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Denitrification – a key process in the global nitrogen cycle and main source of the greenhouse gas N2O – is intricately controlled by O2. While the transition from aerobic respiration to denitrification is well-studied, our understanding of denitrifier communities' responses to cyclic oxic/anoxic shifts, prevalent in natural and engineered systems, is limited. Here, agricultural soil is exposed to repeated cycles of long or short anoxic spells (LA; SA) or constant oxic conditions (Ox). Surprisingly, denitrification and N2O reduction rates are three times greater in Ox than in LA and SA during a final anoxic incubation, despite comparable bacterial biomass and denitrification gene abundances. Metatranscriptomics indicate that LA favors canonical denitrifiers carrying nosZ clade I. Ox instead favors nosZ clade II-carrying partial- or non-denitrifiers, suggesting efficient partnering of the reduction steps among organisms. SA has the slowest denitrification progression and highest accumulation of intermediates, indicating less functional coordination. The findings demonstrate how adaptations of denitrifier communities to varying O2 conditions are tightly linked to the duration of anoxic episodes, emphasizing the importance of knowing an environment's O2 legacy for accurately predicting N2O emissions originating from denitrification. A soil history of constant oxygen exposure enhances N2O reduction rates under anoxia compared to a history of long or short anoxic pulses, highlighting the importance of knowing the oxygen legacy of a soil for accurate N2O emission predictions. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N2O) is a potent greenhouse gas that can be emitted during the biological treatment of wastewater. In this study, a comparison of the long-term performance characteristics and N2O production of sequencing batch reactors (SBR) and continuous stirred tank reactors (CSTR) during nitrite-based denitrification was undertaken. It was found that both reactors had NO2−-N removal efficiencies over 99.9 %, but the N2O-N emissions from the SBR reached ∼2.3 % of the removal nitrite-N load, while in the CSTR it never exceeded 0.1 %. High frequency sampling during one operation cycle of the SBR demonstrated that the N2O accumulation ratio was ∼0.1 % during the feast period, increased to ∼1.9 % in the first five hours of the famine period, and then gradually reached ∼2.3 % at the end of famine. Batch experiments showed that limiting extracellular electron donor is required for N2O accumulation in cells from the SBR-famine period and that cells from the CSTR do not accumulate N2O when either nitrite or carbon is limiting. Another notable difference in the two reactor communities was the high level of accumulation of intracellular granules, most likely polyhydroxybutyrate (PHB), in cells during the feast period in the SBR. Metagenome assembly and binning found that one genome (PRO1), which is a Thauera, accounted for over half the metagenomic reads in both reactors. Neither shifts in gene regulation nor community composition explained the observed differences in reactor performance suggesting some post-transcriptional regulation obligatorily linked to antecedent conditions underly increased N2O production in the SBR. [ABSTRACT FROM AUTHOR]

R. sphaeroides strain 2.4.3, when lacking the cbb3 oxidase, is unable to transition from aerobic respiration to denitrification using cellular respiration as a means of reducing oxygen levels. This is due to an inability to express nirK, the gene encoding nitrite reductase. Under certain photosynthetic conditions this strain can transition from aerobic to nitrate respiration, demonstrating that nirK expression can occur in the absence of a functional [cbb.sub.3] oxidase. If oxygen levels are reduced under non-photosynthetic conditions using low-oxygen gas mixes, nitrite reductase activity is detected at wild-type levels in the strain lacking the oxidase. In addition, co-culture experiments show that incubation of the [cbb.sub.3] deficient strain 2.4.3 with R. sphaeroides 2.4.1, which is nirK deficient but has the high-affinity [cbb.sub.3] oxidase, restores denitrification in sealed-vessel experiments. Taken together these results indicate that high end-point [O.sub.2] levels are the reason why the strain lacking the [cbb.sub.3] oxidase cannot transition from aerobic respiration to denitrification under certain conditions. The protein probably being affected by these [O.sub.2] levels is the transcriptional regulator NnrR. DOI 10.1099/mic.0.038703-0

Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain, nnrV, under control of a previously identified NnrR site, was also identified, paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase, paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome [c.sub.2] together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions. doi: 10.1128/JB.01026-09

Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very. low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression.

The role of cytochrome [c.sub.2], encoded by cycA, and cytochrome [C.sub.Y], encoded by cycY, in electron transfer to the nitrite reductase of Rhodobacter sphaeroides 2.4.3 was investigated using both in vivo and in vitro approaches. Both cycA and cycY were isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability of R. sphaeroides to reduce nitrite. In a cycA--cycY double mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase gene nirK from a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 [s.sup.-1]. was observed for the oxidation of cytochrome [c.sub.2] by nitrite reductase. In contrast, oxidation of [C.sub.Y] only resulted in a turnover of ~ 0.1 [s.sup.-1]. The turnover experiments indicate that [c.sub.2] is a major electron donor to nitrite reductase but [C.sub.Y] is probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition to [C.sub.2], that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in the cycA--cycY double mutant probably results from a change in nirK expression.

The five-coordinate NO-bound heme in cytochrome c' from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor.

Cytochrome c' (Cyt c') is a c-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in Rhodobacter sphaeroides 2.4.3, designated cycP, was isolated and sequenced. Northern blot analysis and [beta]-galactosidase assays demonstrated that cycP transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another Rhodobacter species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt c' levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt c' under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt c' binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt c' has NO reductase activity. Taken together, these results suggest a model where Cyt c' in R. sphaeroides 2.4.3 may shuttle NO to the membrane, where it can be reduced.

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-[1.sup.-/-] mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.

A combination of spectroscopic methods and density functional calculations is used to describe the electronic structure of the axial mutant (Met182Thr) of Rhodobacter sphaeroides nitrite reductase in which the axial methionine is changed to a threonine. The mutation results in a dramatic change in the geometric and electronic structure of the copper site.

Research has been conducted on Rhodobacter sphaeroides strain 2.4.3. The isolation of the 2.4.3 strains carrying prrB gene mutations has been performed in screening for the mutants unable to grow anaerobically on a nitrite containing medium and the results demonstrate the decrease of the gene encoding nitrite reductase in these strains.

Research has been conducted on the nitrite reductases. The kinetic mechanism of the nitrite reduction and the pH dependence on the enzymatic activity and its activation energy have been investigated and the details are reported.

Rhodobacter sphaeroides 2.4.3 has a taxis response to both nitrate and nitrite, as does Agrobacterium tumefaciens. Rhodopseudomonas palustris has a taxis response to nitrite but not nitrate, whereas Rhodobacter sphaeroides 2.4.1 has no taxis response to either nitrite or nitrate.

Research has been conducted on the gene in Rhodobacter sphaeroides 2.4.3 whose expression is controlled by NnrR. The results of the purification and biochemical analyses of the histidine-tagged NnrR indicate that it is a haem-copper protein.

Research elucidates the role played by pseudoazurin protein and nirV gene products in the nitrogen oxide reduction by Rhodobacter sphaeroides. Gene expression studies reveal that nirV is cotranscribed with another gene, nirK, as a part of the regulon NnrR.