Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea.
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ABSTRACT:
SUBMITTER: Ritchie GA
PROVIDER: S-EPMC1178541 | biostudies-other | 1972 Mar
REPOSITORIES: biostudies-other
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Project description:Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N2O). It has been proposed that N2O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH2OH) quantitatively to N2O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH2OH to nitrite ([Formula: see text]) under aerobic conditions. Although we observe [Formula: see text] formation under aerobic conditions, its concentration is not stoichiometric with the NH2OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH2OH to N2O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an FeIII-NH2OH adduct of cyt P460 is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, forming the N-N bond of N2O during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, [Formula: see text] is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH2OH contributes to NO and N2O emissions from nitrifying microorganisms.
| S-EPMC5187719 | biostudies-literature
Project description:UnlabelledNitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production.ImportanceNitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.
| S-EPMC4959225 | biostudies-literature
Project description:Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2. IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on dilution rate and Na2CO3 concentration. IC limitation resulted in a 1.5-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2- and 6.3 fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) to 4.4% (0.25 mM Na2CO3). Transcriptome analysis showed differential expression (p ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3 oxidation from growth and increased N2O production.
2016-03-28 | GSE70988 | GEO
Project description:Nitrosomonas europaeais a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses ofN. europaeato replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3to nitrite, depending on dilution rate and Na2CO3concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (p⤠0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbbgene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest thatN. europaeaadapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3oxidation from growth and increased N2O production. Transcriptome responses of N. europaea in 60 mM NH4+ to suboptimum levels of carbonate (1.0 mM and 0.2 mM) in continuous steady-state culture in a bioreactor.
2016-03-28 | E-GEOD-70988 | biostudies-arrayexpress
Project description:Organohalide-respiring bacteria are key players for the turnover of organohalogens. At sites impacted with chlorinated ethenes, bioremediation promotes reductive dechlorination; however, stoichiometric conversion to environmentally benign ethene is not always achieved. We demonstrate that nitrous oxide (N2O), a compound commonly present in groundwater, inhibits organohalide respiration. N2O concentrations in the low micromolar range decreased dechlorination rates and resulted in incomplete dechlorination of tetrachloroethene (PCE) in Geobacter lovleyi strain SZ and of cis-1,2-dichloroethene ( cDCE) and vinyl chloride (VC) in Dehalococcoides mccartyi strain BAV1 axenic cultures. Presumably, N2O interferes with reductive dechlorination by reacting with super-reduced Co(I)-corrinoids of reductive dehalogenases, which is supported by the finding that N2O did not inhibit corrinoid-independent fumarate-to-succinate reduction in strain SZ. Kinetic analyses revealed a best fit to the noncompetitive Michaelis-Menten inhibition model and determined N2O inhibitory constants, KI, for PCE and cDCE dechlorination of 40.8 ± 3.8 and 21.2 ± 3.5 ?M in strain SZ and strain BAV1, respectively. The lowest KI value of 9.6 ± 0.4 ?M was determined for VC to ethene reductive dechlorination in strain BAV1, suggesting that this crucial dechlorination step for achieving detoxification is most susceptible to N2O inhibition. Groundwater N2O concentrations exceeding 100 ?M are not uncommon, especially in watersheds impacted by nitrate runoff from agricultural sources. Thus, dissolved N2O measurements can inform about cDCE and VC stalls at sites impacted with chlorinated ethenes.
| S-EPMC6944068 | biostudies-literature
Project description:Amt/MEP/Rh proteins are a family of integral membrane proteins implicated in the transport of NH3, CH(2)NH2, and CO2. Whereas Amt/MEP proteins are agreed to transport ammonia (NH3/NH4+), the primary substrate for Rh proteins has been controversial. Initial studies suggested that Rh proteins also transport ammonia, but more recent evidence suggests that they transport CO2. Here we report the first structure of an Rh family member, the Rh protein from the chemolithoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea. This Rh protein exhibits a number of similarities to its Amt cousins, including a trimeric oligomeric state, a central pore with an unusual twin-His site in the middle, and a Phe residue that blocks the channel for small-molecule transport. However, there are some significant differences, the most notable being the presence of an additional cytoplasmic C-terminal alpha-helix, an increased number of internal proline residues along the transmembrane helices, and a specific set of residues that appear to link the C-terminal helix to Phe blockage. This latter linkage suggests a mechanism in which binding of a partner protein to the C terminus could regulate channel opening. Another difference is the absence of the extracellular pi-cation binding site conserved in Amt/Mep structures. Instead, CO2 pressurization experiments identify a CO2 binding site near the intracellular exit of the channel whose residues are highly conserved in all Rh proteins, except those belonging to the Rh30 subfamily. The implications of these findings on the functional role of the human Rh antigens are discussed.
| S-EPMC2148281 | biostudies-literature
Project description:Estimates of regional and global freshwater N2 O emissions have remained inaccurate due to scarce data and complexity of the multiple processes driving N2 O fluxes the focus predominantly being on summer time measurements from emission hot spots, agricultural streams. Here, we present four-season data of N2 O concentrations in the water columns of randomly selected boreal lakes covering a large variation in latitude, lake type, area, depth, water chemistry, and land use cover. Nitrate was the key driver for N2 O dynamics, explaining as much as 78% of the variation of the seasonal mean N2 O concentrations across all lakes. Nitrate concentrations varied among seasons being highest in winter and lowest in summer. Of the surface water samples, 71% were oversaturated with N2 O relative to the atmosphere. Largest oversaturation was measured in winter and lowest in summer stressing the importance to include full year N2 O measurements in annual emission estimates. Including winter data resulted in fourfold annual N2 O emission estimates compared to summer only measurements. Nutrient-rich calcareous and large humic lakes had the highest annual N2 O emissions. Our emission estimates for Finnish and boreal lakes are 0.6 and 29 Gg N2 O-N/year, respectively. The global warming potential of N2 O from lakes cannot be neglected in the boreal landscape, being 35% of that of diffusive CH4 emission in Finnish lakes.
| S-EPMC7078959 | biostudies-literature
Project description:Streams and river networks are increasingly recognized as significant sources for the greenhouse gas nitrous oxide (N2 O). N2 O is a transformation product of nitrogenous compounds in soil, sediment and water. Agricultural areas are considered a particular hotspot for emissions because of the large input of nitrogen (N) fertilizers applied on arable land. However, there is little information on N2 O emissions from forest streams although they constitute a major part of the total stream network globally. Here, we compiled N2 O concentration data from low-order streams (~1,000 observations from 172 stream sites) covering a large geographical gradient in Sweden from the temperate to the boreal zone and representing catchments with various degrees of agriculture and forest coverage. Our results showed that agricultural and forest streams had comparable N2 O concentrations of 1.6 ± 2.1 and 1.3 ± 1.8 µg N/L, respectively (mean ± SD) despite higher total N (TN) concentrations in agricultural streams (1,520 ± 1,640 vs. 780 ± 600 µg N/L). Although clear patterns linking N2 O concentrations and environmental variables were difficult to discern, the percent saturation of N2 O in the streams was positively correlated with stream concentration of TN and negatively correlated with pH. We speculate that the apparent contradiction between lower TN concentration but similar N2 O concentrations in forest streams than in agricultural streams is due to the low pH (<6) in forest soils and streams which affects denitrification and yields higher N2 O emissions. An estimate of the N2 O emission from low-order streams at the national scale revealed that ~1.8 × 109 g N2 O-N are emitted annually in Sweden, with forest streams contributing about 80% of the total stream emission. Hence, our results provide evidence that forest streams can act as substantial N2 O sources in the landscape with 800 × 109 g CO2 -eq emitted annually in Sweden, equivalent to 25% of the total N2 O emissions from the Swedish agricultural sector.
| S-EPMC7027446 | biostudies-literature
Project description:Methane inhibited NH4+ utilization by Nitrosomonas europaea with a Ki of 2mM. O2 consumption was not inhibited. In the absence of NH4+, or with hydrazine as reductant, methane caused nearly a doubling in the rate of O2 uptake. The stimulation was abolished by allylthiourea, a sensitive inhibitor of the oxidation of NH4+. Analysis revealed that methanol was being formed in these experiments, with yields approaching 1 mol of methanol per mol of O2 consumed under certain conditions. When cells were incubated with NH4+ under an atmosphere of 50% methane, 50 microM-methanol was generated in 1 h. It is concluded that methane is an alternative substrate for the NH3-oxidizing enzyme (ammonia mono-oxygenase),m albeit with a much lower affinity than for methane mono-oxygenase of methanotrophs.
| S-EPMC1152006 | biostudies-other
Project description:Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.
| S-EPMC4959501 | biostudies-literature