Nitrite Reductase Gene Research Articles

Ammonium removal by Agrobacterium sp. LAD9 capable of heterotrophic nitrification–aerobic denitrification

Characteristics of ammonium removal by a newly isolated heterotrophic nitrification-aerobic denitrification bacterium Agrobacterium sp. LAD9 were systematically investigated. Succinate and acetate were found to be the most favorable carbon sources for LAD9. Response surface methodology (RSM) analysis demonstrated that maximum removal of ammonium occurred under the conditions with an initial pH of 8.46, C/N ratio of 8.28, temperature of 27.9°C and shaking speed of 150rpm, where temperature and shaking speed produced the largest effect. Further nitrogen balance analysis revealed that 50.1% of nitrogen was removed as gas products and 40.8% was converted to the biomass. Moreover, the occurrence of aerobic denitrification was evidenced by the utilization of nitrite and nitrate as nitrogen sources, and the successful amplifications of membrane bound nitrate reductase and cytochrome cd(1) nitrite reductase genes from strain LAD9. Thus, the nitrogen removal in strain LAD9 was speculated to comply with the mechanism of heterotrophic nitrification coupled with aerobic denitrification (NH(4)(+)-NH(2)OH-NO(2)(-)-N(2)O-N(2)), in which also accompanied with the mutual transformation of nitrite and nitrate. The findings can help in applying appropriate controls over operational parameters in systems involving the use of this kind of strain.

Diversity and Activity of Denitrifiers of Chilean Arid Soil Ecosystems

The Chilean sclerophyllous matorral is a Mediterranean semiarid ecosystem affected by erosion, with low soil fertility, and limited by nitrogen. However, limitation of resources is even more severe for desert soils such as from the Atacama Desert, one of the most extreme arid deserts on Earth. Topsoil organic matter, nitrogen and moisture content were significantly higher in the semiarid soil compared to the desert soil. Although the most significant loss of biologically preferred nitrogen from terrestrial ecosystems occurs via denitrification, virtually nothing is known on the activity and composition of denitrifier communities thriving in arid soils. In this study we explored denitrifier communities from two soils with profoundly distinct edaphic factors. While denitrification activity in the desert soil was below detection limit, the semiarid soil sustained denitrification activity. To elucidate the genetic potential of the soils to sustain denitrification processes we performed community analysis of denitrifiers based on nitrite reductase (nirK and nirS) genes as functional marker genes for this physiological group. Presence of nirK-type denitrifiers in both soils was demonstrated but failure to amplify nirS from the desert soil suggests very low abundance of nirS-type denitrifiers shedding light on the lack of denitrification activity. Phylogenetic analysis showed a very low diversity of nirK with only three distinct genotypes in the desert soil which conditions presumably exert a high selection pressure. While nirK diversity was also limited to only few, albeit distinct genotypes, the semiarid matorral soil showed a surprisingly broad genetic variability of the nirS gene. The Chilean matorral is a shrub land plant community which form vegetational patches stabilizing the soil and increasing its nitrogen and carbon content. These islands of fertility may sustain the development and activity of the overall microbial community and of denitrifiers in particular.

A New Non-Degenerate Primer Pair for the Specific Detection of the Nitrite Reductase Gene nrfA in the Genus Desulfovibrio

Dissimilatory nitrate reduction to ammonia (DNRA) is the process in which nitrate is reduced, via nitrite, to ammonia. Bacteria known to carry out DNRA mainly originate from wastewater treatment plants, where DNRA is a relevant process. The ability to carry out DNRA is phylogenetically widespread, and the gene nrfA, encoding for the key enzyme of the second step of the pathway, could be used as a marker for this process. In this study we developed a new primer pair specific for nrfA in the genus Desulfovibrio. The specificity of the primer pair was tested on DNA from thirteen species of Desulfovibrio and DNA from two wastewater samples. PCR amplifications yielded products of the expected size (850 bp), and sequences obtained from Desulfovibrio strains and environmental sample clone libraries matched the Desulfovibrio nrfA gene. Nevertheless, we found nrfA gene sequences in the environmental samples that are not present in the databases. The new primer set can be used to obtain more sequences of the nrfA gene and improve our knowledge of the DNRA pathway in this genus, e.g. with the aim to improve the wastewater treatment process.

Denitrifying Bacteria from the Genus Rhodanobacter Dominate Bacterial Communities in the Highly Contaminated Subsurface of a Nuclear Legacy Waste Site

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface was investigated at the field scale at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN. The abundance, community composition, and distribution of groundwater microorganisms were examined across the site during two seasonal sampling events. At representative locations, subsurface sediment was also examined from two boreholes, one sampled from the most heavily contaminated area of the site and another from an area with low contamination. A suite of DNA- and RNA-based molecular tools were employed for community characterization, including quantitative PCR of rRNA and nitrite reductase genes, community composition fingerprinting analysis, and high-throughput pyrotag sequencing of rRNA genes. The results demonstrate that pH is a major driver of the subsurface microbial community structure and that denitrifying bacteria from the genus Rhodanobacter (class Gammaproteobacteria) dominate at low pH. The relative abundance of bacteria from this genus was positively correlated with lower-pH conditions, and these bacteria were abundant and active in the most highly contaminated areas. Other factors, such as the concentration of nitrogen species, oxygen level, and sampling season, did not appear to strongly influence the distribution of Rhodanobacter bacteria. The results indicate that these organisms are acid-tolerant denitrifiers, well suited to the acidic, nitrate-rich subsurface conditions, and pH is confirmed as a dominant driver of bacterial community structure in this contaminated subsurface environment.

Molecular Cloning and Characterization of Nitrite Reductase Gene from Sugarbeet

The nitrite reductase (NiR) gene was cloned using RT-PCR and 3′/5′RACE techniques. The cDNA of NiR gene isolated from sugarbeet was 2 014 bp containing a 1 830 bp opening-reading frame (ORF), encoding 599 amino acids. Further comparison showed that NiR gene had high homology to both of Spinacia oleracea NiR gene and Arabidopsis thaliana NiR gene, which was 93%.The predicted NiR protein found to have a hemoprotein beta-component (ferrodoxin-like), and 4Fe-4S region, its 3D structure was predicted by analysis software. Real time PCR analysis showed that when using 0, 10, 20, 30, 40, 50, 80, and 160 mmol L–1 NO3–-N treated for 72 hours, the expression of NiR gene was the highest at 50 mmol L–1;when using 0, 2, 4, 8, 16, 32, 64, and 128 mmol L–1 NH4+-N treated for 48 hours, the expression of NiR gene showed two peaks at 8 mmol L–1 and 64 mmol L–1, re-spectively. Furthermore, in experiments treated with different ratios of NO3–-N to NH4+-N for 48 h, the expression of NiR gene was the highest when the ratio was 80:20. Cycloheximide 9 h treatment experiments showed that NiR gene expression decreased with increasing the treating concentration. NO2–treatments indicated that the maximum expression of NiR gene was induced by 40 mmol L–1 NO2–.

Differential responses of nirK‐ and nirS‐carrying bacteria to denitrifying conditions in the anoxic rice field soil

Denitrification occurs actively in rice field soils. In the present study, the responses of nirK and nirS denitrifier communities to nitrate addition in the anoxic rice soil were determined through molecular analyses of nitrite reductase genes nirK and nirS and 16S rRNA genes. Denitrification occurred rapidly when nitrate was added at the beginning of anoxic incubation (experiment I). The structure of nirK-type denitrifiers did not change; but their abundance as determined by quantitative (real-time) PCR increased in nitrate treatments compared with control. Both the structure and abundance of nirS denitrifiers remained unaffected in experiment I. The rate of denitrification was slowed down when nitrate was added 20 days after the onset of anoxic incubation (experiment II). The structure and abundance of nirK-type denitrifier community did not respond to nitrate addition; but the nirS community changed substantially in this experiment. The copy number of nirS genes increased by an order of magnitude in the treatments of 5 mM and 10 mM nitrate compared with control. The terminal restriction fragment length polymorphism (T-RFLP) analysis of nirS genes revealed that the 100 bp T-RF substantially increased in the nitrate treatments. Cloning and sequence analysis indicated that this T-RF had similarity of up to 90% with Herbaspirillum sp. T-RFLP profiles of the bacterial 16S rRNA genes also showed that Herbaspirillum sp. increased after nitrate amendments. Collectively, the nirK-type denitrifiers were probably active at the beginning of anaerobic incubation, while the nirS denitrifiers, especially those related with Herbaspirillum sp. probably were more active when anaerobic condition was fully developed.

Distribution of typical denitrifying functional genes and diversity of the <i>nirS</i>-encoding bacterial community related to environmental characteristics of river sediments

Abstract. Denitrification in river sediments leads to nitrate removal from the aquatic system; therefore, it is necessary to understand functional diversity of denitrifier communities in the system. Sediment samples (0–25 cm depth) were collected from three typical locations along the Pearl River. The real-time PCR approach was used to measure the abundance of nitrate (narG), nitrite (nirS, nirK and nrfA), and nitrous oxide (nosZ) reductase genes from the sediment samples. Assemblages of nirS, nirK and nosZ indicated that complete denitrification occurred in sediment cores, with the greatest number of gene copies from 5–15 cm depth. Dissimilatory nitrate reduction appeared to be important below 15 cm depth, based on increasing gene copies of narG and nrfA with sediment depth. There was a close match (78–94 %) between the nirS sequences recovered from the Pearl River sediment and those detected in estuarine and marine sediments as well as active sludge, suggesting that the nitrogen source in the Pearl River sediment was affected by domestic sewage inputs and irregular tides. Canonical correspondence analysis indicated that the spatial distribution of denitrifying bacteria was highly correlated with dissolved inorganic nitrogen (including NH4+, NO2− and NO3−) concentrations in sediment. It was concluded that the difference in dissolved inorganic nitrogen concentrations along the sediment profile influenced the distribution of denitrifying genes and the nirS-encoding denitrifier community in the river sediment. In addition, a variety of novel denitrifying bacteria were revealed in the river sediment.

Nitrate removal, communities of denitrifiers and adverse effects in different carbon substrates for use in denitrification beds

Denitrification beds are containers filled with wood by-products that serve as a carbon and energy source to denitrifiers, which reduce nitrate (NO 3 −) from point source discharges into non-reactive dinitrogen (N 2) gas. This study investigates a range of alternative carbon sources and determines rates, mechanisms and factors controlling NO 3 − removal, denitrifying bacterial community, and the adverse effects of these substrates. Experimental barrels (0.2 m 3) filled with either maize cobs, wheat straw, green waste, sawdust, pine woodchips or eucalyptus woodchips were incubated at 16.8 °C or 27.1 °C (outlet temperature), and received NO 3 − enriched water (14.38 mg N L −1 and 17.15 mg N L −1). After 2.5 years of incubation measurements were made of NO 3 −–N removal rates, in vitro denitrification rates (DR), factors limiting denitrification (carbon and nitrate availability, dissolved oxygen, temperature, pH, and concentrations of NO 3 −, nitrite and ammonia), copy number of nitrite reductase ( nirS and nirK) and nitrous oxide reductase ( nosZ) genes, and greenhouse gas production (dissolved nitrous oxide (N 2O) and methane), and carbon (TOC) loss. Microbial denitrification was the main mechanism for NO 3 −–N removal. Nitrate–N removal rates ranged from 1.3 (pine woodchips) to 6.2 g N m −3 d −1 (maize cobs), and were predominantly limited by C availability and temperature ( Q 10 = 1.2) when NO 3 −–N outlet concentrations remained above 1 mg L −1. The NO 3 −–N removal rate did not depend directly on substrate type, but on the quantity of microbially available carbon, which differed between carbon sources. The abundance of denitrifying genes ( nirS, nirK and nosZ) was similar in replicate barrels under cold incubation, but varied substantially under warm incubation, and between substrates. Warm incubation enhanced growth of nirS containing bacteria and bacteria that lacked the nosZ gene, potentially explaining the greater N 2O emission in warmer environments. Maize cob substrate had the highest NO 3 −–N removal rate, but adverse effects include TOC release, dissolved N 2O release and substantial carbon consumption by non-denitrifiers. Woodchips removed less than half of NO 3 − removed by maize cobs, but provided ideal conditions for denitrifying bacteria, and adverse effects were not observed. Therefore we recommend the combination of maize cobs and woodchips to enhance NO 3 − removal while minimizing adverse effects in denitrification beds.

Intron features of key functional genes mediating nitrogen metabolism in marine phytoplankton

Introns are widespread and variable in eukaryotic genomes. Although their histories and functions, or even whether all of them have any function, remain largely unknown, analysis of intron sequences and genomic contexts may shed light on the evolutionary history of genes and organisms. The number and frequency of introns vary widely in the small number of published genomes of protists and algae suggesting that the same is true of the vast diversity of protists and algae that remain uncultivated. The objective of this study were to investigate introns in sequences of functional genes of phytoplankton, both in published genomes and in sequences obtained from environmental clone libraries. We examined the introns of the genes involved in nitrogen uptake and assimilation pathways in the genome sequences of cultivated phytoplankton as well as in environmental clone libraries of nitrate reductases ( NR), nitrite reductase ( NiR), nitrate transporter ( Nrt2) and ammonium transporter ( AMT) genes constructed from pelagic phytoplankton communities in Monterey Bay (CA, USA) and Onslow Bay (NC, USA). Here we describe the most extensive set to date of intron sequences from uncultivated marine algae and report important differences for diatom vs. non-diatom sequences. The majority of the introns in NR, NiR, Nrt2 and AMT from cultured phytoplankton and environmental libraries showed canonical splice patterns. Introns found in diatom-like NR environmental libraries had lower GC content than the respective exons. The green algal-like NR and Nrt2 environmental sequences had introns and exons of much more similar GC content, and both higher than in diatoms. These patterns suggest a different evolutionary history and recent acquisition of diatom introns compared to other algae.

Roles of the transcriptional regulation mediated by the nitrate-responsive cis-element in higher plants

Nitrate is a major nitrogen source for land plants but also acts as a signaling molecule that regulates gene expression, metabolism and plant growth. The genes for nitrate reductase (NR) and nitrite reductase (NIR), which are enzymes in the nitrate assimilation pathway, are typical nitrate-inducible genes. We previously identified the first authentic nitrate-responsive cis-element (NRE) for nitrate-inducible transcription by the analysis of the NIR gene promoter from Arabidopsis. Here we further characterize NRE-mediated regulation using transgenic Arabidopsis plants. First, NRE-mediated regulation is shown to be a primary response to nitrate that is caused by pre-existing components, because the NRE-mediated nitrate-inducible expression of the GUS reporter gene is unaffected by treatment with the protein synthesis inhibitor cycloheximide. Second, we show that NRE-like sequences are present in various dicotyledonous and monocotyledonous NIR gene promoters at similar positions and that they also drive nitrate-inducible expression in Arabidopsis. This suggests that NRE-mediated regulation might be conserved in higher plants. Finally, the NRE-mediated expression of the GUS reporter gene is shown to produce diurnal expression in transgenic Arabidopsis plants. This expression peaked at the beginning of the day and decreased during the day which is very similar to the reported diurnal pattern of the nitrate content, suggesting a role of NRE-mediated regulation in controlling diurnal expression in response to oscillation of the nitrate content over a day/night cycle. These findings further clarify the roles of the NRE-mediated regulatory system in higher plants.

Impact of copper on the diversity, abundance and transcription of nitrite and nitrous oxide reductase genes in an urban European estuary

In this study, the effects of increasing copper (Cu) concentrations on the denitrification pathway and on the diversity of a denitrifier community and gene expression were evaluated in intertidal sandy sediments of the Douro River estuary (north-west Portugal). The results demonstrated that sediment denitrification rates were highly affected by Cu; almost complete inhibition (85%) of the process was observed in sediments amended with 60 μg Cu g(-1) wet wt sediment. Moreover, the addition of Cu stimulated the accumulation of both N(2)O and NO(2)(-) and inhibited the rate of NO(3)(-) uptake. Further, the amendment with even the lowest Cu concentration (4 μg Cu g(-1) wet wt sediment) yielded a drastic decrease in the abundance of nirK, nirS and nosZ (between 79% and 81%) assessed by means of real-time PCR. In agreement, reverse transcription-PCR-denaturing gradient gel electrophoresis analysis of nirK, nirS and nosZ transcripts showed a progressive decrease in the diversity of the transcription products of these genes with an increase of the Cu concentration.

Characterization of Microbial Communities Transforming and Removing Nitrogen in Wetlands

The community structure of bacteria responsible for transformation and removal of nitrogen species from farmyard runoff within two different full-scale integrated constructed wetland (ICW) systems was studied. Microbial communities in litter and sediment components were investigated using denaturing gradient gel electrophoresis (DGGE) and band sequencing of the 16S ribosomal ribonucleic acid gene fragments of putative ammonia-oxidizing bacteria and the nitrite reductase genes (nirK/S) of putative denitrifying bacteria. Findings show that retrieved sequences of ammonia-oxidizing bacteria communities in litter samples were related to Nitrosomonas and Nitrosospira, while those from sediment samples were related to only Nitrosospira. A more diverse denitrifying community was present in the litter compared to the sediment samples. Non-metric multi-dimensional scaling analysis showed that microbial communities were readily distinguishable with respect to site and source (sediment and litter) for the two representative example ICW systems. A comparison of the species composition of the ammonia-oxidizing bacteria and denitrifiers in the systems revealed that the communities were no more similar or dissimilar than if they had been assembled by chance.

Biodegradation of carbazole by the seven Pseudomonas sp. strains and their denitrification potential

Carbazole, one representative of non-alkaline nitrogen heterocyclic compounds, is widespread in the natural environment and harmful to human health. In this research, the seven bacterial strains using carbazole as their sole carbon, nitrogen and energy source were isolated from activated sludge of a coking wastewater treatment plant. All strains efficiently degraded 500 mg/L of carbazole in the medium within 36 h. Based on the DNA sequence and phylogenetic tree analysis, the seven strains were identified as the genera Pseudomonas with different evolutionary pathways. PCR analysis revealed that the seven isolates carried the car gene. Moreover, all of these strains could utilize and transform ammonium and nitrate efficiently, and the six strains except BC043 strain coded the nitrite reductase gene ( nirS) and the nitrous oxide reductase ( nosZ), that indicated their denitrification ability. All these strains may be useful in the bioremediation of environments contaminated by carbazole.

Isolation, Chromosome Assignment, and Expression Assay of Nitrite Reductase Gene and Regulatory Sequence in Wheat

Nitrite reductase is wildly involved in N metabolism in plants and has shown excellent relevance to regeneration poten-tial in the tissue culture of rice(Oryza sativa L.).In this study,in silico and inverse PCR techniques were employed to isolate nitrite reductase encoded gene and its regulatory sequences from wheat(Triticum aestivum L.).This gene was predicted to contain four exons and three introns.The gDNA and cDNA sequences were 2 881 bp and 1 830 bp in length,respectively,and they were both submitted to GenBank under the accession numbers FJ555239 and FJ527909.Its deduced encoding protein product was ap-proximately 65.7 kD,sharing high(more than 60%) identity with other nitrite reductase genes deposited in the NCBI database,especially with those from other monocot cereal crops(more than 80%).The 5′ flanking region was isolated and extended to -2 924 bp(counting from the start code: ATG) through inverse PCR.After induced by 1 mmol L-1 IPTG,a protein of ca.70 kD was obtained in prokaryotic expression vector pET-28a,including a histidine tag of ca.3.8 kD.The highest expression of gene in wheat seedlings was induced by 30 mmol L-1 KNO3 for 1 h.Measurement on nitrite reductase activity showed the enzyme acti-vity increased following the treatment time extension at 5-d intervals under the above same condition.According to the result of AS-PCR analysis,at least one copy of the gene existed on chromosome 6A and 6B each in common wheat.The study provides a basis for functional determination of wheat nitrite reductase gene in subsequent research.

Isolation of Oligotrophic Denitrifiers Carrying Previously Uncharacterized Functional Gene Sequences

Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N(2)O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.

Denitrifying polyphosphate accumulating organisms population and nitrite reductase gene diversity shift in a DEPHANOX-type activated sludge system fed with municipal wastewater

Enhanced biological phosphorus removal (EBPR) is a widely applied method for nutrients removal, although little is known about the key genes regulating the complex biochemical transformations occurring in activated sludge during phosphorus removal. In the present study, the nitrite reductase gene (nirS) diversity and the denitrifying polyphosphate accumulating organisms (DPAOs) population, grown in a bench scale, two-sludge, continuous flow plant, operating for biological anoxic phosphorus removal (DEPHANOX-type), fed with municipal wastewater, were examined by means of physicochemical analyses and the application of molecular techniques. The DEPHANOX configuration highly influenced biomass phosphorus as well as polyhydroxyalkanoates content and facilitated the enrichment of the DPAOs population. The application of double probe fluorescent in situ hybridization (double probe FISH) technique revealed that DPAOs comprised 20% of the total bacterial population. Based on clone libraries construction and nirS gene sequencing analysis, a pronounced shift in denitrifying bacteria diversity was identified during activated sludge acclimatization. Moreover, nirS gene sequences distinct from those detected in any known bacterial strain or environmental clone were identified. This is the first report studying the microbial properties of activated sludge in a DEPHANOX-type system using molecular techniques.

Investigation of Denitrifying Bacteria Communities in Fresh and Aged Municipal Solid Waste Using Nitrite Reductase Genes

Nitrogen removal is of great importance in management of sanitary landfills. To optimize the process of denitrification, a better comprehension of denitrifiers was needed. No molecular studies using function genes with a special focus on diversity and quantity of denitrifying communities in municipal solid waste (MSW) have been reported to date. This research investigated the communities of denitrifying bacteria in MSW samples collected from fresh (3-month-old) and aged (10-year-old) landfill bioreactors. The main methods were denaturing gradient gel electrophoresis and real-time polymerase chain reaction using nirK and nirS as the markers. Results showed that most nirK-containing denitrifiers related to the known Rhizobiales bacteria were from the aged MSW sample, while denitrifiers related to Alcaligenaceae bacteria were all from the fresh one. A clear difference in the diversity of nirK-containing denitrifiers between two MSW samples was observed. About half of the nirS gene sequences obtained was found closely related to the nirS of β-proteobacterium (Burkholderiales and Rhodocyclales) and γ-proteobacterium (Pseudomonadales). The rest of the nirS gene sequences were specific. Real-time polymerase chain reaction results revealed that nirK was more abundant than nirS in the fresh MSW sample, whereas nirS was predominant in the aged one. Aged MSW had more potential denitrifiers to reduce NO3− to NO2− than fresh MSW. This work provided more accurate information about the characteristics of denitrifying communities as a result of using functional gene, rather than using 16S rDNA.

Nitric oxide‐sensitive and ‐insensitive interaction of Bacillus subtilis NsrR with a ResDE‐controlled promoter

NsrR is a nitric oxide (NO)-sensitive transcription repressor that controls NO metabolism in a wide range of bacteria. In Bacillus subtilis, NsrR represses transcription of the nitrite reductase (nasDEF) genes that are under positive control of the ResD-ResE two-component signal transduction system. Derepression is achieved by reaction of NO with NsrR. Unlike some NsrR orthologues that were shown to contain a NO-sensitive [2Fe-2S] cluster, B. subtilis NsrR, when purified anaerobically either from aerobic or from anaerobic Escherichia coli and B. subtilis cultures, contains a [4Fe-4S] cluster. [4Fe-4S]-NsrR binds around the -35 element of the nasD promoter with much higher affinity than apo-NsrR and binding of [4Fe-4S]-NsrR, but not apo-protein, is sensitive to NO. RNA polymerase and phosphorylated ResD make a ternary complex at the nasD promoter and NsrR dissociates the preformed ternary complex. In addition to the -35 region, NsrR binds to two distinct sites of the upstream regulatory region where ResD also binds. These interactions, unlike the high-affinity site binding, do not depend on the NsrR [4Fe-4S] cluster and binding is not sensitive to NO, suggesting a role for apo-NsrR in transcriptional regulation.

Quantification of nitrogen reductase and nitrite reductase genes in soil of thinned and clear-cut Douglas-fir stands by using real-time PCR.

The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH₄-N, NO₃-N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time.