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Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O 2 repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed "bet-hedging," where all cells synthesize the N 2 O reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O 2 or N 2 O. To test this, cells were exposed to gradual O 2 depletion or sudden anoxia and subsequent spikes of O 2 and N 2 O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS , and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e - -acceptor deprivation and show that O 2 spiking leads to bet-hedging, while N 2 O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N 2 O spike had much lower respiration rates than those rescued by the O 2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O 2 . Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy-limited systems. IMPORTANCE Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N 2 O emissions because all cells express NosZ, reducing N 2 O to N 2 , while a minority express NirS + Nor, reducing NO 2 - to N 2 O. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O 2 or N 2 O. We confirm such entrapment and the rescue of all cells by an N 2 O spike but only a fraction by an O 2 spike. The results shed light on the role of O 2 repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic nirS expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of N 2 O, which contribute to anthropogenic climate forcing.

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 NO2(-)). 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 N(2)O reductase (N(2)OR). 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 N(2)OR); or (iii) ancillary factors involved in N(2)O 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 NO(x), 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 N(2)O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N(2)OR.

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 high-resolution 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.

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.