In denitrifying bacteria, nitric oxide (NO) is an electron acceptor and a free intermediate produced during anaerobic respiration. NO is also a signal for transcriptional regulation of the genes encoding nitrite (Nir), nitric oxide (Nor) and nitrous oxide reductases (N2OR). We hypothesise that the timing and strength of the NO signal necessary for full nir expression are key factors in the bet-hedging strategy of Paracoccus denitrificans, and that systems scavenging NO under hypoxia reduce the probability of nir induction. We show that the flavohemoglobin Hmp scavenges NO in aerobic cultures and that hmp is regulated by an NsrR-type repressor. Using a strain with an mCherry-nirS fusion, we found a clear, negative effect of Hmp on initial nir expression. Deletion of norCB eliminated bet-hedging, but the elevated NO levels in co-cultures with the wild type did not abolish bet-hedging in the wild type cells. Our results demonstrate clear roles for Hmp and Nor in regulating the expression of nirS through NO scavenging, while suggesting that the trigger for nir induction is not NO itself, but rather an intracellularly generated derivative. Our findings have important implications for understanding the regulatory network controlling the transition to anaerobic respiration.

Abstract Background Oxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O2 solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO3− is soluble in water, the end-product (N2) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO3−-consumption, resulting in salt accumulation if feeding the bioreactor with NO3− salt. We avoid this with our novel pH–stat approach, where the reactor is supplied with 5 M HNO3 to compensate for the alkalization, thus sustaining NO3−-concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO3− as the N-source and electron acceptor. Results Our fed-batch culture reached 20 g cell dry weight L−1, albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO2/H2CO3 accumulation, thus suppressing pH, leading to a paucity in HNO3-feeding until N2-sparging had removed sufficient CO2. The three free intermediates in the denitrification pathway (NO3− → NO2− → NO → N2O → N2) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO3−-limited. Carefully balancing glucose provision according to the HNO3 injected is thus crucial. Conclusions This work provides a proof of concept, while also identifying CO2/H2CO3 accumulation as a hurdle that must be overcome for further development and optimization of the method.

Farmed soils contribute substantially to global warming by emitting N2O (ref. 1), and mitigation has proved difficult2. Several microbial nitrogen transformations produce N2O, but the only biological sink for N2O is the enzyme NosZ, catalysing the reduction of N2O to N2 (ref. 3). Although strengthening the NosZ activity in soils would reduce N2O emissions, such bioengineering of the soil microbiota is considered challenging4,5. However, we have developed a technology to achieve this, using organic waste as a substrate and vector for N2O-respiring bacteria selected for their capacity to thrive in soil6–8. Here we have analysed the biokinetics of N2O reduction by our most promising N2O-respiring bacterium, Cloacibacterium sp. CB-01, its survival in soil and its effect on N2O emissions in field experiments. Fertilization with waste from biogas production, in which CB-01 had grown aerobically to about 6 × 109 cells per millilitre, reduced N2O emissions by 50–95%, depending on soil type. The strong and long-lasting effect of CB-01 is ascribed to its tenacity in soil, rather than its biokinetic parameters, which were inferior to those of other strains of N2O-respiring bacteria. Scaling our data up to the European level, we find that national anthropogenic N2O emissions could be reduced by 5–20%, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N2O emissions for which other mitigation options are lacking at present.

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.

Microorganisms, including bacteria, archaea, viruses, fungi, and protists, are essential to life on Earth and the functioning of the biosphere. Here, we discuss the key roles of microorganisms in achieving the United Nations Sustainable Development Goals (SDGs), highlighting recent and emerging advances in microbial research and technology that can facilitate our transition toward a sustainable future. Given the central role of microorganisms in the biochemical processing of elements, synthesizing new materials, supporting human health, and facilitating life in managed and natural landscapes, microbial research and technologies are directly or indirectly relevant for achieving each of the SDGs. More importantly, the ubiquitous and global role of microbes means that they present new opportunities for synergistically accelerating progress toward multiple sustainability goals. By effectively managing microbial health, we can achieve solutions that address multiple sustainability targets ranging from climate and human health to food and energy production. Emerging international policy frameworks should reflect the vital importance of microorganisms in achieving a sustainable future.

Heterotrophic nitrifiers continue to be a hiatus in our understanding of the nitrogen cycle. Despite their discovery over 50 years ago, the physiology and environmental role of this enigmatic group remain elusive. The current theory is that heterotrophic nitrifiers are capable of converting ammonia to hydroxylamine, nitrite, nitric oxide, nitrous oxide, and dinitrogen gas via the subsequent actions of nitrification and denitrification. In addition, it was recently suggested that dinitrogen gas may be formed directly from ammonium. Here, we combine complementary high-resolution gas profiles, 15 N isotope labeling studies, and transcriptomics data to show that hydroxylamine is the major product of nitrification in Alcaligenes faecalis . We demonstrated that denitrification and direct ammonium oxidation to dinitrogen gas did not occur under the conditions tested. Our results indicate that A. faecalis is capable of hydroxylamine production from an organic intermediate. These results fundamentally change our understanding of heterotrophic nitrification and have important implications for its biotechnological application.

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors and their active enzymes. Our findings highlight a microbial community enriched for (ligno)cellulose-degrading denitrifiers with key players from the taxa Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota). A wide substrate specificity is observed among the many expressed carbohydrate-active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. IMPORTANCE Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

The inhibition of nitrification by mixing nitrification inhibitors (NI) with fertilizers is emerging as an effective method to reduce fertilizer-induced nitrous oxide (N2O) emissions. The additive 3,4-dimethylpyrazole phosphate (DMPP) apparently inhibits ammonia oxidizing bacteria (AOB) more than ammonia oxidizing archaea (AOA), which dominate the nitrification in alkaline and acid soil, respectively. However, the efficacy of DMPP in terms of nitrogen sources interacting with soil properties remains unclear. We therefore conducted a microcosm experiment using three typical Chinese agricultural soils with contrasting pH values (fluvo-aquic soil, black soil and red soil), which were fertilized with either digestate or urea in conjunction with a range of DMPP concentrations. In the alkaline fluvo-aquic soil, fertilization with either urea or digestate induced a peak in N2O emission (60 μg N kg−1 d−1) coinciding with the rapid nitrification within 3 d following fertilization. DMPP almost eliminated this peak in N2O emission, reducing it by nearly 90%, despite the fact that the nitrification rate was only reduced by 50%. In the acid black soil, only the digestate induced an N2O emission that increased gradually, reaching its maximum (20 μg N kg−1 d−1) after 5–7 d. The nitrification rate and N2O emission were both marginally reduced by DMPP in the black soil, and the N2O yield (N2O-N per NO2−+NO3−-N produced) was exceptionally high at 3.5%, suggesting that the digestate induced heterotrophic denitrification. In the acid red soil, the N2O emission spiked in the digestate and urea treatments at 50 and 10 μg N kg−1 d−1, respectively, and DMPP reduced the rates substantially by nearly 70%. Compared with 0.5% DMPP, the higher concentrations of DMPP (1.0 to 1.5%) did not exert a significantly (P<0.05) better inhibition effect on the N2O emissions in these soils (either with digestate or urea). This study highlights the importance of matching the nitrogen sources, soil properties and NIs to achieve a high efficiency of N2O emission reduction.

Legume cropped farmlands account for substantial N 2 O emissions globally. Legumes are commonly inoculated with N 2 -fixing bacteria, rhizobia, to improve crop yields.

Bioengineering soil metabolism by inoculation is an emerging approach to enhance plant growth and strengthen specific functions such as N 2 O reduction in order to reduce climate forcing. The use of organic wastes as substrates and carriers of microbial biomass has proven to be a viable approach to improving effectiveness and economic viability. A key factor in the success of this approach lies in selection of microbes capable of growth and survival in both organic wastes as well as soils, and which are tolerant of the rapid environmental fluctuations such fertilisations involve. A dual substrate, N 2 O-enrichment experiment, switching between soil and organic waste as substrates has yielded Cloacibacterium isolates which grow well in organic wastes and retain significant N 2 O reduction capacity when applied to soils. However, an understanding of the genetic and phenotypic characteristics utilised by these enrichment winners to dominate under such conditions remains unexplored. Here we have performed a multi-omics examination of the enrichment cultures, using both metagenomics and metaproteomics to probe the genetic basis and expressed proteins which may contribute to the success of Cloacibacterium in the enrichments, and their survival in soil. These omics results show an increase in complex carbohydrate metabolism, chemotaxis and motility genes throughout the enrichment as well as the expression of gliding motility proteins and polysaccharide utilization loci proteins by Cloacibacterium organisms. Taken together this suggests that attachment and complex polysaccharide utilisation may be key processes allowing Cloacibacterium to tolerate the stresses of a changing environment during transfers between digestate and soil.

Farmed soils contribute to global warming primarily by N 2 O-emissions, and mitigation has proven difficult. However, a novel approach with promising results in the laboratory, exploits organic wastes both as substrates and vectors for strains of N 2 O-respiring bacteria (NRB), selected for their ability to survive in soil. Here we demonstrate a strong effect in field experiments: fertilization with waste from biogas-production, in which the strain Cloacibacterium sp. CB-01 had grown aerobically to ∼6*10 9 cells mL -1 , reduced N 2 O-emissions by 50-95 %. The strong and long-lasting effect of CB-01 is ascribed to it’s tenacity in soil, rather than its biokinetic parameters, which were inferior to other NRB-strains. Scaling up to EU level, we find that national anthropogenic N 2 O-emissions can be reduced by 5-20 %, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N 2 O-emissions for which other mitigation options are currently lacking.

Liming of acidic agricultural soils has been proposed as a strategy to mitigate nitrous oxide (N2O) emissions, as increased soil pH reduces the N2O/N-2 product ratio of denitrification. The capacity of different calcareous (calcite and dolomite) and siliceous minerals to increase soil pH and reduce N2O emissions was assessed in a 2-year grassland field experiment. An associated pot experiment was conducted using homogenized field soils for controlling spatial soil variability. Nitrous oxide emissions were highly episodic with emission peaks in response to freezing-thawing and application of NPK fertilizer. Liming with dolomite caused a pH increase from 5.1 to 6.2 and reduced N2O emissions by 30% and 60% after application of NPK fertilizer and freezing-thawing events, respectively. Over the course of the 2-year field trial, N2O emissions were significantly lower in dolomite-limed than non-limed soil (p < .05), although this effect was variable over time. Unexpectedly, no significant reduction of N2O emission was found in the calcite treatment, despite the largest pH increase in all tested minerals. We tentatively attribute this to increased N2O production by overall increase in nitrogen turnover rates (both nitrification and denitrification) following rapid pH increase in the first year after liming. Siliceous materials showed little pH effect and had no significant effect on N2O emissions probably because of their lower buffering capacity and lower cation content. In the pot experiment using soils taken from the field plots 3 years after liming and exposing them to natural freezing-thawing, both calcite (p < .01) and dolomite (p < .05) significantly reduced cumulative N2O emission by 50% and 30%, respectively, relative to the non-limed control. These results demonstrate that the overall effect of liming is to reduce N2O emission, although high lime doses may lead to a transiently enhanced emission.

ABSTRACT Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess nosZ genes and the ability to respire N2O. However, the selective advantage that may favor these nosZ-harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N2O on Nrf-mediated DNRA was examined in a soil isolate, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C2H2, a NosZ inhibitor, with or without N2O, and the results were compared with C2H2-free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N2O. The NO2 −-to-NH4 + reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C2H2-amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possibility of C2H2 involvement was dismissed since the cultures continuously flushed with C2H2/N2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N2O-containing gas. The findings suggest a possibility that the oft-observed nosZ presence in DNRA-catalyzing microorganisms secures an early transcription of their DNRA genes by scavenging N2O, thus enhancing their capacity to compete with denitrifiers at oxic-anoxic interfaces. IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3 − and/or NO2 − to NH4 +. Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.

Nitrous oxide (N2O) emitted from agricultural soils destroys stratospheric ozone and contributes to global warming. A promising approach to reduce emissions is fertilizing the soil using organic wastes augmented by non-denitrifying N2O-reducing bacteria (NNRB). To realize this potential, we need a suite of NNRB strains that fulfill several criteria: efficient reduction of N2O, ability to grow in organic waste, and ability to survive in farmland soil. In this study, we enriched such organisms by sequential anaerobic batch incubations with N2O and reciprocating inoculation between the sterilized substrates of anaerobic manure digestate and soils. 16S rDNA amplicon sequencing and metagenomics analysis showed that a cluster of bacteria containing nosZ genes encoding N2O-reductase, was enriched during the incubation process. Strains of several dominant members were then isolated and characterized, and three of them were found to harbor the nosZ gene but none of the other denitrifying genes, thus qualifying as NNRB. The selected isolates were tested for their capacities to reduce N2O emissions from three different typical Chinese farmland soils. The results indicated the significant mitigation effect of these isolates, even in very acidic red soil. In conclusion, this study demonstrated a strategy to engineer the soil microbiome with promising NNRB with high adaptability to livestock manure digestate as well as different agricultural soils, which would be suitable for developing novel fertilizer for farmland application to efficiently mitigate the N2O emissions from agricultural soils.

Soil chemical properties and microbiome composition impact N2O emission potential, but the relative importance of these factors as determinants of N2O emission in denitrifying systems is rarely tested. In addition, previous linkages between microbiome composition and N2O emission potential rarely demonstrate causality. Here, we determined the relative impact of bacteriome composition (i.e., soil extracted bacterial cells) and soil water extract (i.e., water extractable chemicals and particles below 0.22 µm) on N2O emission potential utilizing an anoxic cell-based assay system. Cells and water extract for assays were sourced from soils with contrasting N2O/N2O + N2 ratios, combined in various combinations and denitrification gas production was measured in response to nitrate addition. Analysis of 16S amplicon sequencing data revealed similarity in composition between extracted and parent soil bacteriomes. Average directionless effects of cell and water extract on N2O/N2O + N2 (Cell: ∆0.17, soil water extract: ∆0.22) and total N2O hypothetically emitted (Cell: ∆2.62 µmol-N, soil water extract: ∆4.14 µmol-N) across two assays indicated water extract is the most important determinant of N2O emissions. Independent pH differences of just 0.6 points impacted N2O/N2O + N2 on par with independent water extract differences, supporting the dominance of this variable in previous studies. However, impacts on overall N2O hypothetically emitted were smaller, suggesting that soil pH manipulation may not necessarily be a successful approach to mitigate emissions. In addition, we observed increased N2O accumulation and emission potential at the end of incubations concomitant with predicted decreases in carbon (C) availability, suggesting that C limitation increases N2O emission transiently with the magnitude of emission dependent on both chemical and bacteriome controls.

Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO3 - , but it often entails accumulation of the cytotoxic intermediate NO2 - and the greenhouse gas N2 O. To explore if these high NO2 - and N2 O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole-genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO2 - , NO, N2 O, N2 and O2 as they depleted O2 and transitioned to denitrification with NO3 - as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO2 - accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO3 - in the narG-containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA-containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N2 O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N2 O reductase expression.

Seed inoculation by plant growth promoting rhizobacteria (PGPRs) is an agronomic practice that stimulates root carbon (C) exudation and nitrogen (N) uptake. Inoculation thus increases and decreases C and N availabilities to denitrifiers in the rhizosphere, respectively. Hence, denitrification rates in the rhizosphere can be positively or negatively influenced by root activity depending on the balance between these two processes. We assumed that inoculation effect on denitrifiers could strongly differ according to soil conditions. Would denitrifiers be mostly limited by C, inoculation would increase denitrifier abundance and activity through increased labile C availability. Would denitrifiers be limited by N rather than C, inoculation would decrease denitrifier abundance and activity through increased competition for N. Here we manipulated denitrification limitation by C and N (i) in a field trial through the use of different fertilization levels, and (ii) in a growth chamber experiment by mimicking root exudate inputs. We analyzed how the effects of maize inoculation by the PGPR Azospirillum lipoferum CRT1 on potential gross and net N2O production rates and NO2−- and N2O-reducer abundances were related to C and N limitation levels. An increase in potential gross (up to +113%) and to a lesser extent net (+37%) N2O production was observed for soils where denitrification was highly limited by C. This was explained by strong and moderate increases in the abundances of NO2−- and N2O-reducers, respectively. In contrast, when denitrification was weakly limited by C, gross and net N2O productions were negatively affected by inoculation (−15 and −40%, respectively). Our results show that the inoculation practice should be evaluated in term of possible increased crop yield but also possible modified N2O emission, paying much attention to cropland soils where denitrifiers are highly limited by C.

The greenhouse gas nitrous oxide (N2O) has strong potential to drive climate change. Soils are a major source of N2O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO3−) to nitrite (NO2−), nitric oxide (NO), N2O, and dinitrogen (N2). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK2 and NnrR transcriptional regulators. To understand the role of FixK2 and NnrR proteins in N2O turnover, we monitored real-time kinetics of NO3−, NO2−, NO, N2O, N2, and oxygen (O2) in a fixK2 and nnrR mutant using a robotized incubation system. We confirmed that FixK2 and NnrR are regulatory determinants essential for NO3− respiration and N2O reduction. Furthermore, we demonstrated that N2O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO3−, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N2O turnover in B. diazoefficiens.

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 O2 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 N2O 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 O2 or N2O. To test this, cells were exposed to gradual O2 depletion or sudden anoxia and subsequent spikes of O2 and N2O. 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 O2 spiking leads to bet-hedging, while N2O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O spike had much lower respiration rates than those rescued by the O2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. 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 N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2- to N2O. 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 O2 or N2O. We confirm such entrapment and the rescue of all cells by an N2O spike but only a fraction by an O2 spike. The results shed light on the role of O2 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 N2O, which contribute to anthropogenic climate forcing.

In anaesthetised rats, 63 raphe-spinal units in nucleus raphe magnus (NRM) have been identified by means of electrophysiological methods and further classified into serotonergic (5-HT) and non-5-HT units according to their conduction velocity and spontaneous discharge rate. All units, except one, showed either excitatory (n = 39) or inhibitory (n = 23) responses to noxious heating of the tail with hot water at 52°C. The threshold temperature was around 44°C and both types of responses increased with stepwise increases in temperature. The excitatory or inhibitory responses of raphe-spinal units induced by noxious heating of the tail correlated well with those elicited by noxious pinching, but did not correlate with the different transmitter populations (5-HT or non-5-HT) of the NRM unit. The effects of morphine or Fentanyl administration on the heat-induced responses of NRM units was observed. The possible involvement of NRM raphe-spinal units in ‘diffuse noxious inhibitory controls’ (DNICs) was discussed, particularly in relation to the ‘lifting of DNICs’ produced by morphine.