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Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H 2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Staphylococcus aureus is a pathogenic bacterium with a widespread distribution that can cause diverse severe diseases. The membrane-bound nitrate reductase NarGHJI serves respiratory function. However, little is known about its contribution to virulence. In this study, we demonstrated that narGHJI disruption results in the downregulation of virulence genes (e.g., RNAIII , agrBDCA , hla , psmα , and psmβ ) and reduces the hemolytic activity of the methicillin-resistant S. aureus (MRSA) strain USA300 LAC. Moreover, we provided evidence that NarGHJI participates in regulating host inflammatory response. A mouse model of subcutaneous abscess and Galleria mellonella survival assay demonstrated that the ΔnarG mutant was significantly less virulent than the wild type. Interestingly, NarGHJI contributes to virulence in an agr -dependent manner, and the role of NarGHJI differs between different S. aureus strains. Our study highlights the novel role of NarGHJI in regulating virulence, thereby providing a new theoretical reference for the prevention and control of S. aureus infection. IMPORTANCE Staphylococcus aureus is a notorious pathogen that poses a great threat to human health. The emergence of drug-resistant strains has significantly increased the difficulty of preventing and treating S. aureus infection and enhanced the pathogenic ability of the bacterium. This indicates the importance of identifying novel pathogenic factors and revealing the regulatory mechanisms through which they regulate virulence. The nitrate reductase NarGHJI is mainly involved in bacterial respiration and denitrification, which can enhance bacterial survival. We demonstrated that narGHJI disruption results in the downregulation of the agr system and agr -dependent virulence genes, suggesting that NarGHJI participates in the regulation of S. aureus virulence in an agr -dependent manner. Moreover, the regulatory approach is strain specific. This study provides a new theoretical reference for the prevention and control of S. aureus infection and reveals new targets for the development of therapeutic drugs., Competing Interests: The authors declare no conflict of interest.

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here, we show that deletion of either dksA1 (one of two dksA homologs in the P. denitrificans genome) or relA / spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA / spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog ( dksA2 ) and relA / spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCE Paracoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here, we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of Δ narQ , the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ , narP , and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo , suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

The nitrogen utilization efficiency of plants varies depending on the plant species. In modern agriculture, nitrogen fertilizer is used to increase crop production, with the amount of fertilizer addition increasing steadily worldwide. This study included the two most used ecotypes of Arabidopsis thaliana, Landsberg erecta (Ler) and Col-0, which were used to identify differences at the molecular level. We found that the efficiency of nitrogen utilization and salt stress resistance differed between these two ecotypes of the same species. We demonstrated distinct salt stress resistance between Ler and Col-0 depending on the differences in nitrate level, which was explained by different regulation of the NIA2 gene expression in these two ecotypes. Our results demonstrate that the genes and promoters regulate expression of these genes and contribute to trait differences. Further studies are required on genes and promoter elements for an improved understanding of the salinity stress resistance mechanism in plants., (© 2022 German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus , but only in few strains of the species Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, in the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O 2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca 2+ -independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca 2+ -dependent enzymes, suggesting convergent evolution., Competing Interests: Conflict of interest—The authors declare no conflicts of interest in regards to this work., (© 2020 Campeciño et al.)

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N 2 O) from nitrate but lost the N 2 O reductase related to the degradation of N 2 O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N 2 O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1-6) exhibiting exceptionally low N 2 O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (-ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4-6, suggesting that the low-N 2 O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.

It is known that adaptive evolution in permanently cold environments drives cold adaptation in enzymes. However, how the relatively high enzyme activities were achieved in cold environments prior to cold adaptation of enzymes is unclear. Here we report that an Antarctic strain of Chlorella vulgaris, called NJ-7, acquired the capability to grow at near 0 °C temperatures and greatly enhanced freezing tolerance after systematic increases in abundance of enzymes/proteins and positive selection of certain genes. Having diverged from the temperate strain UTEX259 of the same species 2.5 (1.1-4.1) to 2.6 (1.0-4.5) Ma, NJ-7 retained the basic mesophilic characteristics and genome structures. Nitrate reductases in the two strains are highly similar in amino acid sequence and optimal temperature, but the NJ-7 one showed significantly higher abundance and activity. Quantitative proteomic analyses indicated that several cryoprotective proteins (LEA), many enzymes involved in carbon metabolism and a large number of other enzymes/proteins, were more abundant in NJ-7 than in UTEX259. Like nitrate reductase, most of these enzymes were not upregulated in response to cold stress. Thus, compensation of low specific activities by increased enzyme abundance appears to be an important strategy for early stage cold adaptation to Antarctica, but such enzymes are mostly not involved in cold acclimation upon transfer from favorable temperatures to near 0 °C temperatures., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In many Thermus thermophilus strains, nitrate respiration is encoded in mobile genetic regions, along with regulatory circuits that modulate its expression based on anoxia and nitrate presence. The oxygen-responsive system has been identified as the product of the dnrST (dnr) operon located immediately upstream of the nar operon (narCGHJIKT), which encodes the nitrate reductase (NR) and nitrate/nitrite transporters. In contrast, the nature of the nitrate sensory system is not known. Here, we analyse the putative nitrate-sensing role of the bicistronic drp operon (drpAB) present downstream of the nar operon in most denitrifying Thermus spp. Expression of drp was found to depend on the master regulator DnrT, whereas the absence of DrpA or DrpB increased the expression of both DnrS and DnrT and, concomitantly, of the NR. Absence of both proteins made expression from the dnr and nar operons independent of nitrate. Polyclonal antisera allowed us to identify DrpA as a periplasmic protein and DrpB as a membrane protein, with capacity to bind to the cytoplasmic membrane. Here, we propose a role for DrpA/DrpB as nitrate sensors during denitrification., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Candidate phyla (CP) are broad phylogenetic clusters of organisms that lack cultured representatives. Included in this fraction is the candidate Parcubacteria superphylum. Specific characteristics that have been ascribed to the Parcubacteria include reduced genome size, limited metabolic potential and exclusive reliance on fermentation for energy acquisition. The study of new environmental niches, such as the marine versus terrestrial subsurface, often expands the understanding of the genetic potential of taxonomic groups. For this reason, we analyzed 12 Parcubacteria single amplified genomes (SAGs) from sediment samples collected within the Challenger Deep of the Mariana Trench, obtained during the Deepsea Challenge (DSC) Expedition. Many of these SAGs are closely related to environmental sequences obtained from deep-sea environments based on 16S rRNA gene similarity and BLAST matches to predicted proteins. DSC SAGs encode features not previously identified in Parcubacteria obtained from other habitats. These include adaptation to oxidative stress, polysaccharide modification and genes associated with respiratory nitrate reduction. The DSC SAGs are also distinguished by relative greater abundance of genes for nucleotide and amino acid biosynthesis, repair of alkylated DNA and the synthesis of mechanosensitive ion channels. These results present an expanded view of the Parcubacteria, among members residing in an ultra-deep hadal environment., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Investigating the environmental influence on the community composition and abundance of denitrifiers in marine sediment ecosystem is essential for understanding of the ecosystem-level controls on the biogeochemical process of denitrification. In the present study, nirK-harboring denitrifying communities in different mud deposit zones of eastern China marginal seas (ECMS) were investigated via clone library analysis. The abundance of three functional genes affiliated with denitrification (narG, nirK, nosZ) was assessed by fluorescent quantitative PCR. The nirK-harboring microbiota were dominated by a few operational taxonomic units (OTUs), which were widely distributed in different sites with each site harboring their unique phylotypes. The mean abundance of nirK was significantly higher than that of narG and nosZ genes, and the abundance of narG was higher than that of nosZ. The inconsistent abundance profile of different functional genes along the process of denitrification might indicate that nitrite reduction occurred independently of denitrification in the mud deposit zones of ECMS, and sedimentary denitrification was accomplished by cooperation of different denitrifying species rather than a single species. Such important information would be missed when targeting only a single denitrifying functional gene. Analysis of correlation between abundance ratios and environmental factors revealed that the response of denitrifiers to environmental factors was not invariable in different mud deposit zones. Our results suggested that a comprehensive analysis of different denitrifying functional genes may gain more information about the dynamics of denitrifying microbiota in marine sediments.

Shewanella, a group of ubiquitous bacteria renowned for respiratory versatility, thrive in environments where various electron acceptors (EAs) of different chemical and physiological characteristics coexist. Despite being extensively studied, we still know surprisingly little about strategies by which multiple EAs and their interaction define ecophysiology of these bacteria. Previously, we showed that nitrite inhibits growth of the genus representative Shewanella oneidensis on fumarate and presumably some other CymA (quinol dehydrogenase)-dependent EAs by reducing cAMP production, which in turn leads to lowered expression of nitrite and fumarate reductases. In this study, we demonstrated that inhibition of fumarate growth by nitrite is also attributable to overproduction of NapB, the cytochrome c subunit of nitrate reductase. Further investigations revealed that excessive NapB per se inhibits growth on all EAs tested, including oxygen. When overproduced, NapB acts as an electron shuttle to dissipate electrons of the quinol pool, likely to extracellullar EAs, because the Mtr system, the major electron transport pathway for extracellular electron transport, is implicated. The study not only sheds light on mechanisms by which certain EAs, especially toxic ones, impact the bacterial ecophysiology, but also provides new insights into how electron shuttle c-type cytochromes regulate multi-branched respiratory networks.

The Crenarchaeon Ignicoccus hospitalis lives in symbiosis with Nanoarchaeum equitans providing essential cell components and nutrients to its symbiont. Ignicoccus hospitalis shows an intriguing morphology that points toward an evolutionary role in driving compartmentalization. Therefore, the bioenergetics of this archaeal host-symbiont system remains a pressing question. To date, the only electron acceptor described for I. hospitalis is elemental sulfur, but the organism comprises genes that encode for enzymes involved in nitrogen metabolism, e.g., one nitrate reductase and two octaheme cytochrome c, Igni&#95;0955 (IhOCC) and Igni&#95;1359. Herein, we detail functional and structural studies of the highly abundant IhOCC, including an X-ray crystal structure at 1.7 Å resolution, the first three-dimensional structure of an archaeal OCC. The trimeric IhOCC is membrane associated and exhibits significant structural and functional differences to previously characterized homologs within the hydroxylamine oxidoreductases (HAOs) and octaheme cytochrome c nitrite reductases (ONRs). The positions and spatial arrangement of the eight hemes are highly conserved, but the axial ligands of the individual hemes 3, 6 and 7 and the protein environment of the active site show significant differences. Most notably, the active site heme 4 lacks porphyrin-tyrosine cross-links present in the HAO family. We show that IhOCC efficiently reduces nitrite and hydroxylamine, with possible relevance to detoxification or energy conservation., Database: Structural data are available in the Protein Data Bank under the accession number 4QO5., (© 2016 Federation of European Biochemical Societies.)

Sensing potential nitrogen-containing respiratory substrates such as nitrate, nitrite, hydroxylamine, nitric oxide (NO) or nitrous oxide (N2 O) in the environment and subsequent upregulation of corresponding catabolic enzymes is essential for many microbial cells. The molecular mechanisms of such adaptive responses are, however, highly diverse in different species. Here, induction of periplasmic nitrate reductase (Nap), cytochrome c nitrite reductase (Nrf) and cytochrome c N2 O reductase (cNos) was investigated in cells of the Epsilonproteobacterium Wolinella succinogenes grown either by fumarate, nitrate or N2 O respiration. Furthermore, fumarate respiration in the presence of various nitrogen compounds or NO-releasing chemicals was examined. Upregulation of each of the Nap, Nrf and cNos enzyme systems was found in response to the presence of nitrate, NO-releasers or N2 O, and the cells were shown to employ three transcription regulators of the Crp-Fnr superfamily (homologues of Campylobacter jejuni NssR), designated NssA, NssB and NssC, to mediate the upregulation of Nap, Nrf and cNos. Analysis of single nss mutants revealed that NssA controls production of the Nap and Nrf systems in fumarate-grown cells, while NssB was required to induce the Nap, Nrf and cNos systems specifically in response to NO-generators. NssC was indispensable for cNos production under any tested condition. The data indicate dedicated signal transduction routes responsive to nitrate, NO and N2 O and imply the presence of an N2 O-sensing mechanism., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world's largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth's most abundant organismal group., Competing Interests: The authors declare no competing financial interests in association with this study.

Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.

Unlabelled: Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp., Importance: The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Nitrogen-metabolizing genes, including nitrogenase (nifH), periplasmic nitrate reductase (napA), and cytochrome cd 1-type nitrite reductase (nirS), were collected from hydrothermal chimney sulfides on 3 middle ocean ridges and compared for the first time. There was a clear phylogenetic distinction of these nifH genes between different hydrothermal ecosystems, which supported the colonization and potential adaptation by different nitrogen fixing microbes in those sulfides. In particular, in sulfides from low-temperature hydrothermal vents of the Southwest Indian Ocean Ridge, the prevalence of nifH genes appears to be attributed to sulfate-reducing bacteria, suggesting their ecological significance. Phylogenetic analysis of nitrate/nitrite reductase genes indicated that nitrate was a critical electron acceptor for sulfur- or metal-oxidizing bacteria in these hydrothermal ecosystems. Our results provided information about the compositions and diversity of the 3 important genes involved in nitrogen fixation and nitrate/nitrite reduction processes in hydrothermal ecosystems and is the first comprehensive genetic repertoire of genes related to potential nitrogen fixation and denitrification processes in various hydrothermal environments.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Amylase inhibitors serve as attractive candidates of defense mechanisms against insect attack. Therefore, the impediment of Helicoverpa armigera digestion can be the effective way of controlling this pest population. Nitrite was found to be a potent mixed non-competitive competitive inhibitor of partially purified α-amylase of H. armigera gut. This observation impelled us to determine the response of nitrite and nitrate reductase (NR) towards H. armigera infestation in nine pigeonpea genotypes (four moderately resistant, three intermediate and two moderately susceptible). The significant upregulation of NR in moderately resistant genotypes after pod borer infestation suggested NR as one of the factors that determine their resistance status against insect attack. The pod borer attack caused greater reduction of nitrate and significant accumulation of nitrite in moderately resistant genotypes. The activity of nitrite reductase (NiR) was also enhanced more in moderately resistant genotypes than moderately susceptible genotypes on account of H. armigera herbivory. Expression of resistance to H. armigera was further revealed when significant negative association between NR, NiR, nitrite and percent pod damage was observed. This is the first report that suggests nitrite to be a potent inhibitor of H. armigera α-amylase and also the involvement of nitrite and NR in providing resistance against H. armigera herbivory., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. In vitro growth assays of the S. Typhimurium 14028 deletion mutants ΔhmpA, ΔnorV and ΔnrfA revealed a growth defect of ΔhmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150 mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150 mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types--periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed.

The nitrogen isotope composition (δ¹⁵N) of plants has potential to provide time-integrated information on nitrogen uptake, assimilation and allocation. Here, we take advantage of existing T-DNA and γ-ray mutant lines of Arabidopsis thaliana to modify whole-plant and organ-level nitrogen isotope composition. Nitrate reductase 2 (nia2), nitrate reductase 1 (nia1) and nitrate transporter (nrt2) mutant lines and the Col-0 wild type were grown hydroponically under steady-state NO₃⁻ conditions at either 100 or 1000 μM NO₃⁻ for 35 days. There were no significant effects on whole-plant discrimination and growth in the assimilatory mutants (nia2 and nia1). Pronounced root vs leaf differences in δ¹⁵N, however, indicated that nia2 had an increased proportion of nitrogen assimilation of NO₃⁻ in leaves while nia1 had an increased proportion of assimilation in roots. These observations are consistent with reported ratios of nia1 and nia2 gene expression levels in leaves and roots. Greater whole-plant discrimination in nrt2 indicated an increase in efflux of unassimilated NO₃⁻ back to the rooting medium. This phenotype was associated with an overall reduction in NO₃⁻ uptake, assimilation and decreased partitioning of NO₃⁻ assimilation to the leaves, presumably because of decreased symplastic intercellular movement of NO₃⁻ in the root. Although the results were more varied than expected, they are interpretable within the context of expected mechanisms of whole-plant and organ-level nitrogen isotope discrimination that indicate variation in nitrogen fluxes, assimilation and allocation between lines., (© 2013 Scandinavian Plant Physiology Society.)

Denitrifiers differ in how they handle the transition from oxic to anoxic respiration, with consequences for NO and N2O emissions. To enable stringent comparisons we defined parameters to describe denitrification regulatory phenotype (DRP) based on accumulation of NO2(-) , NO and N2O, oxic/anoxic growth and transcription of functional genes. Eight Thauera strains were divided into two distinct DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the different denitrification genes. The PO group accumulated nitrite, and no transcription of nirS (encoding nitrite reductase) was detected until all available nitrate (2 mM) was consumed. Addition of a new portion of nitrate to an actively denitrifying culture of a PO strain (T. terpenica) resulted in a transient halt in nitrite reduction, indicating that the electron flow was redirected to nitrate reductase. All eight strains controlled NO at nano-molar concentrations, possibly reflecting the importance of strict control for survival. Transient N2O accumulation differed by two orders of magnitude between strains, indicating that control of N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional genes) and DRP., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein)., (© SBIC 2012)

We have previously illustrated the nitrate/nitrite respiratory pathway of Shewanella oneidensis, which is renowned for its remarkable versatility in respiration. Here we investigated the systems regulating the pathway with a reliable approach which enables characterization of mutants impaired in nitrate/nitrite respiration by guaranteeing biomass. The S. oneidensis genome encodes an Escherichia coli NarQ/NarX homolog SO3981 and two E. coli NarP/NarL homologs SO1860 and SO3982. Results of physiological characterization and mutational analyses demonstrated that S. oneidensis possesses a single two-component system (TCS) for regulation of nitrate/nitrite respiration, consisting of the sensor kinase SO3981(NarQ) and the response regulator SO3982(NarP). The TCS directly controls the transcription of nap and nrfA (genes encoding nitrate and nitrite reductases, respectively) but regulates the former less tightly than the latter. Additionally, phosphorylation at residue 57 of SO3982 is essential for its DNA-binding capacity. At the global control level, Crp is found to regulate expression of narQP as well as nap and nrfA. In contrast to NarP-NarQ, Crp is more essential for nap rather than nrfA.

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ~100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium.

Microorganisms employ diverse mechanisms to withstand physiological stress conditions exerted by reactive or toxic oxygen and nitrogen species such as hydrogen peroxide, organic hydroperoxides, superoxide anions, nitrite, hydroxylamine, nitric oxide or NO-generating compounds. This study identified components of the oxidative and nitrosative stress defence network of Wolinella succinogenes, an exceptional Epsilonproteobacterium that lacks both catalase and haemoglobins. Various gene deletion-insertion mutants were constructed, grown by either fumarate respiration or respiratory nitrate ammonification and subjected to disc diffusion, growth and viability assays under stress conditions. It was demonstrated that mainly two periplasmic multihaem c-type cytochromes, namely cytochrome c peroxidase and cytochrome c nitrite reductase (NrfA), mediated resistance to hydrogen peroxide. Two AhpC-type peroxiredoxin isoenzymes were shown to be involved in protection against different organic hydroperoxides. The phenotypes of two superoxide dismutase mutants lacking either SodB or SodB2 implied that both isoenzymes play important roles in oxygen and superoxide stress defence although they are predicted to reside in the cytoplasm and periplasm respectively. NrfA and a cytoplasmic flavodiiron protein (Fdp) were identified as key components of nitric oxide detoxification. In addition, NrfA (but not the hybrid cluster protein Hcp) was found to mediate resistance to hydroxylamine stress. The results indicate the presence of a robust oxidative and nitrosative stress defence network and identify NrfA as a multifunctional cytochrome c involved in both anaerobic respiration and stress protection., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg²+), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Elevated atmospheric carbon dioxide concentrations ([CO(2) ]) might change the abundance and the function of soil microorganisms in the depth profile of agricultural soils by plant-mediated reactions. The seasonal pattern of abundance and activity of nitrate-reducing bacteria was studied in a Mini-FACE experiment planted with oilseed rape (Brassica napus). Three depths (0-10, 10-20 and 20-30 cm) were sampled. Analyses of the abundances of total (16S rRNA gene) and nitrate-reducing bacteria (narG, napA) revealed strong influences of sampling date and depth, but no [CO(2)] effects. Abundance and activity of nitrate reducers were higher in the top soil layer and decreased with depth but were not related to extractable amounts of nitrogen and carbon in soil. Dry periods reduced abundances of total and nitrate-reducing bacteria, whereas the potential activity of the nitrate reductase enzyme was not affected. Enzyme activity was only weakly correlated to the abundance of nitrate-reducing bacteria but was related to NH(4) (+) and NO(3) (-) concentrations. Our results suggest that in contrast to the observed pronounced seasonal changes, the elevation of atmospheric [CO(2) ] has only a marginal impact on nitrate reducers in the investigated arable ecosystem., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Most of the Shewanella species contain two periplasmic nitrate reductases (NAP-α and NAP-β), which is a unique feature of this genus. In the present study, the physiological function and evolutionary relationship of the two NAP systems were studied in the deep-sea bacterium Shewanella piezotolerans WP3. Both of the WP3 nap gene clusters: nap-α (napD1A1B1C) and nap-β (napD2A2B2) were shown to be involved in nitrate respiration. Phylogenetic analyses suggest that NAP-β originated earlier than NAP-α. Tetraheme cytochromes NapC and CymA were found to be the major electron deliver proteins, and CymA also served as a sole electron transporter towards nitrite reductase. Interestingly, a ΔnapA2 mutant with the single functional NAP-α system showed better growth than the wild-type strain, when grown in nitrate medium, and it had a selective advantage to the wild-type strain. On the basis of these results, we proposed the evolution direction of nitrate respiration system in Shewanella: from a single NAP-β to NAP-β and NAP-α both, followed by the evolution to a single NAP-α. Moreover, the data presented here will be very useful for the designed engineering of Shewanella for more efficient respiring capabilities for environmental bioremediation.

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Bacterial cellular metabolism is renowned for its metabolic diversity and adaptability. However, certain environments present particular challenges. Aerobic metabolism of highly reduced carbon substrates by soil bacteria such as Paracoccus pantotrophus presents one such challenge since it may result in excessive electron delivery to the respiratory redox chain when compared with the availability of terminal oxidant, O2. The level of a periplasmic ubiquinol-dependent nitrate reductase, NAP, is up-regulated in the presence of highly reduced carbon substrates. NAP oxidizes ubiquinol at the periplasmic face of the cytoplasmic membrane and reduces nitrate in the periplasm. Thus its activity counteracts the accumulation of excess reducing equivalents in ubiquinol, thereby maintaining the redox poise of the ubiquinone/ubiquinol pool without contributing to the protonmotive force across the cytoplasmic membrane. Although P. pantotrophus NapAB shows a high level of substrate specificity towards nitrate, the enzyme has also been reported to reduce selenate in spectrophotometric solution assays. This transaction draws on our current knowledge concerning the bacterial respiratory nitrate reductases and extends the application of PFE (protein film electrochemistry) to resolve and quantify the selenate reductase activity of NapAB.

We have obtained draft genomic sequences of PD (partial denitrificant) and CD (complete denitrificant) strains of Thermus thermophilus. Their genomes are similar in size to that of the aerobic strains sequenced to date and probably contain a similar megaplasmid. In the CD strain, the genes encoding a putative cytochrome cd1 Nir (nitrite reductase) and ancillary proteins were clustered with a cytochrome c-dependent Nor (nitric oxide reductase), and with genes that are probably implicated in their regulation. The Nar (nitrate reductase) and associated genes were also clustered and located 7 kb downstream of the genes coding for the Nir. The whole nar-nir-nor denitrification supercluster was identified as part of a variable region of a megaplasmid. No homologues of NosZ were found despite nitrogen balance supports the idea that such activity actually exists.

Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extraradical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. Besides the optimized use in L. bicolor, general consideration was taken to build a vector system with maximum compatibility with other homobasidiomycetes and different transformation techniques., (© 2010 Landes Bioscience)

Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs, which is able to grow on ammonium, as well as nitrate as sole nitrogen source. Based on the complete genome sequence, proteins for nitrate utilization, nitrate transporter (NRT) and nitrate reductase (NR), were predicted to be encoded by the neighboring nuclear genes CMG018C and CMG019C, respectively, but no typical nitrite reductase (NiR) gene was found by similarity searches. On the other hand, two candidate genes for sulfite reductase (SiR) were found, one of which (CMG021C) is located next to the above-noted nitrate-related genes. Given that transcripts of CMG018C, CMG019C and CMG021C accumulate in nitrate-containing media, but are repressed by ammonium, and that SiR and NiR are structurally related enzymes, we hypothesized that the CMG021C gene product functions as an NiR in C. merolae. To test this hypothesis, we developed a method for targeted gene disruption in C. merolae. In support of our hypothesis, we found that a CMG021G null mutant in comparison with the parental strain showed decreased cell growth in nitrate-containing but not in ammonium-containing media. Furthermore, expression of CMG021C in the nirA mutant of a cyanobacterium, Leptolyngbya boryana (formerly Plectonema boryanum), could genetically complement the NiR defect. Immunofluorescent analysis indicated the localization of CMG021C in chloroplasts, and hence we propose an overall scheme for nitrate assimilation in C. merolae.

Campylobacter jejuni is an important human pathogen that causes millions of cases of food-borne enteritis each year. The C. jejuni respiratory chain is highly branched and contains at least four enzymes predicted to contain a metal binding pterin (MPT), with the metal being either molybdenum or tungsten. Also predicted are two separate transport systems, one for molybdenum encoded by modABC and a second for tungsten encoded by tupABC. Both transport systems were mutated and the activities of the four predicted MPT-containing enzymes were assayed in the presence of molybdenum and tungsten in wild-type and mod and tup backgrounds. Results indicate that mod is primarily a molybdenum transporter that can also transport tungsten, while tup is a tungsten-specific transporter. The MPT containing enzymes nitrate reductase, sulphite oxidase, and SN oxide reductase are strict molybdoenzymes while formate dehydrogenase prefers tungsten. A ModE-like protein regulates both transporters, repressing mod in the presence of both molybdenum and tungsten and tup only in the presence of tungsten. Like other ModE proteins, the C. jejuni ModE binds DNA through a helix-turn-helix DNA binding domain, but unlike other members of the ModE family it does not have a metal binding domain.

Bradyrhizobium japonicum utilizes cytochrome cbb(3) oxidase encoded by the fixNOQP operon to support microaerobic respiration under free-living and symbiotic conditions. It has been previously shown that, under denitrifying conditions, inactivation of the cycA gene encoding cytochrome c(550), the electron donor to the Cu-containing nitrite reductase, reduces cbb(3) expression. In order to establish the role of c(550) in electron transport to the cbb(3) oxidase, in this work, we have analyzed cbb(3) expression and activity in the cycA mutant grown under microaerobic or denitrifying conditions. Under denitrifying conditions, mutation of cycA had a negative effect on cytochrome c oxidase activity, heme c (FixP and FixO) and heme b cytochromes as well as expression of a fixP'-'lacZ fusion. Similarly, cbb(3) oxidase was expressed very weakly in a napC mutant lacking the c-type cytochrome, which transfers electrons to the NapAB structural subunit of the periplasmic nitrate reductase. These results suggest that a change in the electron flow through the denitrification pathway may affect the cellular redox state, leading to alterations in cbb(3) expression. In fact, levels of fixP'-'lacZ expression were largely dependent on the oxidized or reduced nature of the carbon source in the medium. Maximal expression observed in cells grown under denitrifying conditions with an oxidized carbon source required the regulatory protein RegR.

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periplasmic nitrate reductase NapA. The composition of the corresponding nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap loci predict accessory proteins such as the iron-sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium Wolinella succinogenes was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a napH deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron-sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CX(3)CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd(1) nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.

Members of the NapC/NrfH family are multihaem c-type cytochromes that exchange electrons with oxidoreductases situated at the outside of the cytoplasmic membrane or in the periplasmic space of many proteobacteria. They form a group of membrane-bound quinol dehydrogenases that are essential components of several electron transport chains, for example those of periplasmic nitrate respiration and respiratory nitrite ammonification. Knowledge of the structure-function relationships of NapC/NrfH proteins is scarce and only one high-resolution structure (Desulfovibrio vulgaris NrfH) is available. In the present study, several Wolinella succinogenes mutants that produce variants of NrfH, the membrane anchor of the cytochrome c nitrite reductase complex, were constructed and characterized in order to improve the understanding of the putative menaquinol-binding site, the maturation and function of the four covalently bound haem c groups and the importance of the N-terminal transmembrane segment. Based on amino acid sequence alignments, a homology model for W. succinogenes NrfH was constructed that underlines the overall conservation of tertiary structure in spite of a low sequence homology. The results support the proposed architecture of the menaquinol-binding site in D. vulgaris NrfH, demonstrate that each histidine residue arranged in one of the four CX(2)CH haem c-binding motifs is essential for NrfH maturation in W. succinogenes, and indicate a limited flexibility towards the length and structure of the transmembrane region.

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c-type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10x and 30x higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.

The enteric bacterium Salmonella enterica serovar Typhimurium is a pathogen that is highly adapted for both intracellular and extracellular survival in a range of oxic and anoxic environments. The cytotoxic radical nitric oxide (NO) is encountered in many of these environments. Protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin (NorV), flavohaemoglobin (HmpA) and cytochrome c nitrite reductase (NrfA), have been shown to reduce NO in vitro. In this work we determined the role of these three enzymes in NO detoxification by Salmonella by assessing the effects of all eight possible combinations of norV, hmpA and nrfA single, double and triple mutations. The mutant strains were cultured and exposed to NO following either glucose fermentation (when nitrite reductase activity is low), or anaerobic respiration (when nitrite reductase activity is high). Wild-type cultures were more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions. Analysis of the mutant strains suggested an important additive role for both NorV and NrfA in both environments, since the norV nrfA mutant could not grow after NO addition. The results also suggested a minor role for HmpA in anaerobic detoxification of NO under the two growth conditions, and a larger role for HmpA in aerobic NO detoxification was confirmed. Activity assays and measurements of NO consumption showed that increased nitrite reductase activity correlates with an elevated capacity for NO reduction by intact cells. Taken together, the results reveal a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, and highlight the influence that growth conditions have on the sensitivity to NO of this pathogenic bacterium.

The control of promoter activity by nitrogen source has recently emerged as an intriguing system for regulated expression of the heterologous genes. The purpose of this work was to investigate whether heterologous gene expression in transgenic Dunaliella salina would be controlled by an inducible promoter. Here we identify that the nitrate reductase (NR) transcripts of D. salina are induced by nitrate but repressed by ammonium. The bar gene integrated into the genome of D. salina is transcribed by a promoter of the NR gene from D. salina and the bar transcripts are induced by nitrate but repressed by ammonium. PPT-resistance of transformants disappears when they are transferred from nitrate-containing medium to ammonium-containing medium. The findings of this study demonstrate that the promoter of the D. salina NR gene can be used to control expression of the heterologous genes in transgenic D. salina.