Nir Gene Research Articles

A missing link in the estuarine nitrogen cycle?: Coupled nitrification-denitrification mediated by suspended particulate matter

In estuarine and coastal ecosystems, the majority of previous studies have considered coupled nitrification-denitrification (CND) processes to be exclusively sediment based, with little focus on suspended particulate matter (SPM) in the water column. Here, we present evidence of CND processes in the water column of Hangzhou Bay, one of the largest macrotidal embayments in the world. Spearman’s correlation analysis showed that SPM was negatively correlated with nitrate (rho = −0.372, P = 0.018) and marker genes for nitrification and denitrification in the water column were detected by quantitative PCR analysis. The results showed that amoA and nir gene abundances strongly correlated with SPM (all P < 0.01) and the ratio of amoA/nir strongly correlated with nitrate (rho = −0.454, P = 0.003). Furthermore, aggregates consisting of nitrifiers and denitrifiers on SPM were also detected by fluorescence in situ hybridization. Illumina MiSeq sequencing further showed that ammonia oxidizers mainly belonged to the genus Nitrosomonas, while the potential denitrifying genera Bradyrhizobium, Comamonas, Thauera, Stenotrophomonas, Acinetobacter, Anaeromyxobacter, Sulfurimonas, Paenibacillus and Sphingobacterium showed significant correlations with SPM (all P < 0.01). This study suggests that SPM may provide a niche for CND processes to occur, which has largely been missing from our understanding of nitrogen cycling in estuarine waters.

Functionalized MoS2 Nanosheets as Multi-Gene Delivery Vehicles for In Vivo Pancreatic Cancer Therapy.

Transition metal dichalcogenides (TMDCs) are categorized as novel two-dimensional (2D) nanomaterials with unique physical and chemical properties, bearing varied applications in medical and materials sciences. However, only a few works report the application of TMDCs for gene therapy in cancer treatment. Here, we engineer a multi-gene delivery system based on functionalized monolayer MoS2, which can co-deliver HDAC1 and KRAS small interfering RNAs (siRNAs) to Panc-1 cancer cells for combinational cancer therapy. The synergistic effect of gene silencing therapy and NIR phototherapy is demonstrated by inhibition of both genes, in vitro cell growth rate, and in vivo tumor volume growth rate, exemplifying pre-eminent anticancer efficacy. This anti-tumor effect is a result of the photothermal effect of MoS2 induced by NIR excitation and inactivation of HDAC1 and KRAS genes, which consequently bring about apoptosis, inhibit migration, and induce cell cycle arrest in the treated Panc-1 cells. Moreover, good biocompatibility and reduced cytotoxicity of MoS2-based nanocarriers enable their metabolism within in vitro and in vivo mouse models over a prolonged duration without any evident ill-effects. In summary, we demonstrate the promising potential of low-toxicity, functionalized MoS2 nanocarriers as a biocompatible gene delivery system for in vivo pancreatic adenocarcinoma therapy.

Effect of NiR gene on in vitro regeneration protocol of indica, japonica, aromatic and wild rice genotypes

Effect of <i>NiR</i> gene on <i>in vitro</i> regeneration protocol of <i>indica</i>, <i>japonica</i>, aromatic and wild rice genotypes

Overall bacterial community composition and abundance of nitrifiers and denitrifiers in a typical macrotidal estuary

Coupled nitrogen cycling processes can alleviate the negative effects of eutrophication caused by excessive nitrogen load in estuarine ecosystems. The abundance and diversity of nitrifiers and denitrifiers across different environmental gradients were examined in the sediment of Hangzhou Bay. Quantitative PCR and Pearson's correlation analyses suggested that the bacterial ammonia-oxidizers (AOB) were the dominant phylotypes capable of ammonia oxidation, while the nirS-encoding denitrifiers predominated in the denitrification process. Simultaneously, nitrite and pH were found to be the two major factors influencing amoA and nir gene abundances, and the distribution of bacterial communities. Moreover, the ratio of nirS/AOB amoA gene abundance showed negative correlation with nitrite concentration. Fluorescence in situ hybridization further demonstrated that AOB and acetate-denitrifying cells were closely connected and formed obvious aggregates in the sediment. Together, all these results provided us a preliminary insight for coupled nitrification-denitrification processes in the sediment of Hangzhou Bay.

Elevated Air Humidity Changes Soil Bacterial Community Structure in the Silver Birch Stand.

Soil microbes play a fundamental role in forest ecosystems and respond rapidly to changes in the environment. Simultaneously with the temperature increase the climate change scenarios also predict an intensified hydrological cycle for the Baltic Sea runoff region. The aim of this study was to assess the effect of elevated air humidity on the top soil microbial community structure of a silver birch (Betula pendula Roth.) stand by using a free air humidity manipulation facility (FAHM). The bacterial community structures of bulk soil and birch rhizosphere were analyzed using high-throughput sequencing of bacteria-specific16S rRNA gene fragments and quantification of denitrification related genes. The increased air humidity altered both bulk soil and rhizosphere bacterial community structures, and changes in the bacterial communities initiated by elevated air humidity were related to modified soil abiotic and biotic variables. Network analysis revealed that variation in soil bacterial community structural units is explained by altered abiotic conditions such as increased pH value in bulk soil, while in rhizosphere the change in absorptive root morphology had a higher effect. Among root morphological traits, the absorptive root diameter was strongest related to the bacterial community structure. The changes in bacterial community structures under elevated air humidity are associated with shifts in C, N, and P turnover as well as mineral weathering processes in soil. Increased air humidity decreased the nir and nosZ gene abundance in the rhizosphere bacterial community. The potential contribution of the denitrification to the N2O emission was not affected by the elevated air humidity in birch stand soil. In addition, the study revealed a strong link between the bacterial community structure, abundance of denitrification related genes, and birch absorptive root morphology in the ecosystem system adaptation to elevated air humidity.

SiRNA Delivery with PEGylated Graphene Oxide Nanosheets for Combined Photothermal and Genetherapy for Pancreatic Cancer.

Since the successful exfoliation of graphene from graphite in 2004, graphene and graphene oxide (GO) have been considered the most promising two-dimensional (2D) nanomaterials with distinguished physical and chemical characteristics and have attracted great attention in many different fields. Graphene oxide is well-known for its distinct physiochemical properties and shows only minimal cytotoxicity compared to carbon nanotubes. Until now, only limited efforts have been invested in utilizing GO for gene therapy in pancreatic cancer treatments. In this study, we utilized multi-functionalized monolayer GO as a gene delivery system to efficiently co-deliver HDAC1 and K-Ras siRNAs (small interfering RNAs targeting the HDAC1 gene and the G12C mutant K-Ras gene, respectively) to specifically target pancreatic cancer cells MIA PaCa-2. The systematic mechanistic elucidation of the dual gene silencing effects indicated the inactivation of both the HDAC1 and the K-Ras gene, thereby causing apoptosis, proliferation inhibition and cell cycle arrest in treated MIA PaCa-2 cells. The synergistic combination of gene silencing and NIR light thermotherapy showed significant anticancer efficacy, inhibiting in vivo tumor volume growth by >80%. Furthermore, GO can be metabolized in the mouse model within a reasonable period of time without obvious side effects. Based on preliminary in vivo application, this study for the first time indicates the promising potential of functionalized GO as a vehicle for gene therapy delivery with low toxicity for the treatment of pancreatic adenocarcinoma.

Morphological and genetic responses of lactobacillus plantarum FQR to nitrite and its practical applications

AbstractIn this article, the growth, morphological, and genetic responses of Lactobacillus plantarum FQR to nitrite were investigated. The growth and degrading ability of the strain to nitrite was delayed; slight changes were also observed on the cell surface. FQR exposed to 0.120% nitrite exhibited rough surfaces with folds, as shown by scanning electron microscopy. Transmission electron microscopy presented numerous white granules in the FQR incubated without nitrite, compared with FQR exposed to 0.045% and 0.120% nitrite. These granules are common energy storage molecules, which decrease with increasing nitrite concentration. In addition, nitrite reductase (nir) gene and membrane‐related genes were activated in varying degrees. A similar trend in relative gene expression indicated that the expression levels of membrane‐related genes (with the exception of lp_1403) in FQR increased significantly (p &lt; .05) in the presence of 0.120% nitrite compared with 0.045% nitrite. However, a marked difference was shown for the nir gene in FQR; the nir gene exhibited a relatively low level of expression when exposed to 0.120% nitrite concentration. In summary, the cell morphology and genetic level of L. plantarum FQR were changed in varying degrees after treatment with different nitrite concentrations.Practical ApplicationsNitrite is an important food additive and nitrite pollution issues of fermented food have become an increasing concern given its potential hazards. L. plantarum has the function of degrading nitrite. However, the detailed mechanisms and pathways involved in the regulation of nitrite in L. plantarum were still unclear. In this article, the growth, morphological, and genetic responses of L. plantarum to nitrite were investigated. It serves as an important reference for future studies of the mechanism of signal transduction in L. plantarum and the mechanism of stress response, and to determine the possible regulating targets. In practice, it can serve as a reference for genetic engineering improvement of L. plantarum used in the fermented food industry reducing nitrite residue.

Gene expression and biomarker discovery of anammox bacteria in different reactors

The multiplicity and heterogeneity of anaerobic ammonia oxidation (anammox) bacteria make their absolute quantification difficult. To reveal the quantitative relationships among the 16S rRNA gene and typical functional genes (nir, hzo and hzs genes) in the anammox process and their quantitative association with nitrogen removal (ammonium and nitrite), the genes were extracted from four reactors and quantified by real-time quantitative PCR (q-PCR). The Pearson correlation coefficients among the associated genes exceeded 0.96 (P<0.01), and the q-PCR results indicated that the relative copy numbers (compared with the level of the hzo gene, which was set as 1) of the hzs gene, nir gene and 16S rRNA gene per cell were 8.00, 25.22 and 157.67, respectively. Nitrogen removal efficiency was directly proportional to the logarithm of the gene concentration, with most of the R2 values being greater than 0.9. The functional genes, especially hzo and hzs, are more suitable to assess anammox activity with the R2 values of fitted equations being greater than 0.96. The mRNA quantity did not correlate with the system nitrogen removal performance directly because of different enzyme activities. This study provided valuable reference data to further reveal the molecular mechanism of anammox metabolism.

Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase

Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV – Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

Isolation and molecular characterization of nitrite reductase (RsNiR) gene under nitrate treatments in radish

Nitrite reductase (NiR, EC1.7.7.1) is a key enzyme that catalyzes the second step of nitrite reduction to ammonium in the process of nitrate assimilation in plants. To study the molecular mechanism underlying nitrate accumulation during plant development, the molecular characterization of the NiR gene (RsNiR) and its expression patterns under different nitrate concentrations and induction periods of nitrate treatments in leaf, root, petiole, stem and lateral root of radish (Raphanus sativus L.) were investigated in this study. Based on our radish transcriptome database, the genomic DNA sequence of RsNiR was isolated with four exons and three introns. The cDNA length was 1756bp containing an open reading frame (ORF) of 1524bp. The deduced protein consists of 506 amino acids, and showed highest identity with NiRs from Arabidopsis. Moreover, using the genomic DNA walking approach, the 843-bp 5’-flanking region upstream of RsNiR was isolated, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and nitrate-responsive cis-element (NRE). With the increasing nitrate concentration, the NR activity (NRA), nitrate contents and RsNiR expression levels exhibited increasing trends in leaf and root, reached a maximum at 30mM nitrate treatment and then decreased. In contrast, there was no obvious relationship among RsNiR expression level, NRA and nitrate content under different induction periods at 30mM nitrate. These results could provide fundamental insight into clarification of the molecular regulation mechanism underlying nitrate assimilation in radish.

Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting

The purpose of this study was to investigate the diversity of denitrifier community during agricultural waste composting. The diversity and dynamics of the denitrifying genes (nirK and nirS) were determined using polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Relationships between physico-chemical parameters and denitrifying genes structures were simultaneously evaluated by redundancy analysis (RDA). Phylogenetic analysis indicated that nirK clones grouped into six clusters and nirS clones into two major clusters, respectively. The results showed a very high diversity of nir gene sequences within composting samples. RDA showed that the nirK and nirS gene structures were significantly related to pH and pile temperature (P < 0.05). Significant amounts of the variation (49.2 and 38.3% for nirK and nirS genes, respectively) were explained by pH and pile temperature, suggesting that those two parameters were the most likely ones to influence, or be influenced by the denitrifiers harboring nirK and nirS genes.

Relating Phylogenetic and Functional Diversity among Denitrifiers and Quantifying their Capacity to Predict Community Functioning

Genetic diversity of phylogenetic or functional markers is widely used as a proxy of microbial diversity. However, it remains unclear to what extent functional diversity (FD), gene sequence diversity and community functioning are linked. For a range of denitrifying bacteria, we analyzed the relationships between (i) the similarity of functional traits evaluated from metabolic profiles (BIOLOG plates) or from N2O accumulation patterns on different carbon sources and (ii) the similarity of phylogenetic (16S rRNA gene) or functional (nir gene) markers. We also calculated different proxies for the diversity of denitrifier community based on taxa richness, phylogenetic (16S rRNA gene) or functional similarities (based either on metabolic profiles or N2O accumulation patterns), and evaluated their performance in inferring the functioning of assembled denitrifying communities. For individual strains, the variation in the 16S rRNA gene sequence was weakly correlated with the variation in metabolic patterns (ρ = 0.35) and was not related to N2O accumulation. The latter was correlated with the similarity of nitrite reductase residues. When nir genes were analyzed separately, the similarity in amino acids coded by the nirS genes was highly correlated with the observed patterns of N2O accumulation (ρ = 0.62), whereas nirK amino acid residues were unrelated to N2O accumulation. For bacterial assemblages, phylogenetic diversity (average similarity among species in a community) and mean community dissimilarity (average distance between species) calculated using 16S rRNA gene sequences, and FD measures associated with metabolic profiles, poorly predicted the variation in the functioning of assembled communities (≤15%). In contrast, the proxies of FD based on N2O accumulation patterns performed better and explained from 23 to 42% of the variation in denitrification. Amongst those, community niche was the best metric, indicating the importance of complementarity for resources in the context of bacterial community functioning.

Response of Nitrous Oxide and Corresponding Bacteria to Managements in an Agricultural Soil

We investigated the linkages between composition and abundance of ammonia –oxidizing bacteria (AOB) and denitrifiers and emissions of N2O in a summer maize (Zea mays L.) field in a calcareous, intensively managed, agricultural soil. The treatments were zero‐N (control), NH4+–based fertilizer N (favoring nitrification), NO3−–based fertilizer N + glucose + soil compaction (NO3−+Glu+Com, favoring denitrification), and urea + straw (U+S, representing conventional farming practices). We observed a substantial period (approximately 1 wk) of high N2O emissions that commenced after N fertilization and irrigation. The highest emissions occurred in the NO3−+Glu+Com treatment. A significant positive relationship was observed between soil NH4+–N concentrations and N2O emissions in the NH4+ and U+S treatments. In general, a positive relationship between N2O emissions and soil water‐filled pore space (WFPS) existed when WFPS reached &gt;55%. Community structure of AOB in the NH4+ treatment during the high N2O emission stage, based on terminal restriction fragment length polymorphism, was similar to that in the control treatment. There were no changes in the denitrifier community structure under NO3−+Glu+Com when 53 times higher N2O emissions occurred than in the control. High abundance of amoA gene copies for AOB and nir gene copies for denitrifiers were detected, however, during the high N2O emission period in the NH4+ and NO3−+Glu+Com treatments, respectively. The results indicate that N2O emissions were related to the abundance of AOB or denitrifiers. It remains unclear, however, whether their community composition can also have an effect on N2O emissions.

Separation of the up-regulated genes under nitrogen starvation from &lt;I&gt;Phaeodactylum tricornutum&lt;/I&gt; by suppression subtractive hy-bridization technology

Sixteen cDNA fragments of Phaeodactylum tricornutum were isolated and identified to be up-regulated under nitrogen starvation by suppression subtractive hybridization (SSH) technology in order to elucidate the molecular mechanism of the diatom nitrogen utilization. Seven of them have high similarity with the functional genes related to the utilization of nitrogen. Northern blotting analysis indicated that 5 genes, nitrate transporter gene (nrt), nitrite reductase gene (nir), ammonium transporter gene (amt), ATP-binding cassette transporter gene (abc), and purine permease gene (pup), were significantly up-regulated under nitrogen starvation.

Estimation of the Diversity of Denitrifying Bacteria in a Membrane Bioreactor by PCR Amplification with nir Gene Probes

Reduction of nitrite by nitrite reductase, encoded by either nirS or nirK genes, is the key step in the denitrification pathway. In this study, nirS and nirK are used as the target genes to detect the nirS- and nirK-containing denitrification bacteria of the activated sludge and membrane biofilm of an intermittent aerated sequencing batch membrane bioreactor (SBMBR) using both the cultured and noncultured methods. Pure cultured method results showed that 44.4% of the isolates contained six different nirS genetic polymorphisms. These nirS-contained isolates were identified as six strains, in which three strains, that is, Paracossus, Thauera, and Acidovorax, are considered as denitrifying bacteria. Only 9.4% of the isolates contained five different nirK genetic polymorphisms, which were further classified into five strains, including the denitrifying genera Mesorphizobium, Paracossus, and Thauera. This implies that 17.5% of the denitrifying bacteria contained the nirK gene. With the cloning method, six and two genetic polymorphisms in the activated sludge were identified as nirS and nirK genes. For the membrane biofilm, on the other hand, nine and four genetic polymorphisms were found to be nirS and nirK genes, respectively. Irregardless of whether the pure cultured method or the gene cloning method was used, the diversity of the nirS genetic polymorphisms was higher than the nirK genetic polymorphisms. Finally, from the phylogenetic analysis, it can be observed that although the genes were obtained from different samples (activated sludge or membrane biofilm) and using different isolation methods (cultured method or gene cloning), their phylogenetic relationship was very similar.

Identification of Acetate- or Methanol-Assimilating Bacteria under Nitrate-Reducing Conditions by Stable-Isotope Probing

Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)C]acetate or [(13)C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).

Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice ( Oryza sativa L.)

Rice varieties vary in their capacity for callus induction, growth, and regeneration. We identified the locus and candidate gene which conferred good callus growth and regenerative ability in the rice variety Koshihikari, a notorious poor rice line for genetic transformation. In addition, we succeeded in establishment of a new selectable marker system using the NiR gene for Agrobacterium-mediated transformation of rice, c.v. “Koshihikari.” The locus was mapped onto chromosome 1, and the nearest RFLP marker was C0178. A total of 500 segregating individuals (BC 6F 2 seeds) were screened for recombination by PCR-based screening and its location narrowed to a 540-kb region that had been sequenced by the International Rice Genome Sequencing Project. One ORF encoded a putative ferredoxin-nitrite reductase (NiR), which has been suggested to be required for callus induction and growth. The growth and regeneration ability of the calli initiated from Koshihikari was improved through integration of the NiR gene from Konansou. Analysis of ORFs and the promoter region of NiR indicated that the promoter region of NiR gene is responsible for growth and regeneration ability of calli in rice, c.v. Koshihikari. We established a NiR selection system for Agrobacterium-mediated transformation in rice, c.v. Koshihikari, by integration of the NiR gene isolated from rice c.v. Konansou. Transgenic rice plants regenerated from selected calli exhibited β-glucuronidase (GUS) activity. A transformation frequency of 9% was obtained. The results indicated that the NiR selection system is devoid of the disadvantages and concerns of using foreign genes (antibiotics and herbicide resistant) for selection.

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems.

Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.

Structural and functional analysis of denitrification genes in Pseudomonas stutzeri A1501

Four gene clusters associated with denitrification were identified in the genome of A1501 strain, nar, nir, nor and nos, including 40 genes totally, which encode proteins for substance transportation, gene regulation and reductases. The three gene clusters, nir, nor and nos are adjacent on chromosome and are far from nar gene cluster. Compared with other denitrifying bacteria, the 40 denitrification genes in A1501 strain compose a complete denitrification catalysis system. In A1501 strain, this system has the following characteristics: (i) only one copy of narK gene is found in nar gene cluster; (ii) a narM gene is present between narK and narG; (iii) two genes, dnarE and orfl are identified at downstream of narX and narL genes, of which dnrE perhaps is a transcriptional factor belonging to FNR family; (iv) there are 16 nir genes in A1501, the most in the known denitrifying bacteria; (v) it is for the first time that norR gene has been found in A1501 and also in Pseudomonas; (vi) nos gene cluster is relatively conservative, with a completely identical composition and arrangement of genome to the reference bacteria strain.

Observation of high seasonal variation in community structure of denitrifying bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene fragments

Temporal and spatial variation of communities of soil denitrifying bacteria at sites receiving mineral fertilizer (60 and 120 kg N ha −1 year −1) and cattle manure (75 and 150 kg N ha −1 year −1) were explored using terminal restriction fragment length polymorphism (T-RFLP) analyses of PCR amplified nitrite reductase ( nirK and nirS) gene fragments. The analyses were done three times during the year: in March, July and October. nirK gene fragments could be amplified in all three months, whereas nirS gene fragments could be amplified only in March. Analysis of similarities in T-RFLP patterns revealed a significant seasonal shift in the community structure of nirK-containing bacteria. Also, sites treated with mineral fertilizer or cattle manure showed different communities of nirK-containing denitrifying bacteria, since the T-RFLP patterns of soils treated with these fertilizers were significantly different. Also, these sites significantly differed from the control plot (no fertilizer treatment), whereas the patterns for low and high N-additions were barely separable from each other. Sequencing and phylogenetic analysis of 54 nirK clones revealed that the major part of the nirK-containing bacteria investigated belonged to a yet uncultivated cluster of denitrifying bacteria.