Plant growth and nitrate absorption and assimilation of two sweet potato cultivars with different N tolerances in response to nitrate supply.

In sweet potato, rational nitrogen (N) assimilation and distribution are conducive to inhibiting vine overgrowth. Nitrate (NO3-) is the main N form absorbed by roots, and cultivar is an important factor affecting N utilization. Herein, a hydroponic experiment was conducted that included four NO3- concentrations of 0 (N0), 4 (N1), 8 (N2) and 16 (N3) mmol L-1 with two cultivars of Jishu26 (J26, N-sensitive) and Xushu32 (X32, N-tolerant). For J26, with increasing NO3- concentrations, the root length and root surface area significantly decreased. However, no significant differences were observed in these parameters for X32. Higher NO3- concentrations upregulated the expression levels of the genes that encode nitrate reductase (NR2), nitrite reductase (NiR2) and nitrate transporter (NRT1.1) in roots for both cultivars. The trends in the activities of NR and NiR were subject to regulation of NR2 and NiR2 transcription, respectively. For both cultivars, N2 increased the N accumulated in leaves, growth points and roots. For J26, N3 further increased the N accumulation in these organs. Under higher NO3- nutrition, compared with X32, J26 exhibited higher expression levels of the NiR2, NR2 and NRT1.1 genes, a higher influx NO3- rate in roots, and higher activities of NR and NiR in leaves and roots. Conclusively, the regulated effects of NO3- supplies on root growth and NO3- utilization were more significant for J26. Under high NO3- conditions, J26 exhibited higher capacities of NO3- absorption and distributed more N in leaves and in growth points, which may contribute to higher growth potential in shoots and more easily cause vine overgrowth.

Nitrite reductase-mimicking catalysis temporally regulating nitric oxide concentration gradient adaptive for antibacterial therapy.

The unique bacterial infection microenvironment (IME) usually requires complicated design of nanomaterials to adapt to IME for enhancing antibacterial therapy. Here, an alternative IME adaptative nitrite reductase-mimicking nanozyme is constructed by in situ growth of ultrasmall copper sulfide clusters on the surface of a nanofibrillar lysozyme assembly (NFLA/CuS NHs), which can temporally regulate nitric oxide (NO) gradient concentration to kill bacteria initially and promote tissue regeneration subsequently. Benefiting from a copper nitrite reductase (CuNIR)-inspired structure with CuS cluster as active center and NFLA as skeleton, NFLA/CuS NHs efficiently boost the catalytic reduction of nitrite to NO. The inherent supramolecular fibrillar networks displays excellent bacterial capture capability, facilitating initial high-concentration NO attacks on the bacteria. The subsequent catalytic release of low-concentration NO by NFLA/CuS NHs-mediated nitrite reduction remarkably promotes cell migration and angiogenesis. This work paves the way for dynamically eliminating MDR bacterial infection and promoting tissue regeneration in a simple and smart way through CuNIR-mimicking catalysis.

Unraveling the impact of perfluorooctanoic acid on sulfur-based autotrophic denitrification process.

PFOA has garnered heightened scrutiny for its impact on denitrification, especially given its frequent detection in secondary effluent discharged from wastewater treatment plants. However, it is still unclear what potential risk PFOA release poses to a typical advanced treatment process, especially the sulfur-based autotrophic denitrification (SAD) process. In this study, different PFOA concentration were tested to explore their impact on denitrification kinetics and microbial dynamic responses of the SAD process. The results showed that an increase PFOA concentration from 0 to 1000 µg/L resulted in a decrease in nitrate removal rate from 9.52 to 7.73 mg-N/L·h. At the same time, it increased nitrite accumulation and N2O emission by 6.11 and 2.03 times, respectively. The inhibitory effect of PFOA on nitrate and nitrite reductase activity in the SAD process was linked to the observed fluctuations in nitrate and nitrite levels. It is noteworthy that nitrite reductase was more vulnerable to the influence of PFOA than nitrate reductase. Furthermore, PFOA showed a significant impact on gene expression and microbial community. Metabolic function prediction revealed a notable decrease in nitrogen metabolism and an increase in sulfur metabolism under PFOA exposure. This study highlights that PFOA has a considerable inhibitory effect on SAD performance.

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases.

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys â†’ T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

The enigmatic enzyme 'amidoxime reducing component' of Lotus japonicus. Characterization, expression, activity in plant tissues, and proposed role as a nitric oxide-forming nitrite reductase.

Human mitochondria contain a molybdoprotein capable of reducing amidoximes using cytochrome b5/cytochrome b5 reductase (Cb/CbR). This 'amidoxime reducing component' (ARC) also reduces nitrite to nitric oxide (NO). In the plant kingdom, distinct functions have been suggested for ARCs. Thus, the single ARC of Chlamydomonas reinhardtii (crARC) reduces nitrite to NO by taking electrons from nitrate reductase (NR). Therefore, it was proposed that a dual NR/crARC system can generate NO under physiological conditions and the crARC was renamed to 'NO-forming nitrite reductase' (NOFNiR). In contrast to this, the two ARC enzymes from Arabidopsis thaliana were not found to produce NO in vitro at physiological nitrite concentrations, suggesting a different, as yet unknown, function in vascular plants. Here, we have investigated the two ARCs of Lotus japonicus (LjARCs) to shed light on this controversy and to examine, for the first time, the distribution of ARCs in plant tissues. The LjARCs are localized in the cytosol and their activities and catalytic efficiencies, which are much higher than those of A. thaliana, are consistent with a role as NOFNiR. LjARCs are prone to S-nitrosylation in vitro by S-nitrosoglutathione and this post-translational modification drastically inhibits their activities. The enzymes are mainly expressed in flowers, seeds and pods, but are absent in nodules. LjARCs are active with NR and Cb/CbR as electron-transferring systems. However, the LjNR mRNA levels in seeds and pods are negligible, whereas our proteomic analyses show that pods contain the two ARCs, Cb and CbR. We conclude that LjARCs may play a role as NOFNiR by receiving electrons from the Cb/CbR system but do not act in combination with NR.

High nitrite accumulation in hydrogenotrophic denitrification at low temperature: Transcriptional regulation and microbial community succession.

High Pressure Hydrogenotrophic Denitrification (HPHD) provided a promising alternative for efficient and clean nitrate removal. In particular, the denitrification rates at low temperature could be compensated by elevated H2 partial pressure. However, nitrite reduction was strongly inhibited while nitrate reduction was barely affected at low temperature. In this study, the nitrate reduction gradually recovered under long-term low temperature stress, while nitrite accumulation increased from 0.1 to 41.0 mg N/L. The activities of the electron transport system (ETS), nitrate reductase (NAR), and nitrite reductase (NIR) decreased by 45.8 %, 27.3 %, and 39.3 %, respectively, as the temperature dropped from 30 °C to 15 °C. Real time quantitative PCR analysis revealed that the denitrifying gene expression rather than gene abundance regulated nitrogen biotransformation. The substantial nitrite accumulation was attributed to the significant up-regulation by 54.7 % of narG gene expression and down-regulation by 73.7 % of nirS gene expression in hydrogenotrophic denitrifiers. In addition, the nirS-gene-bearing denitrifiers were more sensitive to low temperature compared to those bearing nirK gene. The dominant populations shifted from the genera Paracoccus to Hydrogenophaga under long-term low temperature stress. Overall, this study revealed the microbial mechanism of high nitrite accumulation in hydrogenotrophic denitrification at low temperature.

Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in Prokaryotes and eukaryotes.

The microbe-mediated conversion of nitrate (NO3-) to ammonium (NH4+) in the nitrogen cycle has strong implications for soil health and crop productivity. The role of prokaryotes, eukaryotes and their phylogeny, physiology, and genetic regulations are essential for understanding the ecological significance of this empirical process. Several prokaryotes (bacteria and archaea), and a few eukaryotes (fungi and algae) are reported as NO3- reducers under certain conditions. This process involves enzymatic reactions which has been catalysed by nitrate reductases, nitrite reductases, and NH4+-assimilating enzymes. Earlier reports emphasised that single-cell prokaryotic or eukaryotic organisms are responsible for this process, which portrayed a prominent gap. Therefore, this study revisits the similarities and uniqueness of mechanism behind NO3- -reduction to NH4+ in both prokaryotes and eukaryotes. Moreover, phylogenetic, physiological, and genetic regulation also shed light on the evolutionary connections between two systems which could help us to better explain the NO3--reduction mechanisms over time. Reports also revealed that certain transcription factors like NtrC/NtrB and Nit2 have shown a major role in coordinating the expression of NO3- assimilation genes in response to NO3- availability. Overall, this review provides a comprehensive information about the complex fermentative and respiratory dissimilatory nitrate reduction to ammonium (DNRA) processes. Uncovering the complexity of this process across various organisms may further give insight into sustainable nitrogen management practices and might contribute to addressing global environmental challenges.

Effects of sliver nanoparticles on nitrogen removal by the heterotrophic nitrification-aerobic denitrification bacteria Zobellella sp. B307 and their toxicity mechanisms.

Due to the widespread use of sliver nanoparticles (AgNPs), a large amount of AgNPs has inevitably been released into the environment, and there is growing concern about the toxicity of AgNPs to nitrogen-functional bacteria. In addition to traditional anaerobic denitrifying bacteria, heterotrophic nitrification-aerobic denitrification (HNAD) bacteria are also important participants in the nitrogen cycle. However, the mechanisms by which AgNPs influence HNAD bacteria have yet to be explicitly demonstrated. In this study, the inhibitory effects of different concentrations of AgNPs on a HNAD bacteria Zobellella sp. B307 were investigated, and the underlying mechanism was explored by analyzing the antioxidant system and the activities of key denitrifying enzymes. Results showed that AgNPs could inhibit the growth and the HNAD ability of Zobellella sp. B307. AgNPs could accumulate on the surface of bacterial cells and significantly destroyed the cell membrane integrity. Further studies demonstrated that the presence of high concentration of AgNPs could result in the overproduction of reactive oxygen species (ROS) and related oxidative stress in the cells. Furthermore, the catalytic activities of key denitrifying enzymes (nitrate reductase (NAR), nitrite reductase (NIR), and nitrous oxide reductase (N2OR)) were significantly suppressed under exposure to a high concentration of AgNPs (20 mg·L-1), which might be responsible for the inhibited nitrogen removal performance of strain B307. This work could improve our understanding of the inhibitory effect and underlying mechanism of AgNPs on HNAD bacteria.

Inhibitory mechanism of Cr(VI) on sulfur-based denitrification: Bio-toxicity, bio-electron characteristics, and microbial evolution.

Sulfur-based denitrification is a promising technology for efficient nitrogen removal in low-carbon wastewater, while it is easily affected by toxic substances. This study revealed the inhibitory mechanism of Cr(VI) on thiosulfate-based denitrification, including bio-toxicity and bio-electron characteristics response. The activity of nitrite reductase (NIR) was more sensitive to Cr(VI) than that of nitrate reductase (NAR), and NIR was inhibited by 21.32 % and 19.86 % under 5 and 10 mg/L Cr(VI), resulting in 10.12 and 15.62 mg/L of NO2--N accumulation. The biofilm intercepted 36.57 % of chromium extracellularly by increasing 25.78 % of extracellular polymeric substances, thereby protecting microbes from bio-toxicity under 5 mg/L Cr(VI). However, it was unable to resist 20-30 mg/L of Cr(VI) bio-toxicity as 19.95 and 14.29 mg Cr/(g volatile suspended solids) invaded intracellularly, inducing the accumulation of reactive oxygen species by 165.98 % and 169.12 %, which triggered microbial oxidative-stress and damaged the cells. In terms of electron transfer, S2O32- oxidation was inhibited, and parts of electrons were redirected intracellularly to maintain microbial activity, resulting in insufficient electron donors. Meanwhile, the contents of flavin adenine dinucleotide and cytochrome c decreased under 5-30 mg/L Cr(VI), reducing the electron acquisition rate of denitrification. Thermomonas (the dominant genus) possessed denitrification and Cr(VI) resistance abilities, playing an important role in antioxidant stress and biofilm formation. ENVIRONMENTAL IMPLICATION: Sulfur-based denitrification (SBD) is a promising method for nitrate removal in low-carbon wastewater, while toxic heavy metals such as Cr(VI) negatively impair denitrification. This study elucidated Cr(VI) inhibitory mechanisms on SBD, including bio-toxicity response, bio-electron characteristics, and microbial community structure. Higher concentrations Cr(VI) led to intracellular invasion and oxidative stress, evidenced by ROS accumulation. Moreover, Cr(VI) disrupted electron flow by inhibiting thiosulfate oxidation and affecting electron acquisition by denitrifying enzymes. This study provided valuable insights into Cr(VI) toxicity, which is of great significance for improving wastewater treatment technologies and maintaining efficient and stable operation of SBD in the face of complex environmental challenges.

Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria.

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.

A novel mechanism for dissimilatory nitrate reduction to ammonium in Acididesulfobacillus acetoxydans.

The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB) Acididesulfobacillus acetoxydans can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase, A. acetoxydans lacks recognized nitrite reductase genes. In this study, A. acetoxydans was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with A. acetoxydans growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.IMPORTANCENitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by Acididesulfobacillus acetoxydans, an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA). A. acetoxydans was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.

Comparative transcriptome analysis and Arabidopsis thaliana overexpression reveal key genes associated with cadmium transport and distribution in root of two Capsicum annuum cultivars.

The molecular mechanisms underlying high and low cadmium (Cd) accumulation in hot pepper cultivars remain unclear. In this study, comparative transcriptome analysis of root between high-Cd (J) and low-Cd (Z) cultivars was conducted under hydroponic cultivation with 0 and 0.4 mg/L Cd, respectively. The results showed that J enhanced the root uptake of Cd by elevating the expression of Nramp5 and counteracting Cd toxicity by increasing the expression of genes, such as NIR1, GLN1, and IAA9. Z reduced Cd accumulation by enhancing the cell wall lignin synthesis genes PAL, COMT, 4CL, LAC, and POD and the Cd transporters ABC, MTP1, and DTX1. Elevated expression of genes related to sulfur metabolism was observed in Z, potentially contributing to its ability to detoxify Cd. To investigate the function of CaCOMT1, an Arabidopsis thaliana overexpression line (OE-CaCOMT1) was constructed. The results revealed that OE-CaCOMT1 drastically increased the lignin content by 38-42% and reduced the translocation of Cd to the aboveground parts by 32%. This study provides comprehensive insights into the mechanisms underlying Cd accumulation in hot pepper cultivars using transcriptome analysis. Moreover, this study elucidates the critical function of CaCOMT1, providing a theoretical foundation for the production of low-Cd vegetables for food safety.

Exercise Training Decreases Nitrite Concentration in the Heart and Locomotory Muscles of Rats Without Changing the Muscle Nitrate Content.

BACKGROUND: Skeletal muscles are postulated to be a potent regulator of systemic nitric oxide homeostasis. In this study, we aimed to evaluate the impact of physical training on the heart and skeletal muscle nitric oxide bioavailability (judged on the basis of intramuscular nitrite and nitrate) in rats. METHODS AND RESULTS: Rats were trained on a treadmill for 8 weeks, performing mainly endurance running sessions with some sprinting runs. Muscle nitrite (NO2-) and nitrate (NO3-) concentrations were measured using a high-performance liquid chromatography-based method, while amino acids, pyruvate, lactate, and reduced and oxidized glutathione were determined using a liquid chromatography coupled with tandem mass spectrometry technique. The content of muscle nitrite reductases (electron transport chain proteins, myoglobin, and xanthine oxidase) was assessed by western immunoblotting. We found that 8 weeks of endurance training decreased basal NO2- in the locomotory muscles and in the heart, without changes in the basal NO3-. In the slow-twitch oxidative soleus muscle, the decrease in NO2- was already present after the first week of training, and the content of nitrite reductases remained unchanged throughout the entire period of training, except for the electron transport chain protein content, which increased no sooner than after 8 weeks of training. CONCLUSIONS: Muscle NO2- level, opposed to NO3-, decreases in the time course of training. This effect is rapid and already visible in the slow-oxidative soleus after the first week of training. The underlying mechanisms of training-induced muscle NO2- decrease may involve an increase in the oxidative stress, as well as metabolite changes related to an increased muscle anaerobic glycolytic activity contributing to (1) direct chemical reduction of NO2- or (2) activation of muscle nitrite reductases.

Effects of intensive chlorine disinfection on nitrogen and phosphorus removal in WWTPs.

The increased use of disinfection since the pandemic has led to increased effective chlorine concentration in municipal wastewater. Whereas, the specific impacts of active chlorine on nitrogen and phosphorus removal, the mediating communities, and the related metabolic activities in wastewater treatment plants (WWTPs) lack systematic investigation. We systematically analyzed the influences of chlorine disinfection on nitrogen and phosphorus removal activities using activated sludge from five full-scale WWTPs. Results showed that at an active chlorine concentration of 1.0 mg/g-SS, the nitrogen and phosphorus removal systems were not significantly affected. Major effects were observed at 5.0 mg/g-SS, where the nitrogen and phosphorus removal efficiency decreased by 38.9 % and 44.1 %, respectively. At an active chlorine concentration of 10.0 mg/g-SS, the nitrification, denitrification, phosphorus release and uptake activities decreased by 15.1 %, 69.5-95.9 %, 49.6 % and 100 %, respectively. The proportion of dead cells increased by 6.1 folds. Reverse transcriptional quantitative polymerase chain reaction (RT-qPCR) analysis showed remarkable inhibitions on transcriptions of the nitrite oxidoreductase gene (nxrB), the nitrite reductase genes (nirS and nirK), and the nitrite reductase genes (narG). The nitrogen and phosphorus removal activities completely disappeared with an active chlorine concentration of 25.0 mg/g-SS. Results also showed distinct sensitivities of different functional bacteria in the activated sludge. Even different species within the same functional group differ in their susceptibility. This study provides a reference for the understanding of the threshold active chlorine concentration values which may potentially affect biological nitrogen and phosphorus removal in full-scale WWTPs, which are expected to be beneficial for decision-making in WWTPs to counteract the potential impacts of increased active chlorine concentrations in the influent wastewater.

Cyclic di-GMP inhibits nitrate assimilation by impairing the antitermination function of NasT in Pseudomonas putida.

The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.

Incubation study on remediation of nitrate-contaminated soil by Chroococcus sp.

The possibility of using the non-nitrogen-fixing cyanobacterium (Chroococcus sp.) for the reduction of soil nitrate contamination was tested through Petri dish experiments. The application of 0.03, 0.05 and 0.08 mg/cm2 Chroococcus sp. efficiently removed NO3--N from the soil through assimilation of nitrate nutrient and promotion of soil denitrification. At the optimal application dose of 0.05 mg/cm2, 44.06%, 36.89% and 36.17% of NO3--N were removed at initial NO3--N concentrations of 60, 90 and 120 mg/kg, respectively. The polysaccharides released by Chroococcus sp. acted as carbon sources for bacterial denitrification and facilitated the reduction of soil salinity, which significantly (p < 0.05) stimulated the growth of denitrifying bacteria (Hyphomicrobium denitrificans and Hyphomicrobium sp.) as well as significantly (p < 0.05) elevated the activities of nitrate reductase and nitrite reductase by 1.07-1.23 and 1.15-1.22 times, respectively. The application of Chroococcus sp. promoted the dominance of Nocardioides maradonensis in soil microbial community, which resulted in elevated phosphatase activity and increased available phosphorus content. The application of Chroococcus sp. positively regulated the growth of soil bacteria belonging to the genera Chitinophaga, Prevotella and Tumebacillus, which may contribute to increased soil fertility through the production of beneficial enzymes such as invertase, urease and catalase. To date, this is the first study verifying the remediation effect of non-nitrogen-fixing cyanobacteria on nitrate-contaminated soil.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

An unusual active site architecture in cytochrome c nitrite reductase NrfA-1 from Geobacter metallireducens.

Dissimilatory nitrate reduction to ammonia (DNRA) is a central pathway in the biogeochemical nitrogen cycle, allowing for the utilization of nitrate or nitrite as terminal electron acceptors. In contrast to the competing denitrification to N2, a major part of the essential nutrient nitrogen in DNRA is retained within the ecosystem and made available as ammonium to serve as a nitrogen source for other organisms. The second step of DNRA is mediated by the pentahaem cytochrome c nitrite reductase NrfA that catalyzes the six-electron reduction of nitrite to ammonium and is widely distributed among bacteria. A recent crystal structure of an NrfA ortholog from Geobacter lovleyi was the first characterized representative of a novel subclass of NrfA enzymes that lacked the canonical Ca2+ ion close to the active site haem 1. Here, we report the structural and functional characterization of NrfA from the closely related G. metallireducens. We established the recombinant production of catalytically active NrfA with its unique, lysine-coordinated active site haem heterologously in Escherichia coli and determined its three-dimensional structure by X-ray crystallography to 1.9 Å resolution. The structure confirmed GmNrfA as a further calcium-independent NrfA protein, and it also shows an altered active site that contained an unprecedented aspartate residue, D80, close to the substrate-binding site. This residue formed part of a loop that also caused a changed arrangement of the conserved substrate/product channel relative to other NrfA proteins and rendered the protein insensitive to the inhibitor sulphate. To elucidate the relevance of D80, we produced and studied the variants D80A and D80N that showed significantly reduced catalytic activity.

Shifts of active microbial community structure and functions in constructed wetlands responded to continuous decreasing temperature in winter.

Important functions of constructed wetland related to biogeochemical processes are mediated by soil microbes and low-temperature damage is the main limiting factor for microbes in winter. However, the response thresholds for active microbial community and enzyme activities to continuous decreases in temperature remain unclear. In this study, total 90 soil samples were collected every week over a 6-week period to track the dynamics of four enzymes involved in cycles of C, N, P and active bacterial community as field soil temperature decreased continuously from 6.62 °C to 0.55 °C. Enzyme activity changed suddenly when the temperature decreased to 4.83 °C, the nitrite reductase activity reduced by 36.2%, while alkaline phosphatase activity is increased by 396%. The cellulase and urease were only marginally influenced by cold stress. Decreased nitrite reductase activities corresponded with loss of nir-type denitrifiers important for nitrite reduction. For cold stress, N-related bacteria were sensitive species. Whereas increased alkaline phosphatase activity may be due to the fact that P-related bacteria were opportunistic species. Key functional taxa connected with degradation of cellulose promoted species coexistence and microbial network stability. The lower and upper temperature thresholds for community change were 4.85 °C and 6.30 °C, respectively. Collectively, these results revealed that microbial taxa involved in C, N and P cycling respond differently to continuous decreases in temperature and higher than 4.85 °C is an ideal environment to prevent loss of microbial diversity and functions in winter, providing a scientific reference for the targeted isolation and cultivation of key microbial taxa in rhizosphere soil and adjusting temperature range to improve the purification capacity of wetlands during low temperature periods.