Genes Encoding Nitrite Reductase Research Articles

Nitrate assimilation pathway is impacted in young tobacco plants overexpressing a constitutively active nitrate reductase or displaying a defective CLCNt2

BackgroundWe have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function.ResultsWhen grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase.ConclusionsOur data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.

Characteristics of Novel Heterotrophic Nitrification–Aerobic Denitrification Bacteria Bacillus subtilis F4 and Alcaligenes faecalis P4 Isolated from Landfill Leachate Biochemical Treatment System

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are the key functional microorganisms needed to achieve simultaneous nitrification and denitrification (SND). In this study, 25 strains of HN-AD bacteria were successfully isolated from a stable landfill leachate biochemical treatment system, of which 10 strains belonged to Firmicutes and 15 strains belonged to Proteobacteria. Bacillus subtilis F4 and Alcaligenes faecalis P4 displayed good tolerance at a wide range of ammonia nitrogen (NH4+-N) concentrations. When the C/N ratio was 20, the removal rates of ammonia nitrogen were 90.1% and 89.5%, and the chemical oxygen demand (COD) removal rates were 92.4% and 93.9%, respectively. The napA gene encoding periplasmic nitrate reductase (Nap) and the nirS gene encoding nitrite reductase (Nir) were detected, and nitrogen balance showed assimilation and HN-AD was the main nitrogen metabolism mode in both strains. The use of immobilization materials could increase removal rate of ammonia nitrogen by 21.1% and 29.6%, respectively. The research results of this work can provide theoretical basis and technical support for the practical application of HN-AD bacteria to enhance the treatment of high ammonia nitrogen wastewater with high efficiency and low consumption.

Nitrite self-degradation process in radish paocai under the synergistic regulation of prokaryotic microorganisms

Nitrite, as the main hazard in pickle production, can be completely self-degraded through microbial biological enzymatic degradation (BED) and chemical acid degradation (CAD), providing a valuable strategy for the in-situ control of nitrite in pickle production. However, the microbial mechanism of the self-degradation of nitrite in radish paocai, a popular pickle in Asia, remains unclear. Herein, this study aimed to reveal the microbial metabolic pathways and processes regulating the self-degradation of nitrite during the pickling of radish paocai. In the early stage of the pickling process, Enterobacter with nitrate reductase (NarG) might reduce nitrate to form a nitrite peak of 27.61 mg/kg through “truncated denitrification”, which is in excess of the Chinese national standard (20 mg/kg). As nir genes encoding nitrite reductase increased to peak values of 9.8 × 104 copies/μL, BED dominates the self-degradation of nitrite during this early stage, which is performed by prokaryotic microorganisms such as Chryseobacterium and Halobacterium with nitrite reductase (NirK) through “complete denitrification”. Meanwhile, Weissella without nitrite reductase may dominate the pH decrease from 6.7 to 4.3 through diverse organic acids generated by metabolizing dominant reducing sugars, such as glucose and fructose of radish, thereby initiating CAD. Subsequently, the acid-tolerant Limosilactobacillus continued to reduce pH by metabolizing glucose, causing CAD to replace BED to dominate the self-degradation of nitrite until the end of pickling. Thus, the nitrite self-degradation process in radish paocai is under the synergistic regulation of diverse prokaryotic microorganisms.

Effects of native plants on nitrogen cycling microorganisms in soil

Plants modify the nitrogen (N) cycle in soil through plant N uptake, root exudation, and nitrification inhibition resulting from root exudates and changes to the physicochemical properties of soil. Plants with specific traits can be selected to manage N fluxes in the soil following the land application of N-rich wastes thereby reducing losses of N from these systems into the atmosphere (via nitrous oxide) and waterbodies (via nitrate). Previous work has shown that some New Zealand native myrtaceous species can reduce such losses more than other species, but the underlying mechanisms are unknown. It was hypothesized that lower N losses may result from the inhibition of nitrification and denitrification. We aimed to determine the effect of New Zealand native plant species on the abundance of nitrifying and denitrifying microorganisms in the soil using a pot experiment with five native plant species (Carex secta, Coprosma robusta, Kunzea robusta, Leptospermum scoparium, and Metrosideros umbellata) and exotic pasture (Lolium perenne). Half of the pots received fertilisation with N (urea), phosphorus, potassium, and sulphur, while the remainder were unfertilised controls. We quantified the abundance of bacteria, archaea, and the functional genes encoding nitrite reductase (nirK, nirS), nitrous oxide reductase (nosZ), and bacterial and archaeal ammonia monooxygenase (amoA) in the rhizosphere of these plants. Results indicated that plant species had a significant effect on the abundance of nosZ and bacterial amoA. In fertilised soil, the abundance of bacterial amoA was lower under mono- than dicotyledonous species. Similarly, the chemical properties of the soil differed between these groups. Monocotyledonous species took up more N and had lower concentrations of mineral N in the rhizosphere. This indicates that the increased competition for N likely reduced the abundance of amoA and therefore nitrification and nitrate losses. The results support the utilisation of species selection to reduce N losses. In particular, monocotyledonous species may be planted in high-fertility environments to mitigate N contamination of ground- and surface water. Future work should determine the mechanisms of plant specific interaction with ammonia-oxidising bacteria, although plant uptake can explain some of the observed differences.

Diversity of nirS and nirK denitrifying bacteria in rhizosphere and non-rhizosphere soils of halophytes in Ebinur Lake Wetland

There are few studies on denitrification related to the nitrogen cycle in Ebinur Lake wetland. This study aimed to explore the response of the diversity and composition of denitrifying bacteria to the environmental factors in wetland, so as to obtain more information about the community structure of denitrifying bacteria driven by environmental factors. Using the genes encoding nitrite reductase (nirS and nirK) as molecular markers, we analysed the seasonal changes in the diversity of denitrifying bacteria in halophyte soils by high-throughput sequencing technology. The results showed that the diversity of denitrifying bacteria was higher in July and lower in April, showing seasonal changes. The diversity index of denitrifying bacteria in rhizosphere soil was higher than that in non-rhizosphere soil. The diversity of denitrifying bacteria in the Phragmites australis rhizosphere soil was the highest. The diversity of nirK denitrifying bacteria was higher than that of nirS denitrifying bacteria, but the relative abundance was lower than that of nirS denitrifying bacteria. Three-way ANOVA showed that soil types, vegetation types and season had significant effects on the diversity of denitrifying bacteria. Furthermore, redundancy analysis indicated that nitrate was the environmental factor that significantly affected the community structure of nirS denitrifying bacteria in wetland, and electric conductivity, total nitrogen, ammonium and nitrate were the environmental factors that significantly affected the community structure of nirK denitrifying bacteria. These results provide data basis and theoretical support for the dynamic change of diversity of denitrifying bacteria in wetland.

Complete genome sequence of Rhodoferax sp. PAMC 29310 from a marine sediment of the East Siberian Sea.

Rhodoferax sp. PAMC 29310 was isolated from a surface marine sediment of the East Siberian Sea, Arctic. Whole-genome sequencing of the strain Rhodoferax sp. PAMC 29310 was achieved using PacBio RS II and Illumina platform. The resulting complete genome comprised of 4,593,249 base pairs (G+C content of 58.0%) with a single chromosome, 4546 protein-coding genes, 57 tRNAs and 6 rRNA operons. A complete set of genes encoding the enzymes of glycolysis and citric acid cycle were identified. No genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and nitrogenase reductase (nif) were present indicating that strain PAMC 29310 is not capable of fixing of carbon and nitrogen. PAMC 29310 genome contains genes for dissimilatory and assimilatory nitrate reduction. Gene encoding choline dehydrogenase enzyme which functions at the first step in the synthesis of betaine, one of the most effective osmoprotectants, was detected. In particular, among the genomes of the genus Rhodoferax strains, gene encoding nitrite reductase (nirK), which reduces nitrite to nitric oxide and tetA gene encoding tetracycline resistance protein involved in the resistance to tetracycline were identified only in the genome of Rhodoferax sp. PAMC 29310. As the first genome from the strain which was isolated from marine sediment in the genus Rhodoferax, investigation of physiological characteristics based on the complete genome sequences will help understand the adaptation of Rhodoferax sp. PAMC 29310 in the marine sediment.

Imidacloprid Triggers Transcriptomic Changes in Sugarcane (Saccharum officinarum)

Sugarcane, the commercial sugar-yielding crop, frequently suffers serious damage by trunk borers. The neonicotinoid insecticide imidacloprid is often used for disturbing insect nervous system to defense pests, while the molecular regulation of plant response controlling the imidacloprid is still unclear. Here, using the RNA-seq method, we detected the transcription level of sugarcane roots under the imidacloprid treatment for 24 h and 48 h, respectively. Results showed that a total of 1544 and 966 differentially expressed genes (DEGs) were detected under 24 h (1045 up-regulated and 499 down-regulated) and 48 h imidacloprid treatment (539 up-regulated and 427 down-regulated), respectively. Then the accuracy of RNA-seq data was verified by qRT-PCR assay, and a significant positive correlation between RNA-seq data and qRT-PCR results was detected. Crucial genes encoding the peroxidase (Sspon.03G0025980-4P, Sspon.08G0008080-2B) and the ZIM TFs (Sspon.01G0017230-1A, Sspon.01G0042730-3D) were prominently up-regulated in the 24 h treatment, while genes encoding nitrite reductase (Sspon.04G0002730-1A, Sspon.04G0002730-3D) were significantly down-regulated in the 24 h stress. In all, our results not only provide a whole expression analysis of imidacloprid on sugarcane but also are helpful in exploring crucial genes related to plant response on imidacloprid.

Nir gene-based co-occurrence patterns reveal assembly mechanisms of soil denitrifiers in response to fire.

Denitrification causes nitrogen losses from terrestrial ecosystems. The magnitude of nitrogen loss depends on the prevalence of denitrifiers, which show ecological differences if they harbour nirS or nirK genes encoding nitrite reductases with the same biological function. Thus, it is relevant to understand the mechanisms of co-existence of denitrifiers, including their response to environmental filters and competition due to niche similarities. We propose a framework to analyse the co-existence of denitrifiers across multiple assemblages by using nir gene-based co-occurrence networks. We applied it in Mediterranean soils before and during 1 year after an experimental fire. Burning did not modify nir community structure, but significantly impacted co-occurrence patterns. Bacteria with the same nir co-occurred in space, and those with different nir excluded each other, reflecting niche requirements: nirS abundance responded to nitrate and salinity, whereas nirK to iron content. Prior to fire, mutual exclusion between bacteria with the same nir suggested competition due to niche similarities. Burning provoked an immediate rise in mineral nitrogen and erased the signals of competition, which emerged again within days as nir abundances peaked. nir co-occurrence patterns can help infer the assembly mechanisms of denitrifying communities, which control nitrogen losses in the face of ecological disturbance.

N2O production using native nos-deficient denitrifying bacterial strains screened by a genome mining approach

Although N2O is a notorious greenhouse and ozone-depleting gas, N2O is a strong oxidant and can be an energy source by combusting with methane gas. The present study screened N2O-producing denitrifying bacteria by genome mining approach, and the activity and efficiency of NO2− reduction to N2O were examined. Presence and absence of the genes encoding nitrite reductase (nir), nitric oxide reductase (nor), and nitrous oxide reductase (nos) on the 2750 prokaryotic genomes were examined to screen nos-deficient denitrifying bacteria capable of NO2− reduction to N2O. The 131 nos-deficient denitrifying bacterial genomes were screened, and Cupriavidus necator H16 and Propionibacterium freudenreichii showed high activity and efficiency of 15NO2− reduction to 15N2O. Nitrogen removal performance and N2O conversion efficiencies were further investigated in a membrane bioreactor, and C. necator H16 showed 0.18 kg-N m−3 d−1 of NO2− removal rate with 75–95% of N2O conversion efficiencies.

Deep-sea sponge grounds as nutrient sinks: denitrification is common in boreo-Arctic sponges

Abstract. Sponges are commonly known as general nutrient providers for the marine ecosystem, recycling organic matter into various forms of bioavailable nutrients such as ammonium and nitrate. In this study we challenge this view. We show that nutrient removal through microbial denitrification is a common feature in six cold-water sponge species from boreal and Arctic sponge grounds. Denitrification rates were quantified by incubating sponge tissue sections with 15NO3--amended oxygen-saturated seawater, mimicking conditions in pumping sponges, and de-oxygenated seawater, mimicking non-pumping sponges. It was not possible to detect any rates of anaerobic ammonium oxidation (anammox) using incubations with 15NH4+. Denitrification rates of the different sponge species ranged from below detection to 97 nmol N cm−3 sponge d−1 under oxic conditions, and from 24 to 279 nmol N cm−3 sponge d−1 under anoxic conditions. A positive relationship between the highest potential rates of denitrification (in the absence of oxygen) and the species-specific abundances of nirS and nirK genes encoding nitrite reductase, a key enzyme for denitrification, suggests that the denitrifying community in these sponge species is active and prepared for denitrification. The lack of a lag phase in the linear accumulation of the 15N-labelled N2 gas in any of our tissue incubations is another indicator for an active community of denitrifiers in the investigated sponge species. Low rates for coupled nitrification–denitrification indicate that also under oxic conditions, the nitrate used to fuel denitrification rates was derived rather from the ambient seawater than from sponge nitrification. The lack of nifH genes encoding nitrogenase, the key enzyme for nitrogen fixation, shows that the nitrogen cycle is not closed in the sponge grounds. The denitrified nitrogen, no matter its origin, is then no longer available as a nutrient for the marine ecosystem. These results suggest a high potential denitrification capacity of deep-sea sponge grounds based on typical sponge biomass on boreal and Arctic sponge grounds, with areal denitrification rates of 0.6 mmol N m−2 d−1 assuming non-pumping sponges and still 0.3 mmol N m−2 d−1 assuming pumping sponges. This is well within the range of denitrification rates of continental shelf sediments. Anthropogenic impact and global change processes affecting the sponge redox state may thus lead to deep-sea sponge grounds changing their role in marine ecosystem from being mainly nutrient sources to becoming mainly nutrient sinks.

Diversity and abundance of denitrifying and anammox bacteria from the Arabian Sea oxygen minimum zone

Bacterial community involved in nitrogen transformations in the oxygen minimum zones (OMZ) with dissolved oxygen (DO) levels below 0.5 ml L−1 is ascribed to be responsible for the reported 40% losses of fixed nitrogen. The Arabian Sea (AS) OMZ is among the largest suboxic regions accounting for a substantial loss of fixed nitrogen. Sampling was carried out at the Arabian Sea Time Series (ASTS) location (17°0.126′ N, 67°59.772′E) during three different seasons to document the diversity and abundance of both denitrifying and anammox bacteria, based on their molecular markers. Quantitative PCR (qPCR) was done to assess the abundance and distribution of genes encoding nitrite reductase (nirS) in denitrifying bacteria and hydrazine oxidoreductase (hzo) in anammox bacteria. Copy numbers of these genes had their maxima in the core OMZ depths of 250 and 500 m. The preponderance of nirS to the tune of 0.35 × 106 copy numbers L−1 and hzo, of 1.5 × 106 L−1 is indicative of simultaneous denitrification and anammox processes. The abundance of hzo was higher than nirS during all three seasons implying probable dominance of anammox process at this sampling location. Phylogenetic analysis revealed that nirS gene was present in bacteria belonging to Gammaproteobacteria, Alphaproteobacteria and Deltaproteobacteria. All the hzo sequences obtained in this study apparently imply that Candidatus Scalindua is the dominant anammox bacteria at the ASTS location.

Microbial Community and in situ Bioremediation of Groundwater by Nitrate Removal in the Zone of a Radioactive Waste Surface Repository.

The goal of the present work was to investigate the physicochemical and radiochemical conditions and the composition of the microbial community in the groundwater of a suspended surface repository for radioactive waste (Russia) and to determine the possibility of in situ groundwater bioremediation by removal of nitrate ions. Groundwater in the repository area (10-m depth) had elevated concentrations of strontium, tritium, nitrate, sulfate, and bicarbonate ions. High-throughput sequencing of the V3–V4/V4 region of the 16S rRNA gene revealed the presence of members of the phyla Proteobacteria (genera Acidovorax, Simplicispira, Thermomonas, Thiobacillus, Pseudomonas, Brevundimonas, and uncultured Oxalobacteraceae), Firmicutes (genera Bacillus and Paenibacillus), and Actinobacteria (Candidatus Planktophila, Gaiella). Canonical correspondence analysis suggested that major contaminant – nitrate, uranium, and sulfate shaped the composition of groundwater microbial community. Groundwater samples contained culturable aerobic organotrophic, as well as anaerobic fermenting, iron-reducing, and denitrifying bacteria. Pure cultures of 33 bacterial strains belonging to 15 genera were isolated. Members of the genera Pseudomonas, Rhizobium, Cupriavidus, Shewanella, Ensifer, and Thermomonas reduced nitrate to nitrite and/or dinitrogen. Application of specific primers revealed the nirS and nirK genes encoding nitrite reductases in bacteria of the genera Pseudomonas, Rhizobium, and Ensifer. Nitrate reduction by pure bacterial cultures resulted in decreased ambient Eh. Among the organic substrates tested, sodium acetate and milk whey were the best for stimulation of denitrification by the microcosms with groundwater microorganisms. Injection of these substrates into the subterranean horizon (single-well push-pull test) resulted in temporary removal of nitrate ions in the area of the suspended radioactive waste repository and confirmed the possibility for in situ application of this method for bioremediation.

Nitrite-reducing ability is related to growth inhibition by nitrite in Rhodobacter sphaeroides f. sp. denitrificans.

Growth inhibition of Rhodobacter sphaeroides f. sp. denitrificans IL106 by nitrite under anaerobic-light conditions became less pronounced when the gene encoding nitrite reductase was deleted. Growth of another deletion mutant of the genes encoding nitric oxide reductase was severely suppressed by nitrite. Our results suggest that nitrite reductase increases the sensitivity to nitrite through the production of nitric oxide.

Moraxella catarrhalis-produced nitric oxide has dual roles in pathogenicity and clearance of infection in bacterial-host cell co-cultures

In humans, the free radical nitric oxide (NO) is a concentration-dependent multifunctional signaling or toxic molecule that modulates various physiological and pathological processes, and innate immunity against bacterial infections. Because the expression of bacterial genes encoding nitrite reductase (AniA) and NO reductase (NorB) is highly upregulated in biofilms in vitro, it is important to investigate whether bacterial NO-metabolism might subvert host NO signaling and play pathogenic roles during infection. The Moraxella catarrhalis AniA and NorB directly function in production and reduction of NO. Using M. catarrhalis-human bronchial epithelial cell (HBEC) co-cultures, we recently reported AniA/nitrite-dependent cytotoxic effects on HBECs, including altered protein profiles of HBECs and induced HBEC apoptosis, suggesting bacterial nitrite reduction likely dysregulates host cell gene expression. To further clarify whether nitrite reduction-derived NO or nitrite-dependent stimulation of bacterial growth was responsible for adverse effects on HBECs, we monitored bacterial nitrite reduction, levels of NO in co-cultures and resulted dynamic effects on HBEC proliferation and bacterial viability. This study demonstrated that M. catarrhalis nitrite reduction-derived NO was responsible for observed adverse effects on HBECs at mid-to-late stages of infection. More importantly, our data showed that while nitrite promoted bacterial growth and biofilm formation at early hours of infection, nitrite reduction-derived NO was toxic towards M. catarrhalis in maturing biofilms, suggesting nitrite reduction-derived NO might be a possible dualistic mechanism by which M. catarrhalis promotes diseases and spontaneous resolutions.

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase.

The Different Potential of Sponge Bacterial Symbionts in N2 Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes, nirK and nosZ

Nitrogen cycle is a critical biogeochemical process of the oceans. The nitrogen fixation by sponge cyanobacteria was early observed. Until recently, sponges were found to be able to release nitrogen gas. However the gene-level evidence for the role of bacterial symbionts from different species sponges in nitrogen gas release is limited. And meanwhile, the quanitative analysis of nitrogen cycle-related genes of sponge microbial symbionts is relatively lacking. The nirK gene encoding nitrite reductase which catalyzes soluble nitrite into gas NO and nosZ gene encoding nitrous oxide reductase which catalyzes N2O into N2 are two key functional genes in the complete denitrification pathway. In this study, using nirK and nosZ genes as markers, the potential of bacterial symbionts in six species of sponges in the release of N2 was investigated by phylogenetic analysis and real-time qPCR. As a result, totally, 2 OTUs of nirK and 5 OTUs of nosZ genes were detected by gene library-based saturated sequencing. Difference phylogenetic diversity of nirK and nosZ genes were observed at OTU level in sponges. Meanwhile, real-time qPCR analysis showed that Xestospongia testudinaria had the highest abundance of nosZ gene, while Cinachyrella sp. had the greatest abundance of nirK gene. Phylogenetic analysis showed that the nirK and nosZ genes were probably of Alpha-, Beta-, and Gammaproteobacteria origin. The results from this study suggest that the denitrification potential of bacteria varies among sponges because of the different phylogenetic diversity and relative abundance of nosZ and nirK genes in sponges. Totally, both the qualitative and quantitative analyses of nirK and nosZ genes indicated the different potential of sponge bacterial symbionts in the release of nitrogen gas.

Mechanisms of oxygen inhibition of nirK expression in Rhodobacter sphaeroides

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.

Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response.

Nitrate is a major nitrogen source for land plants and also acts as a signaling molecule that induces changes in growth and gene expression. To identify the cis-acting DNA element involved in nitrate-responsive gene expression, we analyzed the promoter of the Arabidopsis gene encoding nitrite reductase (NIR1). A region from positions -188 to -1, relative to the translation start site, was found to contain at least one cis-element necessary for the nitrate-dependent activation of the promoter, in which the activity of nitrate transporter NRT2.1 and/or NRT2.2 plays a critical role. To define this nitrate-responsive cis-element (NRE), we compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif as the putative NRE. A synthetic promoter in which the four copies of a 43-bp sequence containing the motif were fused to the 35S minimal promoter was found to direct nitrate-responsive transcription. Furthermore, mutations within this conserved motif in the native NIR1 promoter markedly reduced the nitrate-responsive activity of the promoter, indicating that the 43-bp sequence is an NRE that is both necessary and sufficient for nitrate-responsive transcription. We also show that both the native NIR1 promoter and the synthetic promoter display a similar level of sensitivity to nitrate, but respond differentially to exogenously supplied glutamine, indicating independent modulation of NIR1 expression by NRE-mediated nitrate induction and feedback repression mediated by other cis-element(s). These findings thus define the presence of multiple cis-elements involved in the nitrogen response in Arabidopsis.

Thioredoxin Reductase Is Essential for Protection ofNeisseria gonorrhoeaeagainst Killing by Nitric Oxide and for Bacterial Growth during Interaction with Cervical Epithelial Cells

In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.

Selenite-reducing capacity of the copper-containing nitrite reductase ofRhizobium sullae

Rhizobium sullae strain HCNT1 contains a nitric oxide-producing nitrite reductase of unknown function due to the absence of a complementary nitric oxide reductase. HCNT1 had the ability to grow on selenite concentrations as high as 50 mM, and during growth, selenite was reduced to the less toxic elemental selenium. An HCNT1 mutant lacking nitrite reductase grew poorly in the presence of 5 mM selenite, was unable to grow in the presence of 25 or 50 mM selenite and also showed no evidence of selenite reduction. A naturally occurring nitrite reductase-deficient R. sullae strain, CC1335, also showed little growth on the higher concentrations of selenite. Mobilization of a plasmid containing the HCNT1 gene encoding nitrite reductase into CC1335 increased its resistance to selenite. To confirm that this ability to grow in the presence of high concentrations of selenite correlated with nitrite reductase activity, a new nitrite reductase-containing strain was isolated from the same location where HCNT1 was isolated. This strain was also resistant to high concentrations of selenite. Inactivation of the gene encoding nitrite reductase in this strain increased selenite sensitivity. These data suggest that the nitrite reductase of R. sullae provides resistance to selenite and offers an explanation for the radically truncated denitrification found uniquely in this bacterium.