Constitutive nitrate- and nitrite reductase activities of Rhizobium in relation to denitrification

Nitrate reductase (NR) activity of R. meliloti SU 47 grown in yeast extract mannitol (YM) medium not containing any nitrate was highest during early stationary phase of growth. The strain did not express nitrite reductase (NiR) activity under this condition. In this medium stationary phase cells of some strains of rhizobia expressed nitrite reductase activity and produced gas from nitrate or nitrite in Durham tubes under anaerobic condition in 72 hrs. The strain SU 47, although, did not produce any gas in 72 hrs, did so and expressed NiR activity when grown for 8 days under anaerobic condition. The data indicated a positive correlation between gas production and NiR activity.     

Assimilatory nitrate reductase in a chlorate-resistant mutant of Escherichia coli

Nitrate reductase was investigated in extracts from cells of a chlorate-resistant mutant strain of E. coli which grew anaerobically on nitrate as the sole source of nitrogen. The nitrate reductase was of particulate nature and reduced chlorate like the nitrate reductase from the wild strain, but in contrast was inhibited only weakly by azide or cyanide. Nitrate reductase activity was found in extracts from the mutant cells grown on nitrate as the sole source of nitrogen, but not in extracts from cells grown in complex nutrient medium. Addition of ammonia also caused a decrease in activity. Accordingly, the nitrate reductase in the chlorateresistant mutant is of the assimilatory type.     

Regulation of nitrate uptake and nitrite efflux in the cyanobacterium Nostoc MAC

Nitrate uptake, nitrite efflux and their regulation have been studied in the cyanobacterium Nostoc MAC. Nitrate uptake as well as nitrite-assimilation-dependent nitrite efflux systems consisted of two distinct phases comprising an initial rapid phase followed by a slower one. Whereas, 3-(3,4-dichlorophenyl)-1-1 dimethyl urea (DCMU), an inhibitor of photosystem II inhibited both nitrate uptake and nitrite efflux significantly, exogenous supply of ATP, however, stimulated both the processes, suggesting that PS II-mediated energy generation plays vital role in regulating both nitrate uptake as well as nitrite efflux. The inhibition of both the processes by p-chloromercuribenzoate (pCMB)¹, N,N dicyclohexylcarbodiimide (DCCD) and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) indicated the involvement of -SH groups and ATP hydrolysis in the regulation of both the processes. Tungstate-treated cells having nonfunctional nitrate reductase although had a significant level of nitrate uptake, yet nitrite efflux by these cells was found to be negligible. These results suggest that (i) nitrate uptake and nitrite efflux processes in Nostoc MAC are energy-dependent; (ii) assimilation of nitrate via nitrate reductase is necessarily required for nitrite efflux to occur; (iii) nitrate uptake and nitrite efflux processes are regulated at different levels by different metabolic inhibitors; and (iv) cations of larger ionic radius facilitate the nitrate uptake and nitrite efflux processes.     

Co-ordination of the nitrate and nitrite assimilation,the glutathione and free radical metabolisms,and the pentose phosphate pathway in Penicillium chrysogenum

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO₃^— and NO₂^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H₂O₂, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.     

Aerobic inhibition of nitrate assimilation in Escherichia coli

Growth of Escherichia coli, strain K-10, in synthetic medium with nitrate as a sole source of nitrogen was not found under aerobic conditions, but found in the medium under anaerobic conditions as well as in synthetic medium with ammonium or nitrite under both aerobic and anaerobic conditions. The presence of nitrate reductase activity in cells indicates that the growth inhibition is due to the inhibition of the enzyme by oxygen and not due to its absence. Conditions of preculture, inoculation size, pH, and composition of the nitratesynthetic medium did not affect the inhibition. All the 37 strains of E. coli tested were grown in the nitrate medium only anaerobically, while 18 strains of Klebsiella-Aerobacter among 22 strains tested were grown in the nitrate medium both aerobically and anaerobically.     

The expression profile of the <Emphasis Type="Italic">Tuber borchii</Emphasis> nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.     

Effect of nitrate reduction on metabolic products and growth of Propionibacterium acidi-propionici

From a culture of P. acidi-propionici (formerly P. arabinosum) in a medium containing lactate and nitrate, a strain with high activity of nitrate reduction was isolated and used in this study. The growth rates on lactate and glucose were not significantly affected by nitrate under anaerobic and aerobic conditions. Addition of nitrate resulted in a decrease of propionate and an increase of acetate and pyruvate produced from lactate and glucose with accumulation of nitrite. In the reaction of cell suspensions in the absence of added nitrogeneous nutrient, a similar result was obtained. Accumulation of large amounts of polysaccharide observed in the presence of glucose under these conditions was not affected by the addition of nitrate. The molar growth yield on lactate but not on glucose in a defined medium was increased in the presence of nitrate. In cell suspensions glycerol, glucose, lactate, and propionate were utilized as electron donors for nitrate reduction to nitrite. Nitrate reduction was also found in cells grown in the absence of nitrate. Cytochrome b, found in the organism, was reduced by lactate and oxidized by nitrate or by fumarate in cell suspensions and in cell-free extracts.     

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47 was studied. In the presence of cyanate assimilatory nitrate reduction activity appeared in the culture after a long lag and a conspicuously low level of nitrite was accumulated. Dissimilatory nitrate reduction activity was low in the intact cells precultured in KCNO of KNO₃ as compared to that in the control. Dissimilatory nitrate reductase activity in the extracts of cells precultured in KCNO was also compared to be low. In the extracts, dissimilatory nitrate reductase activity assayed with increasing concentrations of KCNO confirmed that KCNO acts as an inhibitor of the enzyme.     

The Relationship between Gastric Juice Nitrate/Nitrite Concentrations and Gastric Mucosal Surface pH

Purpose

To investigate gastric juice nitrate/nitrite concentration according to mucosal surface pH extent (area) of gastric corpus intimately contacting the gastric juice.

Materials and Methods

We included ninety-nine patients with dyspepsia. To evaluate gastric mucosal surface pH and its extent, gastric chromosocpy was performed by spraying phenol red dye on the corpus mucosa and estimating the extent of area with color changed. Nitrate/nitrite concentrations and pH of gastric juice were measured by ELISA and pH meter, respectively. Silver staining was done to histologically confirm the presence of Helicobacter pylori.

Results

Intragastric nitrate/nitrite concentrations in patients, showing phenol red staining mucosa were higher than those of unstaining mucosa (p=0.001): the more extensive in the area of phenol red staining area of corpus, the higher gastric juice pH found (r=0.692, p<0.001). Furthermore, the intragastric nitrate/nitrite concentrations correlated positively with gastric juice pH (r=0.481, p<0.001).

Conclusion

The changes of mucosal surface pH and its extent in gastric corpus might affect either pH or nitrate/nitrite level of gastric juice.     

Utilization of nitrate or nitrite as single nitrogen source by Mycobacterium avium.

Twenty L-amino acids and several inorganic compounds were tested individually, as a sole nitrogen source, for ability to support the growth of Mycobacterium avium LM1 serovar 1. Of the amino acids tested, only L-glutamine provided nutritional support comparable to that of ammonium chloride at 1 mM. With either 1 mM potassium nitrate or nitrite substituted for ammonium chloride, similar numbers of CFU were produced. M. avium cells were grown in potassium nitrate or nitrite concentrations of 0.25, 0.5, 1.0, and 2.0 mM, and the medium was assayed for remaining nitrogen compound at several times during growth. Rates of utilization were of first-order kinetics, with nitrite removed more rapidly than nitrate. The rates were approximately 10 times as rapid at 0.25 mM than at 2 mM for either nitrogen source. Nine clinical isolates that included M. avium serovars 1, 4, and 8 and Mycobacterium scrofulaceum serovar 43 were tested for rate of utilization of ammonia, nitrate, or nitrite. Ammonia and nitrite were utilized with first-order kinetics by all strains. Nitrate utilization occurred but was not at the same level for all strains. Clinical tests indicate that M. avium is negative for nitrate reductase; this is because of the rapid reduction of nitrite produced from nitrate.     

Purification and immunologic reactivity ofHymenolepis diminuta surface antigens

The outer part ofHymenolepis diminuta tegument was extracted with 3m KCl. The antigen was adsorbed from the extract on an affinity column containingH. diminuta-infected rat immunoglobulin immobilized on Sepharose 4B and could be eluted with 2.5m urea at pH 2.8. Analysis of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of both the crude extract and material eluted from the column showed that the latter was markedly purified. Double diffusion and immunoelectrophoresis revealed 11 and 4 antigenic components in the crude extract and purified preparation, respectively.     

Nitrate- and nitrite-reducing bacteria in the achlorhydric stomach

The microbial composition of samples of gastric juice from eight achlorhydric patients was determined by aerobic and rigorously anaerobic culture techniques. Bacteria from 16 genera were commonly isolated, but representatives of only three genera, (streptococci, neisseriae and haemophili) were isolated from every patient. Nitrate and nitrite were both reduced by veillonellae, haemophili, staphylococci, corynebacteria, lactobacilli, flavobacteria and fusobacteria, but the potential rate of nitrate reduction by suspensions of veillonellae, Haemophilus parainfluenzae and members of the Enterobacteriaceae were up to ten times more rapid than the rate of nitrite reduction. Conversely, although all Neisseria spp. reduced nitrite only some strains reduced nitrate. Streptococci did not reduce nitrate. Streptococcus sanguis reduced nitrite when grown with haematin; other streptococci did not reduce nitrite. Bacterial nitrate and nitrite reduction were active over the pH range 6-8, similar to the pH range of the achlorhydric stomach. From a knowledge of the composition of the bacterial flora and their potential rates of nitrate and nitrite reduction under prevailing conditions, predictions were made about the tendency of nitrite to accumulate during nitrate reduction. Studies of the transient accumulation of nitrite by mixed cultures of H. parainfluenzae and N. subflava were consistent with these predictions. Haemophili and veillonellae could be responsible for the accumulation of nitrite in the gastric juice of some patients, whereas streptococci and neisseriae would tend to remove nitrite from the stomach as rapidly as it formed.     

Untersuchungen über die Nitritreduktion bei Bacillus cereus

Zusammenfassung Die dissimilatorische Nitritreduktion intakter Zellen und zellfreier Extrakte von Nitrat-adaptierten Zellen von Bac. cereus wurde untersucht. Als einziges lösliches Folgeprodukt wurde Ammoniak nachgewiesen, gasförmige Folgeprodukte werden nicht gebildet. Verschiedene Intermediate des Kohlenhydratabbaus sind als Elektronendonatoren der Nitritreduktion intakter Zellen geeignet. Aus Glucose wird anaerob CO₂ in stöchiometrischem Verhältnis zur eingesetzten Nitrat- oder Nitritmenge gebildet. Die zellfreie Nitritreduktion kann nur NADH und NADPH als Elektronendonatoren verwenden. Aus dem unterschiedlichen Spektrum der Elektronendonatoren von Nitrat- und Nitritreduktion und dem unterschiedlichen Verhalten der beiden Enzyme gegenüber spezifischen Inhibitoren werden vorläufige Schlüsse auf die Kopplung der Nitritreduktase mit den Cytochromen gezogen. Die zellfreie Nitritreduktion wird durch Kupfer(II)-Ionen in kleiner Konzentration gehemmt.

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Summary Cells ofBac. cereus ATCC8035 grown anaerobically in the presence of nitrate contain a dissimilatory nitrite reductase, which under anaerobic conditions reduces nitrite to ammonia in intact cells and cell free extracts. Other soluble intermediates or nitrogen containing gaseous products could not be detected.In an anaerobic system composed of intact cells, glucose and limiting amounts of nitrate or nitrite, 1Mol CO₂ perMol nitrate and 0.5Mol CO₂ perMol nitrite are liberated, suggesting that nitrate and nitrite oxygen is available for glucose oxidation only to the hyponitrite level.Among several inhibitors tested in a cell free nitrite reducing system with NADH as electron donor, the inhibition by Cu^II-ions at low concentrations (2 to 6×10^–6 M) is remarkable.

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Die Arbeit wurde durch Mittel der Deutschen Forschungsgemeinschaft und der Strebelstiftung für Krebs- und Scharlachforschung unterstützt.     

Dissimilatory nitrate reduction in Clostridium tertium

Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium. Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation. The effect was even more dramatic in C. tertium than in C. perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter Unlike C. perfringens, C. tertium responded to the presence of nitrate in the medium with an increased growth rate. A slight increase in the Y_ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of STOUTHAMMER and BETTENHAUSSEN that Y_ATP will vary with the growth rate Thus, C. tertium, like C. perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this trait may be widespread among the nitrate-reducing anaerobes.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

Effect of bioactive fractions of <Emphasis Type="Italic">Citrullus vulgaris</Emphasis> Schrad. leaf extract against <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Aedes aegypti</Emphasis>

The benzene extract of Citrullus vulgaris was tested against Anopheles stephensi and Aedes aegypti for the larvicidal activity and ovicidal properties. The crude benzene extract was found to be more effective against A. stephensi than A. aegypti. The LC₅₀ values were 18.56 and 42.76 ppm respectively. The LC₅₀ values for silica gel fractions (bioactive fractions I, II, III and IV) were 11.32, 14.12, 14.53 and 16.02 ppm respectively. The mean per cent hatchability of the egg rafts were observed after 48 h post treatment. The crude extract of benzene exerted 100% mortality at 250 ppm against A. stephensi and at 300 ppm against A. aegypti. The silica gel fractions I and II afforded 100% mortality at 100 ppm and III and IV exerted the hatchability rate of 4.9 and 5.3% at the same concentration against A. stephensi.     

Isolation and characterization of Russian thistle (Salsola pestifer) pollen allergens

The radioallergosorbent test (RAST) was utilized to identify allergenically active molecules in an extract of Russian thistle (Salsola pestifer) pollen. Two glycoproteins (RT₁ and RT₂) were isolated by ion-exchange chromatography, preparative fiat-bed electrofocusing, and gel filtration chromatography. These highly purified proteins were similar as judged by discontinuous polyacrylamide gel electrophoresis (PAGE), by PAGE in urea-acetate, and by immunodiffusion analysis. In Ouchterlony double-diffusion, rabbit antiserum to crude pollen extract showed a line of identity between RT₁ and RT₂. Each allergen possessed a single polypeptide chain, with molecular weights of 39,000 and 42,000 for RT₁ and RT₂, respectively. Both allergens contained 9.0% ± 0.5% carbohydrate and 10.6% ± 0.2% nitrogen. Both RT₁ and RT₂ eluted from a calibrated Sephadex G-100 column near the ovalbumin marker, but the elution volume of RT₁ was slightly greater than that of RT₂. Amino acid analysis of RT₁ and RT₂ showed that there was no difference in composition between the two proteins. The proteins showed different isoelectric points (RT₁ = 6.7; RT₂ = 6.2). They had similar skin reactivity by Prausnitz-Kustner testing, showed similar potency and identical allergenic qualities as inhibitors in the PAST, and were more potent on a mass basis than crude extract in the inhibition of the reaction between solid-phase crude extract and IgE antibodies in the PAST. RT₁ and RT2 represent 0.014% and 0.010% by mass, respectively, of the pollen. These results suggest that RT₁ and RT₂ are important allergens in Russian thistle pollen and that these proteins are immunologically identical.     

Rapid Testing for Nitrate Reductase Activity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Grown in an Automated Culture System

In order to reduce the time to detection of nitrate reductase activity, which is arguably the most widely used phenotypic trait to differentiate between Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG, the following study was conducted using cultures grown in an automated system. Automated culture systems, which are typically based on liquid medium, have greatly reduced the time-to-recovery of mycobacteria. Yet subsequent testing of isolates for nitrate reductase activity may take several weeks, because culture on solid media is required. Presented here is a procedure to obtain a final result within 24 h for nitrate reductase activity of cultures grown in an automated culture system. Using this procedure, Mycobacterium tuberculosis was rapidly differentiated from Mycobacterium bovis and Mycobacterium bovis BCG.     

The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

Clinical signs,blood gases and acid–base balance after nitrate administration in calves

This work is focused on the course and changes of selected clinical and laboratory findings after nitrate administration in seven calves. The calves were 3–6 months old, were fed a solid diet and had an average body weight of 97 kg. Nitrates were administered intraruminally in a water solution of KNO₃ at a dose of 0.5 g/kg of body weight. We observed the development and course of methaemoglobinaemia (% MtHb) in relation to the emergence of clinical findings, changes in body temperature, breathing and pulse rates, blood gases and acid–base balance. Various time trends as well as methaemoglobinaemia levels were recorded when values increased. The highest values of MtHb were found in the three youngest calves (67.5, 70.9 and 90.3 %), which were consequently treated by 1 % methylene blue solution because of marked clinical findings. Marked changes were observed in breathing and pulse rates, which correlated significantly with methaemoglobinaemia levels (P?<?0.001). Significant effect of nitrate-induced methaemoglobinaemia was found on blood pH (P?<?0.05), pO₂ (P?<?0.001) and O₂ saturation (P?<?0.01). Nitrate administration and formation of methaemoglobin did not affect the metabolic parameters of acid–base balance. Changing MtHb values correlated significantly positively with changes of blood pH (P?<?0.01), breathing and pulse rates (P?<?0.001) and negatively with pCO₂ (P?<?0.05), pO₂ and O₂ saturation (P?<?0.001). The obtained results indicate a markedly different development and course of nitrate poisoning. More distinct changes in clinical findings were not observed until the methaemoglobinaemia levels were over 60 %. Methaemoglobinaemia resulted in failure of oxygen transport and hypoxaemia with a significantly negative correlation of both parameters. The achieved results contribute to the study of acute nitrate/nitrite poisoning in cattle and supplement the present knowledge by significant correlation relationships between methaemoglobinaemia levels and some clinical signs and metabolic indices.