Denitrification in soil is a major source of atmospheric N 2 O. Soil pH appears to exert a strong control on the N 2 O/N 2 product ratio (high ratios at low pH), but the reasons for this are not well understood. To explore the possible mechanisms involved, we conducted an in-depth investigation of the regulation of denitrification in the model organism Paracoccus denitrificans during transition to anoxia both at pH 7 and when challenged with pHs ranging from 6 to 7.5. The kinetics of gas transformations (O 2 , NO, N 2 O, and N 2 ) were monitored using a robotic incubation system. Combined with quantification of gene transcription, this yields highresolution data for direct response patterns to single factors. P. denitrificans demonstrated robustly balanced transitions from O 2 to nitric oxide-based respiration, with NO concentrations in the low nanomolar range and marginal N 2 O production at an optimal pH of 7. Transcription of nosZ (encoding N 2 O reductase) preceded that of nirS and norB (encoding nitrite and NO reductase, respectively) by 5 to 7 h, which was confirmed by observed reduction of externally supplied N 2 O. Reduction of N 2 O was severely inhibited by suboptimal pH. The relative transcription rates of nosZ versus nirS and norB were unaffected by pH, and low pH had a moderate effect on the N 2 O reductase activity in cells with a denitrification proteome assembled at pH 7. We thus concluded that the inhibition occurred during protein synthesis/assembly rather than transcription. The study shed new light on the regulation of the environmentally essential N 2 O reductase and the important role of pH in N 2 O emission.

Denitrifying prokaryotes use NO x as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N 2 O and N 2 , depending on the relative activity of the enzymes catalysing the stepwise reduction of NO 3 − to N 2 O and finally to N 2 . Cultured denitrifying prokaryotes show characteristic transient accumulation of NO 2 − , NO and N 2 O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N 2 O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N 2 O produced (N 2 O/(N 2 +N 2 O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N 2 O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N 2 O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level.

Present-day knowledge on the regulatory biology of denitrification is based on studies of selected model organisms. These show large variations in their potential contribution to NO, NO, and NO accumulation, attributed to lack of genes coding for denitrification reductases, but also to variations in their transcriptional regulation, as well as to post-transcriptional phenomena. To validate the relevance of these observations, there is a need to study a wider range of denitrifiers. We designed an isolation protocol that identifies all possible combinations of truncated denitrification chains (NO/NO/NO/NO/N). Of 176 isolates from two soils (pH 3.7 and 7.4), 30 were denitrifiers sensu stricto, reducing NO to gas, and five capable of NO reduction only. Altogether, 70 isolates performed at least one reduction step, including two DNRA isolates. Gas kinetics and electron flow calculations revealed that several features with potential impact on NO production, reported from model organisms, also exist in these novel isolates, including denitrification bet-hedging and control of NO/NO/NO accumulation. Whole genome sequencing confirmed most truncations but also showed that phenotypes cannot be predicted solely from genetic potential. Interestingly, and opposed to the commonly observed inability to reduce NO under acidic conditions, one isolate identified as Rhodanobacter reduced NO only at low pH.

Denitrifiers differ in how they handle the transition from oxic to anoxic respiration, with consequences for NO and N2O emissions. To enable stringent comparisons we defined parameters to describe denitrification regulatory phenotype (DRP) based on accumulation of NO2(-) , NO and N2O, oxic/anoxic growth and transcription of functional genes. Eight Thauera strains were divided into two distinct DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the different denitrification genes. The PO group accumulated nitrite, and no transcription of nirS (encoding nitrite reductase) was detected until all available nitrate (2 mM) was consumed. Addition of a new portion of nitrate to an actively denitrifying culture of a PO strain (T. terpenica) resulted in a transient halt in nitrite reduction, indicating that the electron flow was redirected to nitrate reductase. All eight strains controlled NO at nano-molar concentrations, possibly reflecting the importance of strict control for survival. Transient N2O accumulation differed by two orders of magnitude between strains, indicating that control of N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional genes) and DRP.

The ability of Agrobacetrium tumefaciens to perform balanced transitions from aerobic to anaerobic respiration was studied by monitoring oxygen depletion, transcription of nirK and norB, and the concentrations of nitrite, nitric oxide (NO) and nitrous oxide in stirred batch cultures with different initial oxygen, nitrate or nitrite concentrations. Nitrate concentrations (0.2-2 mM) did not affect oxygen depletion, nor the oxygen concentration at which denitrification was initiated (1-2 microM). Nitrite (0.2-2 mM), on the other hand, retarded the oxygen depletion as it reached approximately 20 microM, and caused initiation of active denitrification as oxygen concentrations reached 10-17 microM. Unbalanced transitions occurred in treatments with high cell densities (i.e. with rapid transition from oxic to anoxic conditions), seen as NO accumulation to muM concentrations and impeded nitrous oxide production. This phenomenon was most severe in nitrite treatments, and reduced the cells' ability to respire oxygen during subsequent oxic conditions. Transcripts of norB were only detectable during the period with active denitrification. In contrast, nirK transcripts were detected at low levels both before and after this period. The results demonstrate that the transition from aerobic to anaerobic metabolism is a regulatory challenge, with implications for survival and emission of trace gases from denitrifying bacteria.

The hybrid cluster protein, Hcp, contains a 4Fe-2S-2O iron-sulfur-oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N2 O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron-sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp-hcr operon encodes an NADH-dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co-purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.

The reductases performing the four steps of denitrification are controlled by a network of transcriptional regulators and ancillary factors responding to intra- and extracellular signals, amongst which are oxygen and N oxides (NO and ). Although many components of the regulatory network have been identified, there are gaps in our understanding of their role(s) in controlling the expression of the various reductases, in particular the environmentally important N2O reductase (N2OR). We investigated denitrification phenotypes of Paracoccus denitrificans mutants deficient in: (i) regulatory proteins (three FNR-type transcriptional regulators, NarR, NNR and FnrP, and NirI, which is involved in transcription activation of the structural nir cluster); (ii) functional enzymes (NO reductase and N2OR); or (iii) ancillary factors involved in N2O reduction (NirX and NosX). A robotized incubation system allowed us to closely monitor changes in concentrations of oxygen and all gaseous products during the transition from oxic to anoxic respiration. Strains deficient in NO reductase were able to grow during denitrification, despite reaching micromolar concentrations of NO, but were unable to return to oxic respiration. The FnrP mutant showed linear anoxic growth in a medium with nitrate as the sole NOx, but exponential growth was restored by replacing nitrate with nitrite. We interpret this as nitrite limitation, suggesting dual transcriptional control of respiratory nitrate reductase (NAR) by FnrP and NarR. Mutations in either NirX or NosX did not affect the phenotype, but the double mutant lacked the potential to reduce N2O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N2OR.

Current knowledge of denitrification is based on detailed studies of a limited number of organisms. In most cases the importance of these paradigm species in natural ecosystems is questionable. Detailed phenotypic studies of a wider range of prokaryotes, both type strains and dominant denitrifiers isolated from complex systems, will aid the generation of more sophisticated mathematical models for the prediction of NO and N2O emission to the environment. However, in order to facilitate the comparison of a vast range of prokaryotes, phenotypic experiments and functional characteristics included should be standardized. In the present paper, we discuss the term DRP (denitrification regulatory phenotype) for describing a set of phenotypic traits and experimental conditions for the characterization of denitrifying organisms. This is exemplified by the contrasting DRP characteristics of the two well-studied denitrifiers Paracoccus denitrificans and Agrobacterium tumefaciens.