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To elucidate the direct effects of biochar on denitrification metabolism at the cellular level, the global response of model denitrifying soil bacterium (Paracoccus denitrificans) to biochar addition was investigated by physiological, proteomic, and metabolomics analyses. The enhancement effect on denitrification was positively correlated with its pyrolysis temperatures (300-500 °C) and dosages (0.1-1%), regardless of precursors [corn straw (CS) or wheat straw). Moreover, the stimulating effect of CS biochar made at 500 °C (CS-500) was mainly attributed to the bulk particles rather than the released soluble compounds. Without direct contact with cells, bulk CS-500 particles might directly modulate the carbon metabolism by the adsorption of extracellular metabolites. Since carbon flux to storage was shifted to oxidative catabolism and growth assimilation, more share of the produced reducing power was used for nitrogen reduction. Meanwhile, except for nitrate reductase, both protein expressions and enzyme activities of nitrite reductase, nitric oxide reductase, and nitrous oxide reductase were up-regulated. Accordingly, the accumulation of N2O was reduced by 98% due to the optimized electron distribution among denitrifying enzymes. Eventually, the growth rate of Pc. denitrificans enhanced because of the improved energy utilization efficiency. These results updated the regulation mechanism of biochar on denitrification metabolism and N2O mitigation.

Nitric oxide (NO) is generated in some biological systems. Due to its radical character, it exhibits high reactivity, but biological system can manage NO without sustaining any damage to bio-compou...

AIMS The aim of this study was to obtain cold-adapted denitrifying fungi that could be used for bioaugmentation in woodchip bioreactors to remove nitrate from agricultural subsurface drainage water. METHODS AND RESULTS We isolated a total of 91 nitrate-reducing fungal strains belonging to Ascomycota and Mucoromycota from agricultural soil and a woodchip bioreactor under relatively cold conditions (5 and 15°C). When these strains were incubated with 15 N-labelled nitrate, 29 N2 was frequently produced, suggesting the occurrence of co-denitrification (microbially mediated nitrosation). Two strains also produced 30 N2 , indicating their ability to reduce N2 O. Of the 91 nitrate-reducing fungal strains, fungal nitrite reductase gene (nirK) and cytochrome P450 nitric oxide reductase gene (p450nor) were detected by PCR in 34 (37%) and 11 (12%) strains, respectively. Eight strains possessed both nirK and p450nor, further verifying their denitrification capability. In addition, most strains degraded cellulose under denitrification condition. CONCLUSIONS Diverse nitrate-reducing fungi were isolated from soil and a woodchip bioreactor. These fungi reduced nitrate to gaseous N forms at relatively low temperatures. These cold-adapted, cellulose-degrading and nitrate-reducing fungi could support themselves and other denitrifiers in woodchip bioreactors. SIGNIFICANCE AND IMPACT OF THE STUDY The cold-adapted, cellulose-degrading and nitrate-reducing fungi isolated in this study could be useful to enhance nitrate removal in woodchip bioreactors under low-temperature conditions.

Photosynthesis and plant architecture are important factors influencing grain yield in rice (Oryza sativa L.). Here, we identified a high-tillering and dwarf 12 (htd12) mutant and analyzed the effects of the HTD12 mutation on these important factors. HTD12 encodes a 15-cis-ζ-carotene isomerase (Z-ISO) belonging to the nitrite and nitric oxide reductase U (NnrU) protein family, as revealed by positional mapping and transformation experiments. Sequence analysis showed that a single nucleotide transition from guanine (G) to adenine (A) in the 3’ acceptor site between the first intron and second exon of HTD12 alters its mRNA splicing in htd12 plants, resulting in a 49-amino acid deletion that affects carotenoid biosynthesis and photosynthesis. In addition, compared with the wild type, htd12 had significantly lower concentrations of ent-2’-epi-5-deoxystrigol (epi-5DS), a native strigolactone, in both roots and root exudates, resulting in an obvious increase in tiller number and decrease in plant height. These findings indicate that HTD12, the rice homolog of Z-ISO, regulates chloroplast development and photosynthesis by functioning in carotenoid biosynthesis, and modulates plant architecture by affecting strigolactone concentrations.

Membrane-integrated nitric oxide reductases (NOR) catalyze the formation of nitrous oxide (N2O) from two NO molecules using two protons and two electrons at a heme/non-heme iron binuclear center. D...

Recent studies have identified several key intermediates of the nitric oxide reductase (NOR) cycle of flavodiiron proteins (FDPs). These intermediates include, sequentially, a μ-hydroxo diferrous s...

Bacterial wilt, caused by soil‐borne pathogen Ralstonia solanacearum, is a serious disease in many plants such as Solanaceae. To investigate the effects of accumulated nitrogen in soil on the phenotype and pathogenicity of the R. solanacearum, a serial passage experiment (SPE) was designed. Specifically speaking, minimal medium supplied with a slight excess of ammonium sulphate (AS) or ammonium nitrate (AN) was used to simulate the nutrition of soil containing excess nitrogen. During the period of 30 SPE, the phenotype, pathogenicity and relative expression of nitrogen metabolism genes in R. solanacearum were monitored. Phenotypic analysis results illustrated that the colony morphology of R. solanacearum changed after long‐term culture, from high virulence colonies with strong fluidity to small, round non‐mucoid colonies; The strain after prolonged stress of excessive exogenous nitrogen was a no‐virulence phenotype conversion type (PC‐type). The time for a change in colony morphology to occur after exposure to exogenous AS or AN was significantly less than the untreated samples, which treated without exogenous nitrogen. The results of pathogenicity also demonstrated that the cultures treated with exogenous AN or AS reduced virulence more quickly than the control. The disease index of 10 SPE with AN treatment or AS treatment was 89% or 68% lower than that of the control, respectively. In addition, as the incubation time increased, the swimming motility and the number of biofilms formation of the cultures were significantly changed under both treatments in comparison to the untreated samples. Furthermore, the relative expression of the nitric oxide reductase norB gene in the cultures treated with AN was 1.51‐fold higher compared with the control after 30 SPE. These results indicated that excessive nitrogen supply in the environment could accelerate the transformation of R. solanacearum from high virulence wild‐type into a PC‐type, probably for the purpose of adapting to the adverse environment.

Ralstonia solanacearum, which causes bacterial wilt disease of many crops, needs denitrifying respiration to succeed inside its plant host. In the hypoxic environment of plant xylem vessels this pathogen confronts toxic oxidative radicals like nitric oxide (NO), which is generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including Repair of Iron Cluster (RIC) homolog NorA, nitric oxide reductase NorB, and flavohaemoglobin HmpX. During denitrification and tomato pathogenesis and in response to exogenous NO, R. solanacearum upregulated norA, norB, and hmpX. Single mutants lacking ΔnorB, ΔnorA, or ΔhmpX increased expression of many iron and sulfur metabolism genes, suggesting that losing even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining detoxification genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. Loss of norA, norB, and hmpX may be lethal, since the methods used to construct the double mutants did not generate a triple mutant. Aconitase activity assays showed that NorA, HmpX and especially NorB are important for maintaining iron-sulfur cluster proteins. Additionally, plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting that bacterial detoxification of NO reduces pathogen visibility. Thus, R. solanacearum’s three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic oxidative stress during denitrification, for virulence and growth in tomato, and for evading host plant defenses.ImportanceThe soilborne plant pathogen Ralstonia solanacearum (Rs) causes bacterial wilt, a serious and widespread threat to global food security. Rs is metabolically adapted to low oxygen conditions, using denitrifying respiration to survive in the host and cause disease. However, bacterial denitrification and host defenses generate nitric oxide (NO), which is toxic and also alters signaling pathways in both plants and the pathogen. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase NorB, Repair of Iron Centers NorA, and oxidoreductase HmpX. This redundancy, together with analysis of mutants and in-planta dual transcriptomes, indicates that maintaining low NO levels is integral to Rs fitness in tomatoes (because NO damages iron-cluster proteins) and to evading host recognition (because bacterially produced NO can trigger plant defenses).

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- → NO2- → NO → N2O → N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

We report the design, construction, and testing of Escherichia coli-based bioluminescent bioreporters for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX), one of the most prevalent military-grade explosives in use today. These sensor strains are based on a fusion between the promoter of either the hmp (nitric oxide dioxygenase) or the hcp (a high-affinity nitric oxide reductase) E. coli gene, to the microbial bioluminescence luxCDABEG gene cassette. Signal intensity was enhanced in ∆hmp and ∆hcp mutants, and detection sensitivity was improved when the two gene promoters were cloned in tandem. The Photobacterium leiognathi luxCDABEG reporter genes were superior to those of Aliivibrio fischeri in terms of signal intensity, but in most cases inferior in terms of detection sensitivity, due to a higher background signal. Both sensor strains were also induced by additional nitro-organic explosives, as well as by nitrate salts. Sensitive detection of RDX in a solid matrix (either LB agar or sand) was also demonstrated, with the bioreporters encapsulated in 1.5-mm calcium alginate beads. Lowest RDX concentration detected in sand was 1.67 mg/kg sand. The bioreporter strains described herein may serve as a basis for a standoff detection technology of RDX-based explosive devices, including buried landmines.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae. This work was funded by a research grant from MINECO Spain (RTI2018-099860-B-I00) and VIGROB-309 (University of Alicante).

Funding Information: Funding: This study was financially supported by the Portuguese Fundação para a Ciência e Tec-nologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Funding Information: This study was financially supported by the Portuguese Funda??o para a Ci?ncia e Tecnologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union?s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif,-G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release. published

Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

The impacts of metal ions (Cu2+ and Fe3+) on nutrients removal and nitrous oxide (N2O) emission during the simultaneous nitrification, denitrification and phosphorus removal (SNDPR) process were evaluated and the mechanism was further investigated. Results showed that both Cu2+ and Fe3+ addition at moderate concentrations enhanced nitrogen removal by reinforcing the denitrification process. The long-term addition of Fe3+ weakened the removal of phosphorus by 2.3%–11.7%, due to the suppression of polyphosphate-accumulating organisms (PAOs) growth and activity. N2O production and emission factor decreased greatly by the addition of Cu2+. This decrease was caused by the enhancement of nitrite reductase and N2O reductase (N2OR) activities. On the contrary, Fe3+ addition at moderate concentrations stimulated the N2O production. Fe3+ enhanced the nitric oxide reductase (NOR) and increased the NOR/N2OR ratio, leading to the accumulation of N2O. Moreover, addition of Fe3+ caused a reduction in polyhydroxyalkanoates synthesis at the anaerobic stage and renforced glycogen-accumulating organisms (GAOs) activity instead of PAOs activity, resulting in increased N2O accumulation. Addition of high concentrations of metal ions (5 mg/L Cu2+ and 60 mg/L Fe3+ in this study) suppressed the nutrients removal and stimulated the N2O production, partly due to the inhibition of the bacterial activity.

Colwellia sp. Arc7-635, a psychrophilic denitrifying bacterium isolated from Arctic seawater, uses NO3− or NH4+ as the sole nitrogen source to grow at low temperatures. In this article, we describe the complete genome of Colwellia sp. Arc7-635. The genome has one circular chromosome of 4,741,350 bp (38.41 mol% G+C content), consisting of 3841 coding genes, 91 tRNA genes, as well as seven rRNA operons of 16S-23S-5S rRNA, and one operon of 16S-23S-5S-5S rRNA. According to the genomic annotation results, strain Colwellia sp. Arc7-635 encodes a complete denitrifying pathway consisting of genes affiliated with nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. Genes affiliated with nitrate reduction to ammonia including nitrate reductases (NapA and NapB) and nitrite reductases (NirA, NirB, and NirD) were also identified. The whole genome sequences of Arc7-635 provide information that is useful for further clarifying nitrogen metabolisms and facilitate its potential applications in the bioremediation of nitrogen pollutions.

Here, the draft genome of simultaneous nitrification-denitrification strain (SND) Klebsiella sp. KSND revealed possible existence of genes involved in N-assimilation and -dissimilation pathways. The change levels of genes under defined N-sources were analyzed by Quantitative Real-Time PCR. It suggested that NH4+-assimilation via NADP-glutamate dehydrogenase pathway would occur preferentially. NirBD genes were tightly regulated in a lower level, so that nitrite was rapidly consumed for detoxication by denitrification. Three types of nitrate reductase homologues are surprisingly present in KSND, whereas the dominant nitrate reduction for assimilation and denitrification processes mediates by NapA-type nitrate reductase. Nitric oxide reductase homologues FlRd and FlRd-red provide an adequate capacity for NO detoxification. The recombinant hydroxylamine reductase showed high activity in hydroxylamine to generate ammonium, which might contribute to detoxification mechanism in nitrogen cycling. Overall, this study firstly provides valuable insights into the genes expression and enzyme action, which helps understanding the mechanism of SND processes.

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (FeB ). Heme b3 and FeB constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB -less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors., FG and LBM thank FCT/MCTES for the fellowship grants SFRH/BD/52502/2014 and SFRH/BPD/111404/2015, respectively, which are financed by national funds and co-financed by FSE. CMC acknowledges FCT-MCTES funding through project PTDC/BBB-BQB/0129/2014 (FCT/MCTES). This work was supported by the REQUIMTE, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013 and UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265 and POCI-01-0145-FEDER-007728), and also by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged.

Nitrogen removal based on short-cut nitrification (SCN) have attract more attentions, in which stable nitrite accumulation is prerequisite. In this study, different reductive potential was applied to inhibit nitrite oxidizing bacteria for achievement of SCN in aerobic cathode chamber of bioelectrochemical systems with dissolved oxygen concentration of 3.5 mg/L. The results demonstrated that the applied potential facilitated nitrite accumulation with high ammonia oxidation rates. The maximum nitrate accumulation rate of 87.61% was obtained at −800 mV. The abundance of Nitrosomonas and Thauera increased while Nitrospira abundance declined with more negative reductive potentials. The activity of nitric oxide reductase was also evidently inhibited. The above-mentioned three genera were the keystone taxa in co-occurrence network with high degree and closeness centrality. Interestingly, total nitrogen (TN) removal was enhanced simultaneously in the absence of external organic carbon. Reductive potential would be a promising approach for achieving SCN and simultaneously TN removal.

In this study, three up-flow fixed-bed bioreactors were constructed with three different filling ratios (filling volume/effective volume: 30%, 60% and 90%) of polycaprolactone (PCL). Above 98% of nitrate removal efficiency was achieved with low accumulations of nitrite and ammonium for each filling ratio. Low filling ratio of PCL had extensive folds and pores that favored the attachment and growth of microorganisms; however, excessive biomass restrained nitrate specific reduction rate (NaSRR). The most dominant genera were Comamonas (0.80–57.64%), Stenotrophomonas (2.59–54.39%), Acidovorax (7.32–23.55%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (0.30–19.74%) and Thermomonas (0.12–14.58%). Nitrate reductase (EC 1.7.99.4), nitrite reductase (EC 1.7.2.1) and nitric oxide reductase (EC 1.7.2.5) predicted by PICRUSt2 were abundant in high influent nitrate load (NaL). According to the analysis of carbon balance model, the utilization rate (η) of PCL showed a highly positive correlation with influent NaL, indicating reducing filling ratio or HRT might be an effective measure to save cost for nitrate removal.

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The 6 transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA, and IC, and a competing glycogen accumulating organism (GAO) affiliated with Candidatus Competibacter. Strongly elevated expression of nitrite reductase compared to nitrous oxide reductase was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2O accumulation. Surprisingly, while ∼90% of nitrite reductase gene transcripts mapped to the three co-occurring PAO populations, ∼93% of nitric oxide reductase gene transcripts were expressed by the GAO population. This suggests the potential for cooperation between GAOs and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide.Originality-Significance StatementPolyphosphate accumulating organisms (PAOs) affiliated with as-yet-uncultivated Ca. ‘Accumulibacter phosphatis’ are increasingly employed in enhanced biological phosphorus removal (EBPR) processes, a common environmental biotechnology for removing phosphorus from wastewater and thereby preventing detrimental impacts of nutrient pollution. Under anoxic conditions, PAOs have been associated with unusually high emissions of the potent greenhouse gas and denitrification intermediate nitrous oxide. However, the underlying mechanisms and biological controls on incomplete denitrification by denitrifying Accumulibacter, their ecological interactions with understudied glycogen accumulating organisms (GAOs), and patterns of gene expression under anoxic conditions are all poorly understood. Here, we describe genomic features of a previously unrecognized clade of Accumulibacter that is putatively adapted to high rate P uptake under nitrite-driven denitrification and provide evidence that differential gene expression (namely elevated expression of nitrite reductase compared to nitrous oxide reductase) by Accumulibacter is a key control on nitrous oxide production. Moreover, we document genomic and transcriptional potential for cooperation and crossfeeding of the denitrification intermediate nitric oxide between GAOs and PAOs. This is surprising because GAOs are conventionally considered to be competitors to PAOs, and because nitric oxide is toxic to most microorganisms at low concentrations. Taken together, our work provides significant new understanding of metabolic and ecological interactions in EBPR processes that are critical to environmental protection; demonstrates the potential of previously unrecognized crossfeeding of the denitrification intermediate nitric oxide; and expands our understanding of genomic features and clade level diversity of Accumulibacter.

High concentrations of nitrous oxide were recovered from partial nitrification treated leachate in a microbial electrolysis cell (MEC) inoculated with a nosZ-deficient strain of Pseudomonas aeruginosa. N2O conversion efficiencies > 90% were achieved when a potential of 0.8 V was applied to the MEC. The ΔnosZ strain was enriched in the 0.8 V MEC, but Achromobacter dominated the non-current control. Nitric oxide reductase genes were highly expressed by ΔnosZ cells growing in the 0.8 V MEC, consistent with enhanced nitrous oxide production rates. Concentrations of phenazine derivatives and transcripts from phenazine biosynthesis genes were also high in the 0.8 V MEC. Phenazine derivatives are known to act as electron shuttles, enhance biofilm formation, and help ward off competitors, thereby increasing the survivability of the ΔnosZ strain in the MEC. These results show that applied current stabilized growth of the ΔnosZ strain in the reactor and allowed it to sustainably generate high concentrations of nitrous oxide.

Methylomonas sp. ZR1 was an isolated new methanotrophs that could utilize methane and methanol growing fast and synthesizing value added compounds such as lycopene. In this study, the genomic study integrated with the comparative transcriptome analysis were taken to understanding the metabolic characteristic of ZR1 grown on methane and methanol at normal and high temperature regime. Complete Embden-Meyerhof-Parnas pathway (EMP), Entner–Doudoroff pathway (ED), Pentose Phosphate Pathway (PP) and Tricarboxy Acid Cycle (TCA) were found to be operated in ZR1. In addition, the energy saving ppi-dependent EMP enzyme, coupled with the complete and efficient central carbon metabolic network might be responsible for its fast growing nature. Transcript level analysis of the central carbon metabolism indicated that formaldehyde metabolism was a key nod that may be in charge of the carbon conversion efficiency (CCE) divergent of ZR1 grown on methanol and methane. Flexible nitrogen and carotene metabolism pattern were also investigated in ZR1. Nitrogenase genes in ZR1 were found to be highly expressed with methane even in the presence of sufficient nitrate. It appears that, higher lycopene production in ZR1 grown on methane might be attributed to the higher proportion of transcript level of C40 to C30 metabolic gene. Higher transcript level of exopolysaccharides metabolic gene and stress responding proteins indicated that ZR1 was confronted with severer growth stress with methanol than with methane. Additionally, lower transcript level of the TCA cycle, the dramatic high expression level of the nitric oxide reductase and stress responding protein, revealed the imbalance of the central carbon and nitrogen metabolic status, which would result in the worse growth of ZR1 with methanol at 30 °C.

Cytochrome cM was first described in 1994 and its sequence has been found in the genome of manifold cyanobacterial species ever since. Numerous studies have been carried out with the purpose of determining its function, but none of them has given place to conclusive results so far. Many of these studies are based on the assumption that cytochrome cM is a soluble protein located in the thylakoid lumen of cyanobacteria. In this work, we have reevaluated the sequence of cytochrome cM, with our results showing that its most probable 3D structure is strongly similar to that of the C subunit of the bacterial nitric oxide reductase. The potential presence of an α-helix tail, which could locate this protein in the thylakoid membrane, further supports this hypothesis, thus providing a new, unexpected role for this redox protein.

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N2-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.

Over-aeration is usually unavoidable for removing ammonia nitrogen from black-odorous water bodies, which consumes a large amount of organic matter and creates a long-term oxygen-rich environment, limiting the denitrification process in the water. This study aims to investigate the synergistic effect of aerobic denitrifying bacteria and iron-carbon micro-electrolysis (IC-ME) carriers on complete nitrogen removal from the over-aerated black-odorous water. For nitrification, aeration can completely oxidize ammonia nitrogen to nitrate. For denitrification, adding aerobic denitrifying bacteria Pseudomonas ATCC 9027 allowed the nitrate reduction to occur under conditions of excess oxygen and enriched indigenous denitrifying bacteria (1.45% increase in cumulative abundance). These changes brought by Pseudomonas ATCC 9027 reduced the total nitrogen concentration from 12.07 to 7.12 mg/L. Besides, research has shown that IC-ME carriers as electron donors can provide additional electrons and improve the electron transport system activity by 0.07 μg O2/(g protein∙min) in the denitrification process. Enzyme activity test and metagenome analysis revealed that enzymes and genes associated with denitrification were both upregulated with the addition of Pseudomonas ATCC 9027 and IC-ME carriers. Specifically, the improvement for activities of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), nitrous oxide reductase (NOS), and cumulative abundance of denitrification genes was 0.93 μmol NO2−-N/(g·d), 3.74 μmol NO2−-N/(g·d), 0.18 μmol N2O-N/(g·d) and 0.09 μmol N2O-N/(g·d) and 1.48%, respectively. These findings provide an understanding of the cooperative mechanism of aerobic denitrifying bacteria and IC-ME carriers in enhancing nitrogen removal from over-aerated black-odorous water bodies.

Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes., Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae. There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals. IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans, such as hemolytic and uremic syndrome. It has been previously demonstrated that the interplay between EHEC and nitric oxide (NO), a mediator of the host immune innate response, is critical for infection outcome, since NO affects both Shiga toxin (Stx) production and adhesion to enterocytes. In this study, we investigated the role of the NO reductase NorVW in the virulence and fitness of two EHEC strains in a murine model of infection. We determined that the deletion of norVW in the strain O91:H21 B2F1 has no impact on its virulence, whereas it reduces the ability of the strain O157:H7 620 to persist in the mouse gut and to produce Stx. We also revealed that the fitness defect of strain 620 &Delta, norVW is strongly attenuated when mice are treated with an NO synthase inhibitor. Altogether, these results demonstrate that the NO reductase NorVW participates in EHEC resistance against NO produced by the host and promotes virulence through the modulation of Stx synthesis. The contribution of NorVW in the EHEC infectious process is, however, strain-dependent and suggests that the EHEC response to nitrosative stress is complex and multifactorial.

AIMS: Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase), and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB, and nosZ in anaerobic growth, nitrate reduction, biofilm formation, and virulence. METHODS AND RESULTS: Four in-frame deletion mutants in virulent F. columnare strain 94–081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94–081, FcΔnasA, FcΔnirS, and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS, and FcΔnosZ compared to parent strain 94–081. Under anaerobic conditions, FcΔnasA, FcΔnorB, and FcΔnosZ formed significantly less biofilm than the wild type strain at 24 and 96 hours, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated in catfish, whereas FcΔnasA, FcΔnorB, and FcΔnosZ had similar virulence to FcWT. CONCLUSIONS: These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate potential for F. columnare to grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that a FcΔnirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease.

Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions., Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Legume-rhizobia symbiotic associations have beneficial effects on food security and nutrition, health and climate change. Hypoxia induced by flooding produces nitric oxide (NO) in nodules from soybean plants cultivated in nitrate-containing soils. As NO is a strong inhibitor of nitrogenase expression and activity, this negatively impacts symbiotic nitrogen fixation in soybean and limits crop production. In Bradyrhizobium diazoefficiens, denitrification is the main process involved in NO formation by soybean flooded nodules. In addition to denitrification, nitrate assimilation is another source of NO in free-living B. diazoefficiens cells and a single domain hemoglobin (Bjgb) has been shown to have a role in NO detoxification during nitrate-dependent growth. However, the involvement of Bjgb in protecting nitrogenase against NO in soybean nodules remains unclear. In this work, we have investigated the effect of inoculation of soybean plants with a bjgb mutant on biological nitrogen fixation. By analyzing the proportion of N in shoots derived from N2-fixation using the 15N isotope dilution technique, we found that plants inoculated with the bjgb mutant strain had higher tolerance to flooding than those inoculated with the parental strain. Similarly, reduction of nitrogenase activity and nifH expression by flooding was less pronounced in bjgb than in WT nodules. These beneficial effects are probably due to the reduction of NO accumulation in bjgb flooded nodules compared to the wild-type nodules. This decrease is caused by an induction of expression and activity of the denitrifying NO reductase enzyme in bjgb bacteroids. As bjgb deficiency promotes NO-tolerance, the negative effect of NO on nitrogenase is partially prevented and thus demonstrates that inoculation of soybean plants with the B. diazoefficiens bjgb mutant confers protection of symbiotic nitrogen fixation during flooding., This work was supported by the European Regional Development Fund (ERDF) co-financed grants from Ministerio de Economia y Competitividad, Spain [Grant Numbers: AGL2013-45087-R and AGL2017-85676R (MD)]; the Junta de Andalucia [Grant Number: PE2012-AGR1968 (EB)]; the Biotechnology and Biological Sciences Research Council [Grant Number: BB/M00256X/1 (AG)]; and the Royal Society International Exchanges Program, United Kingdom [Grant Number: IE140222 (AG and MD)]. AS was financed by a FPU contract from Ministerio de Educación, Cultura y Deporte (FPU14/04527).

Flavo-diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N(2)O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N(2)O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS(1)·S(2)), were found to differ substantially, +17 cm(−1) for the diiron mononitrosyl and +60 cm(−1) for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mossbauer hyperfine parameters and DFT calculations confirmed a semibridging NO(−) ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO(−) ligands, which could initiate N-N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N(2)O.

Development of low-temperature biological nitrogen removal processes is of scientific and engineering importance. Cold-adapted heterotrophic nitrifying and aerobic denitrifying (HNAD) bacteria have attracted increasing interest. However, the nitrogen metabolism and cold adaption mechanisms of HNAD bacteria remain unclear. In this article, a novel cold-adapted HNAD-capable bacterium, Pseudomonas indoloxydans YY-1, was isolated. Analyses of draft whole-genome features indicated that strain YY-1 was capable of complete dissimilatory nitrate reduction, ammonium assimilation, and cyanate decomposition. The gene cluster of napABCDE and gene norR, which encode for the periplasmic nitrate reductase and nitric oxide reductase transcription regulator, were identified in the YY-1 genome. Adenosine triphosphate levels increased fivefold, and polysaccharide content significantly rose in the extracellular polymeric substances of strain YY-1 when temperature decreased from 25 °C to 5 °C. Comparative transcriptional profiles of the strain grown at 25 °C and 10 °C revealed that the genes involved in tricarboxylic acid cycle, cytochrome reductase, transhydrogenase, and adenosine triphosphate synthesis were overexpressed, whereas the genes that encod for nicotinamide adenine dinucleotide dehydrogenase, cytochrome reductase, and the functional proteins of nitrate assimilation were downregulated. For ammonium assimilation of strain YY-1 at 10 °C, transcriptional data revealed the overexpression of glutamate dehydrogenase and glutamate synthase genes. This study highlights the potential nitrogen metabolic diversity of HNAD bacteria and expands the understanding of physiological and transcriptional strategies of cold adaption of those bacteria.

Bacterial NO reductases (NOR) catalyze the reduction of NO into N2O, either as a step in denitrification or as a detoxification mechanism. cNOR from Paracoccus (P.) denitrificans is expressed from the norCBQDEF operon, but only the NorB and NorC proteins are found in the purified NOR complex. Here, we established a new purification method for the P. denitrificans cNOR via a His-tag using heterologous expression in E. coli. The His-tagged enzyme is both structurally and functionally very similar to non-tagged cNOR. We were also able to express and purify cNOR from the structural genes norCB only, in absence of the accessory genes norQDEF. The produced protein is a stable NorCB complex containing all hemes and it can bind gaseous ligands (CO) to heme b3, but it is catalytically inactive. We show that this deficient cNOR lacks the non-heme iron cofactor FeB. Mutational analysis of the nor gene cluster revealed that it is the norQ and norD genes that are essential to form functional cNOR. NorQ belongs to the family of MoxR P-loop AAA+ ATPases, which are in general considered to facilitate enzyme activation processes often involving metal insertion. Our data indicates that NorQ and NorD work together in order to facilitate non-heme Fe insertion. This is noteworthy since in many cases Fe cofactor binding occurs spontaneously. We further suggest a model for NorQ/D-facilitated metal insertion into cNOR.

The fungicide chlorothalonil (CHT) has been widely used in the tea orchard due to its high-efficiency and sterilization. It has been reported that repeated application of CHT inhibits soil nitrification process. However, the acute impact of CHT on soil denitrification and associated N2O emissions is unclear. This study evaluated nitrate (NO3−) removal, denitrifying gene abundance and denitrifying enzyme activity of tea orchard soil after a 72-h-exposure to CHT. It was found that increasing CHT from 5 to 25 mg kg−1 suppressed the NO3− removal efficiency from 74.6% to 54.1%, but increased N2O emissions from 23.1% to 94.8%. Following treatment with 25 mg kg−1 of CHT, the abundances of the nirK, nirS and nosZ genes were reduced by 31.6%, 22.1%, and 50.7%, respectively. Alternatively, the declines of the electron transport system activity (ETSA) value and adenosine triphosphate (ATP) content suggested that CHT had an inhibitory effect on microbial metabolism. Enzyme activity studies further revealed that the decrease of nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR) activities was the main reason for the suppression of denitrification by CHT. Furthermore, positive correlations were observed between denitrifying reductase activity and the intracellular metabolism, indicating that the decrease in microbial metabolism should also be responsible for the inhibitory effect of CHT on the denitrifying process. Overall, it was found that the acute exposure of soil to CHT could inhibit the denitrification process and significantly increase N2O emissions, which might result in destruction of the soil nitrogen cycle and exacerbation of global warming.

Microbial denitrification is a main source of nitrous oxide (N2O) emissions which have strong greenhouse effect and destroy stratospheric ozone. Though the importance of sulfide driven chemoautotrophic denitrification has been recognized, its contribution to N2O emissions in nature remains elusive. We built up long-term sulfide-added microcosms with sediments from two freshwater lakes. Chemistry analysis confirmed sulfide could drive nitrate respiration in long term. N2O accumulated to over 1.5% of nitrate load in both microcosms after long-term sulfide addition, which was up to 12.9 times higher than N2O accumulation without sulfide addition. Metagenomes were extracted and sequenced during microcosm incubations. 16 S rRNA genes of Thiobacillus and Defluviimonas were gradually enriched. The nitric oxide reductase with c-type cytochromes as electron donors (cNorB) increased in abundance, while the nitric oxide reductase receiving electrons from quinols (qNorB) decreased in abundance. cnorB genes similar to Thiobacillus were enriched in both microcosms. In parallel, enrichment was observed for enzymes involved in sulfur oxidation, which supplied electrons to nitrate respiration, and enzymes involved in Calvin Cycle, which sustained autotrophic cell growth, implying the coupling relationship between carbon, nitrogen and sulfur cycling processes. Our results suggested sulfur pollution considerably increased N2O emissions in natural environments.

As bridge in global cycles of carbon, nitrogen, and sulfur, sulfate-reducing bacteria (SRB) play more and more important role under various environments, especially the saline-alkali environments with significant increase in area caused by human activities. Sulfate reduction can be inhibited by environmental nitrate. However, how SRB cope with environmental nitrate stress in these extreme environments still remain unclear. Here, after a long-term enrichment of sediment from saline-alkali Qinghai Lake of China using anaerobic filter reactors, nitrate was added to evaluate the response of SRB. With the increase in nitrate concentrations, the inhibition on sulfate reduction was gradually observed. Interestingly, extension of hydraulic retention time can relieve the inhibition caused by high nitrate concentration. Mass balance analysis showed that nitrate reduction is prior to sulfate reduction. Further metatranscriptomic analysis shows that, genes of nitrite reductase (periplasmic cytochrome c nitrite reductase gene) and energy metabolisms (lactate dehydrogenase, formate dehydrogenase, pyruvate:ferredoxin-oxidoreductase, and fumarate reductase genes) in SRB was down-regulated, challenging the long-held opinion that up-regulation of these genes can relieve the nitrate inhibition. Most importantly, the nitrate addition activated the denitrification pathway in denitrifying bacteria (DB) via significantly up-regulating the expression of the corresponding genes (nitrite reductase, nitric oxide reductase c subunit, nitric oxide reductase activation protein and nitrous oxide reductase genes), quickly reducing the environmental nitrate and relieving the nitrate inhibition on SRB. Our findings unravel that in response to environmental nitrate stress, haloalkaliphilic SRB show dependency on DB, and expand our knowledge of microbial relationship during sulfur and nitrogen cycles.

Marinobacter hydrocarbonoclasticus nitric oxide reductase, c NOR, is an integral membrane protein composed of two subunits with different roles, NorC (electron transfer) and NorB (catalytic) that receives electrons from the soluble cytochrome c 552 and reduces nitric oxide to nitrous oxide in the denitrification pathway. The solvent-exposed domain of NorC, harboring a c -type heme was heterologously produced, along with its physiological electron donor, cytochrome c 552 . These two proteins were spectroscopically characterized and shown to be similar to the native proteins, both being low-spin and Met-His coordinated, with the soluble domain of NorC presenting some additional features of a high-spin heme, which is consistent with the higher solvent accessibility of its heme and weaker coordination of the methionine axial ligand. The electron transfer complex between the two proteins has a 1:1 stoichiometry, and an upper limit for the dissociation constant was estimated by 1 H NMR titration to be 1.2 ± 0.4 μM. Electrochemical techniques were used to characterize the interaction between the proteins, and a model structure of the complex was obtained by molecular docking. The electrochemical observations point to the modulation of the NorC reduction potential by the presence of NorB, tuning its ability to receive electrons from cytochrome c 552 .

This work was funded by research grant from the MINECO Spain (CTM2013-43147-R) and Generalitat Valenciana (ACIF 2016/077).

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O2, and found that the pKa of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

The potential for aerobic NO2- removal by Alcaligenes faecalis strain NR was investigated. 35 mg/L of NO2--N was removed by strain NR under aerobic conditions in the presence of NH4+. 15N-labeling experiment demonstrated that N2O and N2 were possible products during the aerobic nitrite removal process by strain NR. The key enzyme genes of nirK, norB and nosZ, which regulate the aerobic nitrite denitrification process, were successfully amplified from strain NR. The gene sequence analysis indicates that copper-containing nitrite reductase (NIRK) and periplasmic nitrous oxide reductase (NOSZ) were both hydrophilic protein and the transmembrane structures were absent, while nitric oxide reductase large subunit (NORB) was a hydrophobic and transmembrane protein. According to the three-dimensional structure and binding site analysis, the bulky and hydrophobic methionine residue proximity to the nitrite binding sites of NIRK was speculated to be related to the oxygen tolerance of NIRK from strain NR.

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0.70 μmol/g of the total protein, which showed a single band on a SDS-PAGE and Western blot. The direct electrochemistry of CYP55B1 in dihexadecylphosphate (DHP) film was realized with an electric potential at −0.47 V at the scan rate of 1 V s−1. We studied the in vitro interaction between P450 55B1 and NO by the fluorescence spectrometric method. The results show that the fluorescence intensity of iron-porphyrin in P450 55B1 changes gradually with the addition of NO. The fluorescence intensity change values against NO concentrations were plotted, and it showed a linear range of NO from 0 to 22.5 μM with a sensitivity of 0.15 μM/AU and a detection limit of 0.15 μM.

N-acyl-homoserine lactones (AHLs) serve as quorum-sensing signals, which control a number of bacterial processes in many proteobacteria. Here we report the effects of exogenous short-chain AHL on the denitrifying process of Paracoccus denitrificans, which are capable of aerobic and anaerobic growth by utilizing nitrate. The denitrification activity of these cells was monitored by measuring denitrification products (including nitrate, nitrite, and nitrous oxide), and the individual messenger ribonucleic acid (mRNA) levels of nitrate, nitrite, nitric oxide and nitrous oxide reductases. The results indicated that 2μmol/L C6-homoserine lactone (HSL) has little effect on cell density under either anaerobic or aerobic culture conditions, and the nitrate reduction activity appeared slightly affected by N-hexanoyl-DL-homoserine lactone (C6-HSL). However, exogenous C6-HSL significantly affected the transcription of nitrite reductase and nitric oxide reductase genes in P. denitrificans regardless of the presence of oxygen, and N2O accumulation activity in P. denitrificans was suppressed by C6-HSL under aerobic condition. In contrast, exogenous C6-HSL stimulated the production of N2O under anaerobic condition, suggesting that the regulation of denitrification by quorum sensing may be important in N2O release.

Halomonas halodenitrificans nitric oxide reductase (NOR) is the membrane-bound heterodimer complex of NorC, which contains a low-spin heme c center, and NorB, which contains a low-spin heme b center, a high-spin heme b3 center, and a non-heme FeB center. The soluble domain of NorC, NorC* (ΔMet1-Val37) was heterologously expressed in Escherichia coli using expression plasmids harboring the truncated norC gene deleted of its 84 5'-terminal nucleotides. Analogous scission of the N-terminal helix as the membrane anchor took place when the whole norC gene was used. NorC* exhibited spectra typical of a low-spin heme c. In addition, NorC* functioned as the acceptor of an electron from a cytochrome c isolated from the periplasm of H. halodenitrificans and small reducing reagents. The redox potential of NorC* shifted ca. 40mV in the negative direction from that of NorC. Unlike NorC, recombinant NorB was not heterologously expressed. However, recombinant NOR (rNOR) could be expressed in E. coli by using a plasmid harboring all genes in the nor operon, norCBQDX, from which the three hairpin loops (mRNA) were deleted, and by using the ccm genes for the maturation of C-type heme. rNOR exhibited the same spectroscopic properties and reactivity to NO and O2 as NOR, although its enzymatic activity toward NO was considerably decreased. These results on the expression of rNOR and NorC* will allow us to develop more profound studies on the properties of the four Fe centers and the reaction mechanism of NOR from this halophilic denitrifying bacterium.

The goal of this study was to elucidate the mechanisms of nitrous oxide (N2O) production from a bioreactor for partial nitrification (PN). Ammonia-oxidizing bacteria (AOB) enriched from a sequencing batch reactor (SBR) were subjected to N2O production pathway tests. The N2O pathway test was initiated by supplying an inorganic medium to ensure an initial NH4+-N concentration of 160 mg-N/L, followed by 15NO2– (20 mg-N/L) and dual 15NH2OH (each 17 mg-N/L) spikings to quantify isotopologs of gaseous N2O (44N2O, 45N2O, and 46N2O). N2O production was boosted by 15NH2OH spiking, causing exponential increases in mRNA transcription levels of AOB functional genes encoding hydroxylamine oxidoreductase (haoA), nitrite reductase (nirK), and nitric oxide reductase (norB) genes. Predominant production of 45N2O among N2O isotopologs (46% of total produced N2O) indicated that coupling of 15NH2OH with 14NO2– produced N2O via N-nitrosation hybrid reaction as a predominant pathway. Abiotic hybrid N2O production was also obse...

NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.