitrogen nutrition, nitrate reduction and seasonal variation on oxalate synthesis in leaves of Napier-bajra hybrid (Pennisetum purpureum × P. glaucum)

Oxalate may cause hypocalcaemia or formation of urinary calculi in animals with prolonged grazing of Napier grass (Pennisetum purpureum) × pearl millet (bajra, P. glaucum) hybrid (NBH). We investigated the influence of nitrate metabolism, nitrogen (N) nutrition, N forms and seasonal variation on oxalate accumulation in leaves of NBH in a field experiment in Ludhiana, India. The experiment was a randomised block design with three N sources (nitrate, amide and ammonium), three application rates (50, 75 and 100 kg N/ha), four seasons (summer, monsoon, autumn, pre-winter) and three replicates. Applied N nutrition induced oxalate synthesis and activities of nitrate reductase (NR) and nitrite reductase (NiR) enzymes. A positive association of N nutrition with both oxalate accumulation and nitrate-reducing enzymes was found. Nitrate-N increased oxalate accumulation and NiR activity more than ammonium and amide. A differential effect of seasons on NR and NiR activities, as well as on oxalate accumulation, was observed. Among different harvest seasons, NR and NiR activities were positively associated with oxalate accumulation in summer and the monsoon season. These results suggest that N fertilisation, particularly in nitrate form, is associated with upregulation of nitrate-reducing enzymes, leading to oxalate accumulation in NBH leaves.

Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis.

Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets.

Studies on pine ectendomycorrhizae in nurseries

Fungus isolates from ectendomycorrhizae of <i>Pinus sylvestris</i> seedlings showed uniform morphological, physiological and mycorrhizal features. Studies on hyphal ultrastucture suggest that the fungus is an <i>Ascomyceles</i>. Growth of cultures in vitro and formation of ectendoroycorrhizae in the nursery were stimulated both by Ca compounds and urea. Strains of the fungus showed considerable ability to synthesize nitrate reductase. The Studies provided evidence of a permanent occurrence of N<sub>2</sub>-fixing <i>Bacillus polymyxa</i> in the ectendomycorrhizosphere.

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg2+ contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants.

Influence of Salinity on the Nitrogen Metabolism of Cordyline fruticosa

Irrigated agriculture depends on adequate and quality water supplies. As the level of salt increases in an irrigation source, the quality of that water for plant growth decreases. Crop plants are usually nonhalophytes that tolerate only moderate salt concentrations. Under salinity, they accumulate salt in their aboveground organs and, to a smaller extent, in roots. High salinity reduces plant growth and leaf area, which prejudices quality, especially in ornamental crops. The study of the behavior of ornamental plants that are tolerant to saline waters can be an advantage in areas with poor quality waters. The aim of this work is to study the influence of the fertigation water salinity on the nitrogen metabolism of Cordyline fruticosa var. ‘Red Edge,’ known as ti plant, Hawaiian ti, cordyline, or good luck plant. Four nutrient solutions with different electrical conductivity (EC) levels were applied: T1 (1.5 dS m−1), T2 (2.5 dS m−1), T3 (3.5 dS m−1), and T4 (4.5 dS m−1), obtained with the application of different amounts of sodium chloride (NaCl) to the fertigation water. As salinity increases, nitrogen concentration in leaves does not change, but it increases in roots. In leaves, nitrate reductase (NR) in vivo activity declines when salinity increases; when a substrate [nitrate (NO3 −)] or a cofactor [molybdenum (Mo)] is added, NR activity increases. Potential NR activity (NO3 − + Mo) shows no significant differences between treatments; this indicates that NR synthesis is not affected by salinity. So, the diminution of the NR in vivo activity suggests a NO3 − and Mo level metabolic imbalance. In roots, Mo is more restrictive of the NR activity than the NO3 − substrate level. There are more amino acids in T1 than in T4, but it seems that there are no significant differences in T2 and T3. No significant differences are observed in the protein content; this indicates that amino acid diminution is due to the maintenance of the protein content. In conclusion, it can be said that the increased activity of NR in roots may constitute part of an adaptation strategy of the plant to an increasing salinity in the growing medium.

Catabolite Repression Control ofnapF(Periplasmic Nitrate Reductase) Operon Expression inEscherichia coliK-12

Escherichia coli, a facultative aerobe, expresses two distinct respiratory nitrate reductases. The periplasmic NapABC enzyme likely functions during growth in nitrate-limited environments, whereas the membrane-bound NarGHI enzyme functions during growth in nitrate-rich environments. Maximal expression of the napFDAGHBC operon encoding periplasmic nitrate reductase results from synergistic transcription activation by the Fnr and phospho-NarP proteins, acting in response to anaerobiosis and nitrate or nitrite, respectively. Here, we report that, during anaerobic growth with no added nitrate, less-preferred carbon sources stimulated napF operon expression by as much as fourfold relative to glucose. Deletion analysis identified a cyclic AMP receptor protein (Crp) binding site upstream of the NarP and Fnr sites as being required for this stimulation. The napD and nrfA operon control regions from Shewanella spp. also have apparent Crp and Fnr sites, and expression from the Shewanella oneidensis nrfA control region cloned in E. coli was subject to catabolite repression. In contrast, the carbon source had relatively little effect on expression of the narGHJI operon encoding membrane-bound nitrate reductase under any growth condition tested. Carbon source oxidation state had no influence on synthesis of either nitrate reductase. The results suggest that the Fnr and Crp proteins may act synergistically to enhance NapABC synthesis during growth with poor carbon sources to help obtain energy from low levels of nitrate.

Kinetics of uptake of phosphates and nitrates by marine multicellular algae Gelidium latifolium (Grev.) Born. et Thur.

We studied nonstationary kinetics of the uptake of phosphates and nitrates by the red marine algae Gelidium latifolium (Grev.) Born et Thur. and calculated constants of the Michaelis-Menten equation for these elements. In the area of 0-3 microM, the kinetics of phosphate consumption had the following coefficients: maximum rate of uptake 0.8 micromol/(g x h), constant of half-saturation 1.745 microM. For nitrate nitrogen at 0-30 microM, an adaptive strategy of uptake kinetics was noted with change of the equation parameters with time: after 1 h, the maximum rate of uptake was 5.1 micromol/(g x h) and constant of half-saturation 19 gM, while within 2 h, the maximum rate of uptake significantly increased. This could be related to the synthesis of nitrate reductase. Coupled with the uptake of nitrates, nonstationary kinetics of the release of nitrates in the surrounding medium had a one-peak pattern: the maximum concentration of nitrites in the medium and the time of its achievement increased with the initial concentration of nitrates. The maximum concentration of nitrites was 6 to 14% of the initial concentration in the medium.

Structured model for denitrifier diauxic growth

We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.

Molecular characterization and transcriptional regulation of nitrate reductase in a ruminal bacterium, Selenomonas ruminantium.

Nitrate reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced. The 6.4 kbp nar gene consisted of narG, H, J, and I in this order. The deduced amino acid sequences of these subunits resembled those of membrane-bound nitrate reductase-A reported for Escherichia coli. It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon). The level of intracellular nar-mRNA was higher when S. ruminantium was grown with nitrate than when grown without nitrate, suggesting that nar transcription is enhanced by nitrate. The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply. Therefore, NaR synthesis in S. ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons. S. ruminantium reduced nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription. Addition of fumarate stimulated nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction.

Carbon supply and 2-oxoglutarate effects on expression of nitrate reductase and nitrogen-regulated genes in Synechococcus sp. strain PCC 7942

Synthesis of nitrate reductase in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942 took place at a slow rate when the cells were incubated without a supply of inorganic carbon, but addition to these cells of CO 2/bicarbonate or, in a Synechococcus strain transformed with a gene encoding a 2-oxoglutarate permease, 2-oxoglutarate stimulated expression of the enzyme. Induction by 2-oxoglutarate was also observed at the mRNA level for two nitrogen-regulated genes, nir and amt1, but not for the photosystem II D1 protein-encoding gene psbA1. Our results are consistent with a role of 2-oxoglutarate in nitrogen control in cyanobacteria.

A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

A cytochrome c (NarC) is encoded as the first gene of the operon for nitrate respiration in Thermus thermophilus. NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR). The α and δ subunits (NarG, NarJ) of the NR were constitutively expressed in narC::kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes. Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T. thermophilus.

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters.

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413.

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.

Cloning, sequencing and characterization of Fnr from Klebsiella pneumoniae.

The transcription factor Fnr (fumarate nitrate reductase regulator) globally regulates gene expression in response to oxygen deprivation in Escherichia coli. We report here the cloning and sequencing of the fnr gene from the facultative anaerobic bacterium Klebsiella pneumoniae M5al, another member of the enteric bacteria. The deduced amino acid sequence of K. pneumoniae fnr showed very high similarity (98% amino acid identity) to the Fnr protein from E. coli and contained the four essential cysteine residues which are presumed to build the oxygen-sensing [4Fe4S]+2 center. Transfer of the K. pneumoniae gene to a fnr mutant of E. coli complemented the mutation and permitted synthesis of nitrate reductase and fumarate reductase during anaerobic growth. A gene fusion between K. pneumoniae fnr and glutathione S-transferase was constructed and expressed in E. coli under anaerobic conditions in order to make the protein available in preparative amounts. The overproduced protein was purified by glutathione-Sepharose 4B affinity chromatography in the absence of oxygen, and biochemically characterized.

Novel growth characteristics and high rates of nitrate reduction of an Escherichia coli strain, LCB2048, that expresses only a periplasmic nitrate reductase.

Escherichia coli strain LCB2048 is a double mutant defective in the synthesis of the two membrane-associated nitrate reductases A and Z. This strain can grow anaerobically on a non-fermentable carbon source, glycerol, in the presence of nitrate even in media supplemented with high concentrations of tungstate. This growth was totally dependent upon a highly active, periplasmic nitrate reductase (Nap). Due to the presence of a previously unreported narL mutation, synthesis of the periplasmic nitrate reductase by this strain was induced during anaerobic growth by nitrate. We have also demonstrated that methyl viologen is an ineffective electron donor to Nap: its use leads to an underestimation of the contribution of Nap activity to the rate of nitrate reduction in vivo.