Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity.

Nitrous oxide production by Escherichia coli seems to result from the reduction of NO2- by NO3- reductase. This hypothesis is consistent with previous observations and with the observation that molybdenum was required for both NO3- reduction and N2O production. Several E. coli NO3- reductase mutants were assayed for both N2O-producing and NO3--reducing activity. The hypothesized role of NO3- reductase is supported by the correlation of these two activities. Nitrate reduction to NH4+ enhanced growth, but NO2- reduction to N2O apparently did not. Therefore, this process differs significantly from respiratory denitrification.     

Dissimilatory Reduction of NO(2) to NH(4) and N(2)O by a Soil Citrobacter sp

Dissimilatory reduction of NO(2) to N(2)O and NH(4) by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N(2)O production by this mechanism. In batch cultures with defined media, NO(2) reduction to NH(4) was favored by high glucose and low NO(3) concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO(3) concentrations. With succinate as the energy source, little or no NO(2) was reduced to NH(4) but N(2)O was produced. Resting cell suspensions reduced NO(2) simultaneously to N(2)O and free extracellular NH(4). Chloramphenicol prevented the induction of N(2)O-producing activity. The K(m) for NO(2) reduction to N(2)O was estimated to be 0.9 mM NO(2), yet the apparent K(m) for overall NO(2) reduction was considerably lower, no greater than 0.04 mM NO(2). Activities for N(2)O and NH(4) production increased markedly after depletion of NO(3) from the media. Amendment with NO(3) inhibited N(2)O and NH(4) production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH(4) but not of N(2)O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N(2)O at rates equal to the wild type. These observations suggest that N(2)O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO(2) to NH(4).     

Production of nitrous oxide from nitrite in Klebsiella pneumoniae: mutants altered in nitrogen metabolism.

Under anaerobic conditions, Klebsiella pneumoniae reduced nitrite (NO2-), yielding nitrous oxide (N2O) and ammonium ions (NH4+) as products. Nitrous oxide formation accounted for about 5% of the total NO2- reduced, and NH4+ production accounted for the remainder. Glucose and pyruvate were the electron donors for NO2- reduction to N2O by whole cells, whereas glucose, NADH, and NADPH were found to be the electron donors when cell extracts were used. On the one hand, formate failed to serve as an electron donor for NO2- reduction to N2O and NH4+, whereas on the other hand, formate was the best electron donor for nitrate reduction in either whole cells or cell extracts. Mutants that are defective in the reduction of NO2- to NH4+ were isolated, and these strains were found to produce N2O at rates comparable to that of the parent strain. These results suggest that the nitrite reductase producing N2O is distinct from that producing NH4+. Nitrous oxide production from nitric oxide (NO) occurred in all mutants tested, at rates comparable to that of the parent strain. This result suggests that NO reduction to N2O, which also uses NADH as the electron donor, is independent of the protein(s) catalyzing the reduction of NO2- to N2O.     

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 microM 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.     

Detergent inhibition of nitric-oxide reductase activity

Gas chromatography revealed that exposure of extracts of the denitrifiers 'Achromobacter cycloclastes', Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N2O, and thus concomitantly inhibited reduction of NO2- to N2O. After exposure of extracts to Triton X-100, the ratio of H+ consumed to NO2- added decreased from approx. 2.0 (for untreated extracts) to approx. 1.5, which indicated that NO2- was reduced to NO by the treated extracts. Addition of a CHAPS-soluble extract (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P. denitrificans restored capacity for reduction of NO2- on to N2O. Exposure to either the NO that accumulated from reduction of NO2- or to enthetic NO transiently inhibited rates of NO2- reduction in Triton X-100-treated extracts. Use of an Oxides of Nitrogen analyzer indicated that only 5-33% of NO2- reduced by untreated extracts appeared in the stripping gas as NO, whereas 80-95% of NO2- reduced by Triton X-100-treated extracts was recovered as NO.     

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation.

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.     

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures.

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Temporary low oxygen conditions for the formation of nitrate reductase and nitrous oxide reductase by denitrifying Pseudomonas sp. G59

Formation of nitrate reductase (NaR) and nitrous oxide reductase (N2OR) by a Pseudomonas sp. G59 did not occur in aerobic or anaerobic conditions, but was observed in a microaerobic incubation in which an anaerobically grown culture was agitated in a sealed vessel initially containing 20 kPa oxygen in the headspace. During the microaerobic incubation, the oxygen concentration in the headspace decreased and dissolved oxygen reached 0.1-0.2 kPa. NaR activity was detected immediately and N2OR activity after 3 h of incubation irrespective of the presence or absence of NO3- or N2O. In the presence of NO3-, NO2- was accumulated as a major product, but N2O was observed in low concentrations only after N2OR appeared. After microaerobic incubation for 3 h, N2OR formation continued even anaerobically in an atmosphere of N2O. In contrast, Escherichia coli formed NaR not only microaerobically but also anaerobically. However, NaR formation by E. coli was inhibited by sodium fluoride under anaerobic, but not under microaerobic conditions. The Pseudomonas culture did not possess fermentative activity. It is suggested that the dependence on microaerobiosis for the formation of these reductases by the Pseudomonas culture was due to an inability to produce energy anaerobically until these anaerobic respiratory enzymes were formed.     

Improvement of protoplast regeneration by growing yeast cells hypertonically

N2O was produced during the reduction of NO2- by resting cells of Lactobacillus lactis TS4. At an initial NO2- concentration of 69 micrograms/ml, the rate of N2O production was 1.97 nmol/min per mg of protein, and the recovery of reduced NO2- -N as N2O-N after 24 h was 77%. Higher initial NO2- concentrations decreased both the rate of production of N2O and the recovery of reduced NO2- -N. CO2 production increased during NO2- reduction.     

Production and consumption of nitric oxide by three methanotrophic bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N(2)O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 x 10(-17) mol of NO cell(-1) day(-1), mostly after a culture became O(2) limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O(2), and required CH(4). Denitrification (methanol-supported N(2)O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd(1) and Cu nitrite reductases, NO reductase, and N(2)O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O(2) and nitrate availability occur.     

Improvement of Protoplast Regeneration by Growing Yeast Cells Hypertonically

N2O was produced during the reduction of NO2- by resting cells of Lactobacillus lactis TS4. At an initial NO2- concentration of 69 micrograms/ml, the rate of N2O production was 1.97 nmol/min per mg of protein, and the recovery of reduced NO2- -N as N2O-N after 24 h was 77%. Higher initial NO2- concentrations decreased both the rate of production of N2O and the recovery of reduced NO2- -N. CO2 production increased during NO2- reduction.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Nitric oxide homeostasis in Salmonella typhimurium: roles of respiratory nitrate reductase and flavohemoglobin

Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO(2)(-)) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO(2)(-) of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO(2)(-) by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.     

Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem

Quantification of harmful nitrous oxide (N(2)O) emissions from soils is essential for mitigation measures. An important N(2)O producing and reducing process in soils is denitrification, which shows deceased rates at low pH. No clear relationship between N(2)O emissions and soil pH has yet been established because also the relative contribution of N(2)O as the denitrification end product decreases with pH. Our aim was to show the net effect of soil pH on N(2)O production and emission. Therefore, experiments were designed to investigate the effects of pH on NO(3)(-) reduction, N(2)O production and reduction and N(2) production in incubations with pH values set between 4 and 7. Furthermore, field measurements of soil pH and N(2)O emissions were carried out. In incubations, NO(3)(-) reduction and N(2) production rates increased with pH and net N(2)O production rate was highest at pH 5. N(2)O reduction to N(2) was halted until NO(3)(-) was depleted at low pH values, resulting in a built up of N(2)O. As a consequence, N(2)O:N(2) production ratio decreased exponentially with pH. N(2)O reduction appeared therefore more important than N(2)O production in explaining net N(2)O production rates. In the field, a negative exponential relationship for soil pH against N(2)O emissions was observed. Soil pH could therefore be used as a predictive tool for average N(2)O emissions in the studied ecosystem. The occurrence of low pH spots may explain N(2)O emission hotspot occurrence. Future studies should focus on the mechanism behind small scale soil pH variability and the effect of manipulating the pH of soils.     

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

Microscale biosensor for measurement of volatile fatty acids in anoxic environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO(-)(3) to N(2)O. A culture of denitrifying bacteria deficient in N(2)O reductase was immobilized in front of the tip of an electrochemical N(2)O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N(2)O sensor. The signal of the sensor, which corresponded to the rate of N(2)O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O(2), N(2)O, NO(2)(-), H(2)S, and NO(-)(3). Interference from NO(-)(3) was low and could be quantified and accounted for. The detection limit was equivalent to about 1 microM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32 per thousand. The functioning of the sensor over a temperature span of 7 to 30 degrees C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5 degrees C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.