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Fumarate and nitrate reduction regulatory protein (Fnr)-a global transcriptional regulator-can directly or indirectly regulate many genes in different metabolic pathways at the top of the bacterial transcription regulation network. The present study explored the regulatory mechanism of Fnr-mediated nitrite degradation in Lactobacillus plantarum WU14 through gene transcription and expression analysis of oxygen sensing and nir operon expression regulation by Fnr. The interaction and the mechanism of transcriptional regulation between Fnr and GlnR were also examined under nitrite stress. After Fnr and GlnR purification by glutathione S-transferase tags, they were successfully expressed in Escherichia coli by constructing an expression vector. The results of electrophoresis mobility shift assay and qRT-PCR indicated that Fnr specifically bound to the PglnR and Pnir promoters and regulated the expression of nitrite reductase (Nir) and GlnR. After 6-12 h of culture, the expressions of fnr and nir under anaerobic conditions were higher than under aerobic conditions; the expression of these two genes increased with sodium nitrite (NaNO 2 ) addition during aerobic culture. Overall, the present study indicated that Fnr not only directly participated in the expression of Nir and GlnR but also indirectly regulated the expression of Nir through GlnR regulation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis. The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at -87 to -84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended -10 promoter, it lacks a reasonable -35 element. Genetic analysis and structural simulations predicted that the absence of the -35 element might be compensated by interactions between σ A and αCTD and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified, and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors, including the ubiquitous response regulators of two-component regulatory systems, particularly in Gram-positive bacteria.

Cu-containing nitrite reductases that convert NO 2 - to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br 2D NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br 2D NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br 2D NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br 2D NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br 2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO 2 - ) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462 T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462 T . Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.

Studies have shown that the addition of biochar to agricultural soils has the potential to mitigate climate change by decreasing nitrous oxide (N 2 O) emissions resulting from denitrification. Rice paddy field soils have been known to have strong denitrifying activity, but the response of microbes to biochar for weakening denitrification in rice paddy field soils is not well known. In this work, compared with the chemical fertilizer alone, the chemical fertilizer + 20 t hm -2 biochar fertilizer slightly decreased denitrifying the nitrite reductase activity (S-NiR) and N 2 O emission without statistic difference, whereas the chemical fertilizer + 40 t hm -2 biochar significantly boosted them. The abundance of nir-denitrifiers contributed to S-NiR and N 2 O emission, especially nirS-denitrifiers, rather than the variation of community structure. Pearson correlation analysis showed that NO 2 - -N was a key factor for controlling the abundance of nir-denitrifiers, S-NiR and N 2 O emission. The biochar addition fertilization treatments strongly shaped the community structure of nirK-denitrifiers, while the community structure of nirS-denitrifiers remained relatively stable. In addition, Paracoccus and Sinorhizobium were revealed to be as the predominant lineage of nirS- and nirK-containing denitrifiers, respectively. Distance-based redundancy analysis (db-RDA) showed that changes in the nir-denitrifier community structure were significantly related to soil organic carbon, NO 3 - -N, and total phosphorus. Our findings suggest that, although the nirS- and nirK-denitrifiers are both controlling nitrite reductase, their responses to biochar addition fertilization treatments showed significant discrepancies of diversity, abundance, and contribution to N 2 O and S-NiR in a paddy soil.

Shewanella oneidensis, a metal reducer and facultative anaerobe, expresses a large number of c-type cytochromes, many of which function as anaerobic reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. Two c-type cytochrome reductases also possess atypical heme-binding sites, the NrfA nitrite reductase (CXXCK) and the SirA sulfite reductase (CX 12 NKGCH). S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). SirE located in the sir gene cluster is required for the maturation of SirA, but not NrfA. Here we show that maturation of SirA requires the combined function of the two apocytochrome c chaperones CcmI and SirG. Loss of either protein resulted in decreased sulfite reductase. Furthermore, SirA was not detected in a mutant that lacked both chaperones, perhaps due to misfolding or instability. These results suggest that CcmI interacts with SirEFG during SirA maturation, and with CcmF during maturation of NrfA. Additionally, we show that CRP regulates expression of sirA via the newly identified transcriptional regulatory protein, SirR.

Denitrifying bacteria is a driver of nitrogen removal process in wastewater treatment ecosystem. However, the geographical characteristics of denitrifying bacterial communities associated with activated sludge from diverse wastewater treatment plants (WWTPs) are still unclear. Here, quantitative PCR and next-generation sequencing of the nirS gene were applied to characterize the abundance and denitrifying bacterial communities from 18 geographically distributed WWTPs. The results showed that the nirS abundance ranged from 4.6 × 10 2 to 2.4 × 10 3 copies per ng DNA, while nirS-type denitrifying bacterial populations were diverse and distinct from activated sludge communities. Among WWTPs, total nitrogen removal efficiencies varied from 25.8 to 84%, which was positively correlated with diversity indices, whereas abundance-based coverage estimator index decreased with an increase in latitude. The dominant phyla across all samples were proteobacteria, accounting for 46.23% (ranging from 17.98 to 87.07%) of the sequences. Eight of the 22 genera detected were dominant: Thauera sp., Alicycliphilus sp., and Pseudomonas sp., etc. Based on network analysis, the coexistence and interaction between dominant genera may be vital for regulating the nitrogen and carbon removal behaviors. Multivariate statistical analysis revealed that both geographic location and wastewater factors concurrently govern the distribution patterns of nirS-type denitrifying bacterial community harbored in WWTPs. Taking together, these results from the present study provide novel insights into the nirS gene abundance and nirS-type denitrifying bacterial community composition in geographically distributed WWTPs. Moreover, the knowledge gained will improve the operation and management of WWTPs for nitrogen removal.

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k cat =3.9(4) s -1 ) lower than that of wt SmNir (k cat =240(50) s -1 ). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N δ1 H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the N δ1 H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The potential for aerobic NO 2 - removal by Alcaligenes faecalis strain NR was investigated. 35 mg/L of NO 2 - -N was removed by strain NR under aerobic conditions in the presence of NH 4 + . 15 N-labeling experiment demonstrated that N 2 O and N 2 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.

The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N 2 O to molecular nitrogen (N 2 ) by four membrane-localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine-tuned stable and transient protein-protein interactions. Recently, an interactomic approach was used to determine the exact protein-protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane-localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N 2 O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N 2 O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein-protein complexes and interactions involved in the assembly and function of multi-enzyme complexes., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To reflect the importance of nitrite reductase (NIR) in the environment, we studied its distribution., Methods: The sequences of NIR were searched in the sequenced genome database at NCBI based on previous reported NIR sequences. The sequence similarity was done by multiple sequence alignment, and phylogenetic relationship was evaluated via constructing the phylogenetic tree. Furthermore, their distribution in the marine metagenome was studied by metagenomics., Results: The homologues of these two enzymes were 397 and 812 strains in sequenced genome, and the proportion was 8 and 15.7 percent, respectively. Almost all of archaea containing type II NIR. They have high identity by multiple sequence alignment analysis. The cofactor, the substrate and the cooper binding sites in type II were high conserved, suggesting that these enzymes had the specific function in denitrification. Phylogenetic analysis showed the two enzymes may have the common ancestor. In marine metagenome analysis, type I and II have 6 and 35 reads per 100000 reads, respectively, the two types of NIRs have the biggest proportion at the tropical south pacific area., Conclusion: Collectively, we suggested NIR, especially type II, play a key role in bioremediation of nitrogen contamination.

Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ 54 , was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.

The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory., (Copyright © 2016. Published by Elsevier Ltd.)

Denitrification transforms nitrogen applied as fertilizer and emits N 2 O, which is a potent greenhouse gas. Very little is known about the identities of abundant and active denitrifiers in agricultural soils. We coupled DNA stable-isotope probing (DNA-SIP) with flow-through reactors (FTRs) to detect active agricultural soil denitrifiers. The FTRs were incubated with nitrate and 13 C 6 -glucose under anoxic conditions and sampled at multiple time points. Labelled DNA from active microorganisms was analyzed by 16S rRNA gene fingerprinting, amplicon and shotgun metagenomic sequencing. Taxonomic representation of heavy fractions was consistent across sites and time points, including Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus and Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas) and Actinobacteria (4%; Streptomycetaceae). Most nitrite-reductase reads from heavy DNA annotated to the copper-containing form (nirK). Assigned taxonomies of active denitrifiers based on reads matching the nirK gene were comparable to those obtained through nitric oxide (norB) and RNA polymerase (rpoB) annotations but not the nitrous oxide reductase gene (nosZ). Analysis of recovered metagenomes from heavy DNA demonstrated extensive nirK sequence family diversity, including novel taxonomic groups that are not captured by existing primers., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Unlabelled: Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed., Importance: Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa. The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Copper-containing nitrite reductases (CuNiRs) catalyze the reduction of nitrite to nitric oxide, a key step in the denitrification process that maintains balance between organic and inorganic nitrogen. Despite their importance, their functioning is not well understood. In this work, we carry out first-principles calculations and show that the available structural data are consistent only with a single mechanism. For this mechanism, we determine the activation energies, transition states, and minimum energy pathways of CuNiR. The calculations lead to an updated enzymatic mechanism and resolve several controversial issues. In particular, our work identifies the origins of the two protons necessary for the enzymatic function and shows that the transformation from the initial O-coordination of substrate to the final N-coordination of product is achieved by electron transfer from T1 copper to T2 copper, rather than by the previously reported side-on coordination of a NO intermediate, which only takes place in the reduced enzyme. We also examine the role of structural change in the critical residue Asp(98), reported in one experimental study, and find that while the structural change affects the energetics of substrate attachment and product release at the T2 copper reaction center, it does not significantly affect the activation energy and reaction pathways of the nitrite reduction process.

Nitrite reductase (NIR) catalyses the reduction of nitrite to nitric oxide (NO) in the denitrification pathway. In Pseudomonas aeruginosa, expression of the gene encoding NIR (nirS) is induced by NO and is under control of the NO-sensing regulator DNR (dissimilatory nitrate respiration regulator). Because DNR is under control of the oxygen-sensing regulator ANR (anaerobic regulator of arginine deiminase and nitrate reductase), nirS is expressed only under low oxygen and anaerobic conditions. Both ANR and DNR are FNR (fumarate and nitrate reductase regulator)-type regulators and recognize the consensus FNR-binding motif. The motif of the nirS promoter is thought to be recognized only by DNR, and not by ANR. Here, mutant strains expressing either ANR or DNR were constructed and used to analyse the role of ANR and DNR in the activation of nirS expression. Analysis of transcriptional activity by microarray and quantitative reverse transcription polymerase chain reaction revealed that nirS is transcribed under low oxygen conditions in an ANR-dependent manner, although the expression level was 10-fold lower than that of the DNR-dependent expression. An artificial promoter containing the FNR-binding motif of the nirS promoter was also twofold upregulated by ANR. These results indicate that low-level expression of NIR in the presence of nitrite may provide NO as a trigger for the full expression of denitrification genes when oxygen is depleted., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2 tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2 (-)) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2 tensions. Anoxic resting-cell assays and measurements of instantaneous NO2 (-) reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bacillus megaterium NCT-2 is a novel bacterium that can utilize nitrate as its only nitrogen source for growth.The nitrate assimilation related genes that are involved in this process would be expected to be crucial. However, little is known about the genomic background of this bacterium,let alone the sequences of the nitrate assimilation related genes. In order to further investigate the nitrate assimilation function of the NCT-2, genome sequencing was performed.After obtaining the fine map of the NCT-2 genome, which was submitted to the NCBI GenBank (AHTF00000000), the sequences of the nitrate assimilation related genes (the nitrate reductase electron transfer subunit nasB and the nitrate reductase catalytic subunit nasC, the nitrite reductase [NAD(P)H]large subunit nasD and the nitrite reductase [NAD(P)H] small subunit nasE, and the glutamine synthetase glnA) were identified.Multiple alignments were performed to find out the sequence identities of the nitrate assimilation related genes to that of their similar species. Through KEGG signaling mapping search, the nitrate assimilation related genes were revealed to be located in the nitrogen metabolism signaling pathway. The putative 3D protein structures of these genes were modeled by SWISS MODEL, and shown to be highly similar to the nitrate assimilation related genes in the PDB database. Finally, the sequence validity of the nitrate assimilation related genes was verified by PCR with specifically designed primers.

We investigated the diversity and community composition of denitrifying bacteria in surface water from the Yellow River estuary. Our results indicated that the diversity of the denitrifying community in freshwater based on the nirK gene was higher than that in seawater. Furthermore, phylogenetic analysis suggested that the bacteria community could be distributed into eight clusters (Clusters I to VIII). Redundancy analysis (RDA) revealed that community compositions were related to multiple environment factors, such as salinity and nitrate concentration. The results of the present study have provided a novel insight into the denitrifying community in water columns in estuaries.

Denitrification is a major biological process to reduce nitrate to molecular nitrogen (N2). In shallow eutrophic lakes, this process can remove the largest portion of fixed nitrogen and plays an important role in self-purification of this ecosystem. To understand the structure of denitrifying communities in a shallow eutrophic lake, denitrifier communities in four sub-lakes of East Lake in Wuhan, China, were explored by restriction fragment length polymorphisms (RFLP) analysis and sequencing of nirS gene clone libraries. nirS is a functional marker gene for denitrification encoding cytochrome cd 1-containing nitrite reductase, which catalyzes the reduction of nitrite to nitric oxide. Both RFLP fingerprints clustering analysis and phylogeny analysis based on the amino acid sequences of NirS revealed that NirS-type communities in East Lake sediment could be roughly divided into three clusters. Cluster I accounted for 74-82 % of clones from the moderately eutrophic sub-lakes Tuan, Tang Ling, and Guo Zheng. Cluster II accounted for 76 % of the communities in hypertrophic sub-lake Miao Lake and cluster III as a minor group (7 % of the total), mainly presented in Miao Lake. Phylogenetic analysis revealed that cluster I was related to the reference clones from a broad range of ecological environments, and clusters II and III were more phylogenetically related to the reference clones from entrophic environments. Canonical correspondence analysis indicated that total nitrogen, total phosphate, total organic carbon, and NH4-N and NO2-N were important environmental factors affecting the dispersion of NirS-type denitrifier in the sediments. Cluster I showed a weak relationship with the nutrient content, while cluster II and III were positively related with the nutrient content. Principal coordinates analysis indicated that NirS-type communities from Tuan Lake, Tang Ling Lake, and Guo Zheng Lake sediments were divergent from those found in river, estuary sediment, and forest soil but similar to communities in constructed wetland sediment despite large geographic distances. The communities from the hypertrophic sub-lake Miao Lake deviated from other sub-lakes and the reference communities and clustered independently. Our results support the argument that environmental factors regulate the composition and distribution of the functional bacterial groups.

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd1-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d(1) heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d1 heme for holo-cytochrome cd(1) assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.

Anaerobic ammonium oxidation (anammox) and denitrification are two important processes responsible for nitrogen loss; monitoring of microbial communities carrying out these two processes offers a unique opportunity to understand the microbial nitrogen cycle. The aim of the current study was to characterize community structures and distribution of anammox and nirS-encoding nitrite-reducing bacteria in surface sediments of the northern South China Sea (SCS). The consistent phylogenetic results of three biomarkers of anammox bacteria, including 16S rRNA, hzo, and Scalindua-nirS genes, showed that Scalindua-like bacteria were the only anammox group presenting in surface sediments of the SCS. However, a relatively high micro-diversity was found within this group, including several SCS habitat-specific phylotypes, Candidatus "Scalindua zhenghei". Comparing to 16S rRNA gene, hzo and Scalindua-nirS genes provided a relatively higher resolution to elucidate anammox bacteria. For the nirS-encoding nitrite-reducing bacteria, the detected nirS gene sequences were closely related to various marine nirS denitrifiers, especially those which originated from coastal and estuarine sediments with a much higher diversity than anammox bacteria. Anammox bacterial communities shifted along with the seawater depth, while nirS-encoding nitrite-reducing bacteria did not. Although nirS-encoding nitrite-reducing bacteria have a much higher abundance and diversity than anammox bacteria, they showed similar abundance variation patterns in research sites, suggesting the two microbial groups might be affected by the similar environmental factors. The significant correlations among the abundance of the two microbial groups with the molar ratio of NH4 (+) to (NO2 (-) + NO3 (-)), pH, and organic matters of sediments strongly supported this hypothesis.

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

Adaptation to oxygen deficiency is essential for virulence and persistence of Brucella inside the host. The flexibility of this bacterium with respect to oxygen depletion is remarkable, since Brucella suis can use an oxygen-dependent transcriptional regulator of the FnrN family, two high-oxygen-affinity terminal oxidases, and a complete denitrification pathway to resist various conditions of oxygen deficiency. Moreover, our previous results suggested that oxidative respiration and denitrification can be simultaneously used by B. suis under microaerobiosis. The requirement of a functional cytochrome bd ubiquinol oxidase for nitrite reductase expression evidenced the linkage of these two pathways, and the central role of the two-component system RegB/RegA in the coordinated control of both respiratory systems was demonstrated. We propose a scheme for global regulation of B. suis respiratory pathways by the transcriptional regulator RegA, which postulates a role for the cytochrome bd ubiquinol oxidase in redox signal transmission to the histidine sensor kinase RegB. More importantly, RegA was found to be essential for B. suis persistence in vivo within oxygen-limited target organs. It is conceivable that RegA acts as a controller of numerous systems involved in the establishment of the persistent state, characteristic of chronic infections by Brucella.

NirD is part of the nitrite reductase complex NirBD that catalyses the reduction of nitrite to NH(3) in nitrate assimilation and anaerobic respiration. The crystal structure analysis of NirD from Mycobacterium tuberculosis shows a double β-sandwich fold. NirD is related in three-dimensional structure and sequence to the Rieske proteins; however, it does not contain any Fe-S cluster or other cofactors that might be involved in electron transfer. A cysteine residue at the protein surface, conserved in NirD homologues lacking the iron-sulfur cluster might be important for the interaction with NirB and possibly stabilize one of the Fe-S centers in this subunit., (Copyright © 2012 Wiley Periodicals, Inc.)

'Candidatus Methylomirabilis oxyfera'; is a polygon-shaped bacterium that was shown to have the unique ability to couple anaerobic methane oxidation to denitrification, through a newly discovered intra-aerobic pathway. Recently, the complete genome of Methylomirabilis oxyfera was assembled into a 2.7-Mb circular single chromosome by metagenomic sequencing. The genome of M. oxyfera revealed the full potential to perform both methane oxidation and the conversion of nitrite via nitric oxide into oxygen and dinitrogen gas. In this study, we show by immunogold localization that key enzymes from both methane- and nitrite-converting pathways are indeed present in single M. oxyfera cells. Antisera targeting the particulate methane monooxygenase (pMMO) and the cd(1) nitrite reductase (NirS) were raised and used for immunogold localization in both single- and double-labelling experiments. Our previous studies have shown that M. oxyfera does not develop pMMO-containing intracytoplasmic membranes as is observed in classical proteobacterial methanotrophs. Our results suggest that in M. oxyfera, the pMMO and NirS enzymes localized to the cytoplasmic membrane and periplasm, respectively. Further, double-labelling showed co-occurrence of pMMO and NirS in single M. oxyfera cells., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The impact of fertilization regimes on sequential denitrifying communities was investigated in a rice paddy field with 17 years continuous fertilization, located in Taoyuan Agro-ecosystem Research Station (110°72″ E, 28°52″ N), China. The diversity, community composition, and size of denitrifying genes of narG, qnorB, and nosZ were determined using molecular tools including terminal restriction fragment length polymorphism, quantitative polymerase chain reaction (qPCR), cloning, and sequencing analysis. Soil samples were collected from the plots with no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). UR and BM caused marked increase in the community size of the denitrifying genes; however, BMR resulted in the highest abundance. The community size of narG was the most affected by the fertilization regimes, while qnorB was the least. Fertilization also induced some shifts in the composition of denitrifying genes, but the responses of different genes varied. However, fertilization regimes caused no significant changes to the diversity of the denitrifying genes. Potential denitrification activity (PDA) was significantly correlated with the abundance of narG and nosZ rather than qnorB, but there were no such correlations between PDA and the composition and diversity of denitrifying communities. Conclusively, long-term fertilization significantly affected denitrifying community size and composition, but not diversity. Among the sequential denitrifying genes, narG was the most, while qnorB was the least sensitive communities to fertilization regimes.

The abundance of denitrifying bacteria in soil has been determined primarily by the conventional most probable number (MPN) method. We have developed a single-cell identification technique that is culture-independent, direct in situ PCR, to enumerate denitrifying bacteria in soils. The specificity of this method was evaluated with six species of denitrifying bacteria using nirK as the target gene; Escherichia coli was used as a negative control. Almost all (97.3%-100%) of the nirK-type denitrifying bacteria (Agromonas oligotrophica, Alcaligenes faecalis, Achromobacter denitrificans, Bradyrhizobium japonicum, and Pseudomonas chlororaphis) were detected by direct in situ PCR, whereas no E. coli cells and only a few cells (2.4%) of nirS-type denitrifying bacteria (Pseudomonas aeruginosa) were detected. Numbers of denitrifying bacteria in upland and paddy soil samples quantified by this method were 3.3 × 10(8) to 2.6 × 10(9) cells g(-1) dry soil. These values are approximately 1,000 to 300,000 times higher than those estimated by the MPN method. These results suggest that direct in situ PCR is a better tool for quantifying denitrifying bacteria in soil than the conventional MPN method.

The cytochrome cd₁ nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d₁ heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, ΔnirF, showed that this gene is essential for cd₁ assembly and consequently for denitrification, which was restored when the ΔnirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d₁ heme. It is concluded that His41 of NirF, which aligns with His200 of the d₁ heme domain of cd₁, is essential both for this binding and for the production of d₁ heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed., (© 2010 The Authors Journal compilation © 2010 FEBS.)

The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes.

R. sphaeroides strain 2.4.3, when lacking the cbb(3) oxidase, is unable to transition from aerobic respiration to denitrification using cellular respiration as a means of reducing oxygen levels. This is due to an inability to express nirK, the gene encoding nitrite reductase. Under certain photosynthetic conditions this strain can transition from aerobic to nitrate respiration, demonstrating that nirK expression can occur in the absence of a functional cbb(3) oxidase. If oxygen levels are reduced under non-photosynthetic conditions using low-oxygen gas mixes, nitrite reductase activity is detected at wild-type levels in the strain lacking the oxidase. In addition, co-culture experiments show that incubation of the cbb(3) deficient strain 2.4.3 with R. sphaeroides 2.4.1, which is nirK deficient but has the high-affinity cbb(3) oxidase, restores denitrification in sealed-vessel experiments. Taken together these results indicate that high end-point O(2) levels are the reason why the strain lacking the cbb(3) oxidase cannot transition from aerobic respiration to denitrification under certain conditions. The protein probably being affected by these O(2) levels is the transcriptional regulator NnrR.

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

In Bradyrhizobium japonicum the napEDABC, nirK, norCBQD and nosRZDYFLX genes, which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide, respectively, are required for denitrification. Microaerobic induction of these genes depends on fixLJ and fixK2, whose products form the FixLJ-FixK2 regulatory cascade. In B. japonicum, a second oxygen-responsive regulatory cascade mediated by the nitrogen fixation regulatory protein, NifA, has been described. In this study, we show that disruption of nifA caused a growth defect in B. japonicum cells, when grown under denitrifying conditions, and decreased activity of periplasmic nitrate and nitrite reductase enzymes was also observed. Furthermore, expression of napE-lacZ, nirK-lacZ or norC-lacZ transcriptional fusions, as well as levels of nirK transcripts were significantly reduced in the nifA mutant after incubation under nitrate-respiring conditions. Haem c staining analyses revealed that NifA is required for full synthesis of the NapC and NorC proteins, which are required for denitrification. A B. japonicum rpoN1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor sigma54, was able to grow anaerobically with nitrate as terminal electron acceptor and showed wild-type levels of nitrate and nitrite reductase activities. We propose that the nitrogen fixation regulatory protein, NifA, is involved in the maximal expression of the denitrification genes in B. japonicum. This influence is independent of sigma54.

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (Delta narGH) and nitrite reductase (Delta nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to Delta nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.

The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.

W-band (95 GHz) HYSCORE and pulse ENDOR are used to characterize the nitrosyl d(1) heme complex (d(1)NO) of cd(1) nitrite reductase from Pseudomonas aeruginosa in the wild type and the Y10F mutant. The spectra and the derived (14)N hyperfine and quadrupole interactions were found to be the same for wt and Y10F. This suggests that Tyr10 does not influence the NO ligand orientation in the reduced state in solution. This study is the first application of HYSCORE at high fields and shows its potential for characterizing low gamma nuclei with large hyperfine couplings.

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for approximately 30% of N2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n + 1) process rate data and log(n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc(2)155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DeltaglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

The soluble domain (residues 483-913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 A. The crystal belonged to space group P2(1)3, with unit-cell parameters a = b = c = 108.63 A. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V(M) of 2.1 A(3) Da(-1).

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

Three c-type cytochromes were identified in Neisseria meningitidis, based on predictions from genome sequences, that were hypothesized to be involved in electron transport to terminal electron acceptor reductases for oxygen (the cytochrome cbb(3) oxidase) and nitrite (the nitrite reductase, AniA). Mutants were generated by allelic exchange with disrupted copies of the genes encoding these cytochromes and the phenotypes of the resultant mutants analysed. It was found that cytochrome c(5) is required for in vivo nitrite reductase activity, whereas cytochromes c(x) and c(4) are both required for efficient growth using oxygen as an electron acceptor. Mutants in c(x), c(4), and c(x)+c(4) have a decreased capacity to reduce oxygen, but there is a background oxygen-reduction activity, indicating that there may be other routes for electron transfer from the cytochrome bc(1) complex to the cytochrome cbb(3) oxidase, whereas cytochrome c(5) appears to be the sole route of electrons to the nitrite reductase in N. meningitidis. Interestingly, cytochrome c(x) is highly similar to a domain of copper nitrite reductases from various proteobacteria, whereas cytochrome c(5) has high identity with a domain of the cytochrome cbb(3) oxidase of Neisseria gonorrhoeae, yet these two proteins function in oxygen respiration and nitrite respiration, respectively. This highlights a limitation of predicting protein function from similarity to known proteins, i.e. very closely related protein domains in different organisms can have different redox partners.

In the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245, nitric oxide produced by denitrification could be a signal involved in stimulation of root branching, and the dissimilatory nitrite reductase gene nirK is upregulated on wheat roots. Here, it was found that Sp245 did not contain one copy of nirK but two (named nirK1 and nirK2), localized on two different plasmids, including one plasmid prone to rearrangements. Their deduced protein sequences displayed 99.2% identity but their promoter regions and upstream genetic environment differed. Phylogenetic studies revealed that nirK1 and nirK2 clustered next to most beta-proteobacterial sequences rather than in the vicinity of other Azospirillum spp. and most alpha-proteobacterial sequences, regardless of whether DNA or deduced protein sequences were used. This points to past horizontal gene transfers. Analysis of the number of nonsynonymous and synonymous substitutions per site indicated that nirK has been subjected to neutral selection in bacteria. The use of transcriptional fusions with egfp, encoding an enhanced green fluorescent protein variant, revealed that both nirK1 and nirK2 promoter regions were upregulated in vitro under microaerobiosis or the presence of nitrite as well as on wheat roots. The analysis of nirK1 and nirK2 mutants revealed that the two genes were functional. Overall, results suggest that nirK has been acquired horizontally by A. brasilense Sp245 from a distant relative and underwent subsequent duplication; however, both paralogs remained functional and retained their upregulation by the plant partner.

Recently, the structure of a Cu-containing nitrite reductase (NiR) from Hyphomicrobium denitrificans (HdNiR) has been reported, establishing the existence of a new family of Cu-NiR where an additional type 1 Cu (T1Cu) containing cupredoxin domain is located at the N-terminus (Nojiri et al. in Proc. Natl. Acad. Sci. USA 104:4315-4320, 2007). HdNiR retains the well-characterised coupled T1Cu-type 2 Cu (T2Cu) core, where the T2Cu catalytic site is also built utilising ligands from neighbouring monomers. We have undertaken a genome analysis and found the wide occurrence of these NiRs, with members clustering in two groups, one showing an amino acid sequence similarity of around 80% with HdNiR, and a second group, including the NiR from the extremophile Acidothermus cellulolyticus, clustering around 50% similarity to HdNiR. This is reminiscent of the difference observed between the blue (Alcaligenes xylosoxidans) and green (Achromobacter cycloclastes and Alcaligenes faecalis) NiRs which have been extensively studied and may indicate that these also form two distinct subclasses of the new family. Genome analysis also showed the presence of Cu-NiRs with a C-terminal extension of 160-190 residues containing a class I cytochrome c domain with a characteristic beta-sheet extension. Currently no structural information exists for any member of this family. Genome analysis suggests the widespread occurrence of these novel NiRs with representatives in the alpha, beta and gamma subclasses of the Proteobacteria and in two species of the fungus Aspergillus. We selected the enzyme from Ralstonia pickettii for comparative modelling and produced a plausible structure highlighting an electron transfer mode in which the cytochrome c haem at the C-terminus can come within 16-A reach of the T1Cu centre of the T1Cu-T2Cu core.