Absence Of Nitrite Research Articles

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence, conserved residues, and proton hyperfine structure.

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S=1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (kET(intra)) in the presence of nitrite and also the apparent catalytic rate constants (kcat) of HydNIR decrease gradually with increasing pH in the range of pH 4.5–7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.

Peroxidase- and nitrite-dependent metabolism of the anthracycline anticancer agents daunorubicin and doxorubicin.

Oxidation of the anticancer anthracyclines doxorubicin (DXR) and daunorubicin (DNR) by lactoperoxidase(LPO)/H(2)O(2) and horseradish peroxidase(HRP)/H(2)O(2) systems in the presence and absence of nitrite (NO(2)(-)) has been investigated using spectrophotometric and EPR techniques. We report that LPO/H(2)O(2)/NO(2)(-) causes rapid and irreversible loss of anthracyclines' absorption bands, suggesting oxidative degradation of their chromophores. Both the initial rate and the extent of oxidation are dependent on both NO(2)(-) concentration and pH. The initial rate decreases when the pH is changed from 7 to 5, and the reaction virtually stops at pH 5. Oxidation of a model hydroquinone compound, 2,5-di-tert-butylhydroquinone, by LPO/H(2)O(2) is also dependent on NO(2)(-); however, in contrast to DNR and DXR, this oxidation is most efficient at pH 5, indicating that LPO/H(2)O(2)/NO(2)(-) is capable of efficiently oxidizing simple hydroquinones even in the neutral form. Oxidation of anthracyclines by HRP/H(2)O(2)/NO(2)(-) is substantially less efficient relative to that by LPO/H(2)O(2)/NO(2)(-) at either pH 5 or pH 7, most likely due to the lower rate of NO(2)(-) metabolism by HRP/H(2)O(2). EPR measurements show that interaction of anthracyclines and 2,5-di-tert-butylhydroquinone with LPO/H(2)O(2)/NO(2)(-) generates the corresponding semiquinone radicals presumably via one-electron oxidation of their hydroquinone moieties. The possible role of the (*)NO(2) radical, a putative LPO metabolite of NO(2)(-), in oxidation of these compounds is discussed. Because in vivo the anthracyclines may co-localize with peroxidases, H(2)O(2), and NO(2)(-) in tissues, their oxidation via the proposed mechanism is likely. These observations reveal a novel, peroxidase- and nitrite-dependent mechanism for the oxidative transformation of the anticancer anthracyclines, which may be pertinent to their biological activities in vivo.

Role of NtcB in activation of nitrate assimilation genes in the cyanobacterium Synechocystis sp. strain PCC 6803.

In Synechocystis sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units, nrtABCD-narB and nirA, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in Synechococcus sp. strain PCC 7942. The slr0395 gene, which encodes a protein 47% identical to Synechococcus NtcB, was identified as the Synechocystis ntcB gene, on the basis of the inability of an slr0395 mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While Synechococcus NtcB strictly requires nitrite for its action, Synechocystis NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the Synechococcus ntcB mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the Synechocystis ntcB mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in Synechocystis. Activities of the nitrate assimilation enzymes in the Synechocystis ntcB mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.

Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. Catalysis by nicotine and trimethylamine.

Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H2O2 and Cl(-). We have found that various (2'-deoxy)nucleosides react with HOCl to form chlorinated (2'-deoxy)nucleosides, including novel 8-chloro(2'-deoxy)guanosine, 5-chloro(2'-deoxy)cytidine, and 8-chloro(2'-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitro-guanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro- or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2'-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2'-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G-463A polymorphism of the MPO gene, which strongly reduces myeloperoxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits.

Antimicrobial and cytotoxic activity of hypochlorous acid: interactions with taurine and nitrite.

HOCl, a major bactericidal product of neutrophil MPO-halide system reacts with taurine to form taurine chloramine (TauCl), a less toxic anti-inflammatory mediator. Recently, it has been reported that HOCl may also react with nitrite (NO2-), a major end-product of nitric oxide (NO) metabolism, to form very active oxidant, nitryl chloride (NO2Cl). The present study was conducted to elucidate the effect of nitrite on bactericidal and some immunoregulatory properties of HOCl and TauCl. TauCl was prepared from NaOCl and taurine. The reaction was carried out at pH 5.0 and pH 7.4, in the presence or absence of nitrite. All reactions were monitored by UV absorption spectra. Bactericidal activity of HOCl and TauCl in the presence of nitrite was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the effect of the compounds on activity of inflammatory cells, murine peritoneal neutrophils (PMN) and macrophages were used. The cells were activated in vitro with either LPS, IFN-gamma or zymosan and the production of following mediators was measured: reactive oxygen species using luminol-dependent chemiluminescence; nitric oxide by Griess reaction; TNF-alpha using capture ELISA. In addition, we tested the effect of HOCl and TauCl on activity of myeloperoxidase (MPO). At physiological pH nitrite reacts with HOCl but not with TauCl. This reaction was abolished in the presence of taurine. Nitrite prevented HOCl-mediated bacterial killing, inhibition of MPO activity, cellular cytotoxicity and inhibition of TNF-alpha production. Nitrite did not affect any activity of TauCl. We have shown that nitrite may react in vitro with HOCl but not with TauCl, to form new biologically active product(s). We did not confirm the hypothesis that a product of HOCl reaction with nitrite is more toxic than HOCl. To the contrary, we found that nitrite diminished bactericidal and immunoregulatory properties of HOCl. In vivo, nitrite will also compete with taurine for reaction with PMN-released HOCl. Nevertheless, due to high concentration of taurine in PMN cytosol, formation of TauCl will be a major regulatory mechanism of MPO-halide-system.

Synergistic lethal combination of nitrite and acid pH on a verotoxin-negative strain of Escherichia coli O157

This study was concerned with the possible consequences of reducing the nitrite concentration of a fermented sausage environment on the survival of the pathogen E. coli O157:H45, a verotoxin-negative relative of E. coli O157:H7. A liquid medium, FM, was constructed with a liquid phase, a w and pH similar to fermented sausage. Survival of E. coli O157:H45 in FM depended on both pH and nitrite concentration. In trials in which the pH was decreased by growing Pediococcus acidilactici in FM, survival of E. coli O157:H45 was clearly dependent on nitrite concentration; at least 100 ppm nitrite was required to inhibit growth and the number of survivors after 2 days with 200 ppm nitrite was 1000-fold less than in the absence of nitrite. In laboratory-scale sausage fermented with P. acidilactici, E. coli O157:H45 failed to grow in the absence of nitrite and the numbers slowly declined over 14 days. However, the rate of decline was much faster with nitrite present even at 50 ppm; at 200 ppm nitrite, the E. coli O157:H45 population declined 100 times faster than in the absence of nitrite.

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase: ROLE OF CARBONATE ANION RADICAL

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

The pH-dependent changes of intramolecular electron transfer on copper-containing nitrite reductase.

Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3.

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

Lactoperoxidase-catalyzed oxidation of the anticancer agent mitoxantrone by nitrogen dioxide (NO2.) radicals.

Mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl] amino]-9,10-anthracenedione, MXH2] is a novel anticancer agent frequently employed in the chemotherapy of leukemia and breast cancer. Earlier studies have shown that metabolic oxidation to reactive 1,4-quinone or/and 5,8-diiminequinone intermediates may be an important mechanism of activation of this agent, pertinent to its cytotoxic action in vivo. Here we report that in the presence of nitrite ions (NO2-), MXH2 undergoes oxidation by the mammalian enzyme lactoperoxidase (LPO) and hydrogen peroxide and that the process proceeds at a rate that is proportional to NO2- concentration. In contrast, when MXH2 was exposed to LPO/H2O2 in the absence of nitrite, oxidation of the drug was either completely absent or markedly inhibited. These experiments were carried out using concentrated solutions of MXH2 (approximately 100 microM) at near neutral pH where dimers of the drug predominate. We propose that oxidation of MXH2 is mediated by an LPO/ H2O2 metabolite of NO2-, most likely the .NO2 radical. Because in mitoxantrone therapy the drug is administered intravenously, it is directly exposed to nitrogen oxides and other free radicals produced by blood components. It is therefore possible that the ability of mitoxantrone to react with the nitrogen dioxide radical may be relevant to the biological action of the drug in vivo.

Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event.

Cleavage of Cα-Cβ Bonds of Lignin Model Compounds by Nitrite and Nitric Acid

Nitrous acid treatment of kraft pulp has been proposed as a non-chlorine bleaching method. The degradation mechanism of lignin by aqueous nitric acid containing nitrite was studied using lignin model compounds. The Cα-Cβ bonds of nonphenolic β-O-4 type compounds were cleaved during the treatment with nitric acid containing nitrite to give veratraldehyde, whereas they were stable and converted to the corresponding stereo isomers in the absence of nitrite. Nitration of aromatic rings and oxidation of side chains were discussed using some monomeric model compounds. Veratrylglycerol-β-guaiacyl ether labeled with deuterium at the Cα position gave deuterium-labeled veratraldehyde, but did not give any dimeric α-carbonyl compounds. The mechanism proposed previously for acidic oxygenation was discussed further concerning the acid-promoted conversion of nonphenolic lignin units into α, β-unsaturated structures, followed by the oxidative cleavage of the Cα-Cβ bonds.

Optical nitrite sensor based on a potential-sensitive dye and a nitrite-selective carrier

A membrane responsive to nitrite has been developed which is composed of plasticized PVC, the anion carrier benzylbis(triphenylphosphine) palladium(II)chloride, and the potential-sensitive dye (PSD) rhodamine B octadecyl ester perchlorate. On exposure to nitrite, fluorescence intensity increases, while the wavelengths of both the excitation and emission maxima remain unchanged. The sensor membrane exhibits its highest sensitivity to nitrite in the 5 to 5000 mg l–1 range, and the detection limit is 0.5 mg l–1. The signal change on exposure to 100 mmol l–1 nitrite is as high as +95%. The effect of pH is significant: from pH 5.0 to pH 9.0 and in the absence of nitrite, the fluorescence intensity changes almost linearly by around –9% per pH unit. In addition, the sensor is cross-sensitive to pH: the relative signal change from plain buffer to 1 mmol l–1 nitrite is smaller by 65% at pH 9.0 than at pH 5.0. The selectivity coefficients relative to nitrite were determined by the separate solution method at pH 7.13 and were found to be 8 × 10–3 for nitrate, 1.6 × 10–3 for chloride, 8 × 10–4 for hydrogencarbonate, and 3 × 10–4 for sulfate. The lifetime of the sensor membrane is limited by leaching of the PSD, which is in the range of 1–3% h–1.

Growth of Listeria monocytogenes on vacuum-packed cooked meats: effects of pH, aw, nitrite and ascorbate

Slice of cooked meats were inoculated with Listeria monocytogenes strain Murray B, vacuum-packed and stored at either 0 or 5°C. Decreases in pH (6.9−5.9) and a w (0.993–0.960; adjusted with sodium chloride) of the cooked meats increased the lag time and reduced the growth rate at 5°C. The type of meat (beef, pork, chicken or turkey) had no effect on the growth of the organism after allowance was made for pH. Sodium tripolyphosphate (0.3%) increased growth by increasing the pH of the cooked meat. Sodium nitrite reduced the growth rate and increased the lag time. Three μM of residual undissociated nitrite doubled the time taken for a 3 log increase in numbers of L. monocytogenes. The effectiveness of nitrite was significantly increased by sodium ascorbate (0.042%). In the absence of nitrite, ascorbate had no detectable effect on growth. The extent of growth at 0°C was similarly influenced by the interaction of pH, a w, nitrite and ascorbate, and was considerably less than at 5°C. Quadratic equations were developed to describe some of the combined effects of pH, a w and residual nitrite on lag, growth rate and time for a 3 log increase in numbers of L. monocytogenes.

Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase

The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to ammonia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c-type cytochrome with a predicted M(r) for the unprocessed product of 53,788. Cleavage of the putative signal peptide at Ala-26 would result in a mature, periplasmic cytochrome of M(r) 50,580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS-polyacrylamide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in-phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In-phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping. Expression from the FNR-dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite-sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP-cAMP complex.

Effect of Sodium Nitrite on Growth of Shigella flexneri

A partial factorial design study of the effect of NaNO2 (0, 100, 200, 1000 ppm) in combination with NaCl (0.5, 2.5, 4.0%), pH (7.5, 6.5, 5.5), and temperature (37, 28, 19°C) on growth of Shigella flexneri is reported. Experiments were done aerobically in brain-heart infusion medium, using an inoculum of 1 × 103 CFU/ml. Growth curves were fitted from plate count data by the Gompertz equation; exponential growth rates, lag times, generation times, and maximum populations were derived for all variable combinations. In the absence of nitrite, the organism grew well under all test conditions at 37 and 28°C but did not grow at 19°C at pH 5.5 nor at pH 7.5 with 4% NaCl. Nitrite did not affect growth in media of pH 7.5 at 37 and 28°C. At pH 6.5 growth was inhibited by 1000 ppm NaNO2. The organism failed to grow at 19°C at all nitrite levels in the presence of 2.5 or 4.0% NaCl. The inhibitory effect of nitrite was much greater in media of pH 5.5 and increased with increasing salt levels. More inhibition was apparent at 28 than at 37°C. While lack of growth was used as a paradigm of the effect of nitrite on S. flexneri, nitrite also increased the lag and generation times and decreased the exponential growth rate. Results indicated that NaNO2 in combinations with low temperature, low pH, and high salt content can effectively inhibit the growth of S. flexneri.

EFFECTS OF NITRITE AND SULFANILAMIDE ON THE 2-THIOBARBITURIC ACID (TBA) VALUES IN AQUEOUS MODEL AND CURED MEAT SYSTEMS

The absorbance of the 2-thiobarbituric acid-malonaldehyde (TBA-MA) complex at 532 nm for aqueous model systems and cured meats were measured. Addition of sulfanilamide played a beneficial role in evaluating the oxidative state of cured meats, prepared with the addition of ≧ 100 ppm NaNO2, in that it prevented the underestimation of the TBA values. However, in the absence of nitrite, sulfanilamide may interact with malonaldehyde to produce a 1-amino-3-iminopropene derivative. Fluorescence spectroscopic studies showed an excitation maximum at 395 nm and an emission maximum at 460 nm for the adduct of malonaldehyde and sulfanilamide. Therefore, the TBA test for the evaluation of the oxidative state of cured meats may only be used when the content of nitrite in the cure or final product is known.

Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria

Operon fusion strains and mutants of Escherichia coli K-12 lacking the NADH-dependent nitrite reductase have been used to determine the regulation and physiological roles of two independent pathways for nitrite reduction to ammonia. Both the formate- and NADH-dependent pathways (Nrf and Nir, respectively) were totally repressed during aerobic growth, partially active during anaerobic growth in the absence of nitrite and further induced anaerobically by nitrite. Both were dependent upon a functional Fnr protein (a transcription activator of genes for anaerobic respiration). During anaerobic growth in the presence of nitrate, the Nir pathway was fully induced but Nrf was strongly repressed. Mutants defective in the NarL protein, which induces transcription of nitrate reductase genes but represses fumarate reductase genes in the presence of nitrate, were derepressed for Nrf activity during growth with nitrate, but the Nir enzyme was less active. The synthesis of Nrf components was also sensitive to glucose repression and weak activation by NarL during growth in the absence of nitrate. These data indicate that the Nir pathway provides a mechanism for detoxifying nitrite formed in the cytoplasm as a product of nitrate reduction. In contrast, the electrogenic reduction of nitrite by the Nrf pathway provides a secondary source of energy during anaerobic growth and is consequently repressed by the NarL protein when the thermodynamically more favourable electron acceptor, nitrate, is available. Two short DNA sequences, 5'-TACCAT-3' and 5'-CTCCTT-3', were found in the promoters of operons known to be activated or repressed by the NarL protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional control of the cysG gene of Escherichia coli K‐12 during aerobic and anaerobic growth

The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter. No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.