Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Structure and Function of Methanogens along a Short-Term Restoration Chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Impact of Long-Term Fertilization on the Composition of Denitrifier Communities Based on Nitrite Reductase Analyses in a Paddy Soil

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

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Effect of Sulfadiazine on Abundance and Diversity of Denitrifying Bacteria by Determining nirK and nirS Genes in Two Arable Soils

Sulfadiazine (SDZ) is an antibiotic frequently used in agricultural husbandry. Via manuring of excrements of medicated animals, the drug reaches the soil and might impair important biochemical transformation processes performed by microbes, e.g., the nitrogen turnover. We studied the effect of pig manure and SDZ-spiked pig manure on denitrifying bacteria by quantifying nirK and nirS nitrite reductase genes in two arable soils. Addition of manure entailed mainly an increase of nirK-harboring denitrifiers in both soils, whereas in the SDZ-amended treatments, primarily the nirS denitrifiers increased in abundance after the bioavailable SDZ had declined. However, the community composition of nirS nitrite reducers investigated by denaturing gradient gel electrophoresis did not change despite the observed alterations in abundance.     

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2–6 times more abundant than those revealed with the conventional primers. RNA- and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.     

Effect of pH and dissolved organic matter on the abundance of nirK and nirS denitrifiers in spruce forest soil

Acid N depositions in the Bohemian Forest during the second half of the last century caused enormous soil acidification which led to the leaching of essential nutrients including nitrates. We investigated the effect of dissolved organic matter (DOM) and pH on the abundance of 16S RDNA, nirK and nirS gene copies in four spruce forest sites. Soil samples for molecular based quantification (qPCR) were taken from the organic litter and humus layers. The amounts of dissolved organic carbon (DOC) and dissolved nitrogen (DN) were much lower in highly acidified soils. We found a strong correlation between nirK denitrifiers and the amount of available P (r = 0.83, p < 0.001), which suggested a higher nutrient sensitivity of this group of denitrifying bacteria. Additionally, we found that correlations between the amount of nirK denitrifiers and DOC and pH are exponentional showing two important threshold values, being 4.8 mol kg^?1 and 5, respectively. The amount of nirK denitrifiers rapidly decreased below these values. The amount of nirK and nirS denitrifiers was higher in the organic litter horizon than the organic humus horizon at all sampling sites.     

Distribution of denitrifying bacterial communities in the stratified water column and sediment–water interface in two freshwater lakes and the Baltic Sea

We have studied the distribution and community composition of denitrifying bacteria in the stratified water column and at the sediment–water interface in lakes Plußsee and Schöhsee, and a near-shore site in the Baltic Sea in Germany. Although environmental changes induced by the stratification of the water column in marine environments are known to affect specific populations of denitrifying bacteria, little information is available for stratified freshwater lakes and brackish water. The aim of the present study was to fill this gap and to demonstrate specific distribution patterns of denitrifying bacteria in specific aquatic habitats using two functional markers for the nitrite reductase (nirK and nirS genes) as a proxy for the communities. The leading question to be answered was whether communities containing the genes nirK and nirS have similar, identical, or different distribution patterns, and occupy the same or different ecological niches. The genes nirK and nirS were analyzed by PCR amplification with specific primers followed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequence analysis. Overall, nirS-denitrifiers were more diverse than nirK-denitrifiers. Denitrifying communities in sediments were clearly different from those in the water column in all aquatic systems, regardless of the gene analyzed. A differential distribution of denitrifying assemblages was observed for each particular site. In the Baltic Sea and Lake Plußsee, nirK-denitrifiers were more diverse throughout the water column, while nirS-denitrifiers were more diverse in the sediment. In Lake Schöhsee, nirS-denitrifiers showed high diversity across the whole water body. Habitat-specific clusters of nirS sequences were observed for the freshwater lakes, while nirK sequences from both freshwater lakes and the Baltic Sea were found in common phylogenetic clusters. These results demonstrated differences in the distribution of bacteria containing nirS and those containing nirK indicating that both types of denitrifiers apparently occupy different ecological niches.     

Salinity Decreases Nitrite Reductase Gene Diversity in Denitrifying Bacteria of Wastewater Treatment Systems

Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant nirK clone was related to nirK of the genus Alcaligenes.     

Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots

An analysis of the molecular diversity of N₂ fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N₂ fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.     

Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Denitrifier Community Composition along a Nitrate and Salinity Gradient in a Coastal Aquifer

Nitrogen flux into the coastal environment via submarine groundwater discharge may be modulated by microbial processes such as denitrification, but the spatial scales at which microbial communities act and vary are not well understood. In this study, we examined the denitrifying community within the beach aquifer at Huntington Beach, California, where high-nitrate groundwater is a persistent feature. Nitrite reductase-encoding gene fragments (nirK and nirS), responsible for the key step in the denitrification pathway, were PCR amplified, cloned, and sequenced from DNAs extracted from aquifer sediments collected along a cross-shore transect, where groundwater ranged in salinity from 8 to 34 practical salinity units and in nitrate concentration from 0.5 to 330 μM. We found taxonomically rich and novel communities, with all nirK clones exhibiting <85% identity and nirS clones exhibiting <92% identity at the amino acid level to those of cultivated denitrifiers and other environmental clones in the database. Unique communities were found at each site, despite being located within 40 m of each other, suggesting that the spatial scale at which denitrifier diversity and community composition vary is small. Statistical analyses of nir sequences using the Monte Carlo-based program ∫-Libshuff confirmed that some populations were indeed distinct, although further sequencing would be required to fully characterize the highly diverse denitrifying communities at this site.     

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.     

Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico

To understand the composition and structure of denitrifying communities in the oxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) and Pseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp. strain G-179 (98 to 99%), Bradyrhizobium japonicum (91%), Blastobacter denitrificans (83%), and Alcaligenes xylosoxidans (96%). The rest of the clones, however, were less than 80% similar to nirS and nirK sequences available in sequence databases. The results of a principal-component analysis (PCA) based on the percentage of OTUs and biogeochemical data indicated that the nitrate concentration and oxygen have an effect on the denitrifying communities. The communities at the stations in oxygen-deficient zones were more similar than the communities at the stations in the oxygenated zone. The denitrifying communities were more similar at the stations that were closer together and had similar nitrate levels. Also, the results of PCA based on biogeochemical properties suggest that geographic location and biogeochemical conditions, especially the nitrate and oxygen levels, appear to be the key factors that control the structure of denitrifying communities.     

不同季节艾比湖湿地盐角草根际nirS-型与nirK-型反硝化细菌群落组成分析

旨在了解艾比湖湿地盐生植物盐角草根际与非根际中不同类型反硝化细菌的分布及其随季节变化情况,为温带干旱地区荒漠盐化生态系统的代表-艾比湖湿地在生态植被恢复过程中,由微生物推动的土壤氮素循环过程提供数据支撑。采集了艾比湖湿地夏、秋、春三个季节的盐角草根际和非根际土壤样本,通过高通量测序技术,比较分析了nirS-型和nirK-型两种类型的反硝化细菌的多样性和群落结构特点;利用RDA (redundancy analysis)探究了土壤理化因素对反硝化细菌多样性及群落结构的影响。艾比湖湿地盐角草根际与非根际中,nirS-型和nirK-型反硝化细菌多样性最高的为秋季根际土壤样本;各土壤样本中的反硝化细菌多样性均呈现根际>非根际。盐角草各土壤样本中的nirS-型反硝化细菌在门分类水平上隶属于变形菌门(Proteobacteria),厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),而nirK-型反硝化细菌在门水平上分类仅包括了Proteobacteria和Firmicutes。Proteobacteria在各土壤样本中的占比均较高;其中Gamma-Proteobacteria的盐单胞菌属(Halomonas)和假单胞菌属(Pseudomonas)是各土壤样本所共有的nirS-型反硝化菌的优势菌属,但它们在每个土壤样本中的相对丰度各有差异。Alpha-Proteobacteria的根瘤菌属(Rhizobium)是盐角草各土壤样本中较为广泛存在的nirK-型反硝化细菌。艾比湖湿地盐角草各土壤样本中的反硝化细菌群落结构存在着一定的差异。RDA结果显示含水量、有机质、全氮和铵态氮等对各土壤样本中的nirS-型反硝化细菌的多样性影响较大,含水量、有机质、全氮、碱解氮等是nirK-型反硝化细菌多样性的主要影响因素。土壤电导率、全磷、全钾、全氮和碱解氮协同影响nirS-型反硝化细菌的群落结构,有机质、速效钾、速效磷、pH和硝态氮是nirK-型反硝化细菌群落结构组成的主要影响因素。艾比湖湿地反硝化细菌呈现季节性变化,nirS-型和nirK-型反硝化细菌以不同的主要菌属,共同推进湿地反硝化作用。而对于湿地生态系统的保护,则需要进行长期而广泛的土壤状态评估和土壤反硝化微生物菌群的动态监测。     

Nitrite Reductase Genes (nirK and nirS) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

Cryoturbated peat circles (that is, bare surface soil mixed by frost action; pH 3–4) in the Russian discontinuous permafrost tundra are nitrate-rich ‘hotspots'' of nitrous oxide (N₂O) emissions in arctic ecosystems, whereas adjacent unturbated peat areas are not. N₂O was produced and subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated but not in those with unturbated peat soil. Nitrate, nitrite and acetylene stimulated net N₂O production of both soils in anoxic microcosms, indicating denitrification as the source of N₂O. Up to 500 and 10 μ nitrate stimulated denitrification in cryoturbated and unturbated peat soils, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification were 28 and 18 nmol N₂O g_DW⁻¹ h⁻¹, for cryoturbated and unturbated peat soils, respectively. Barcoded amplicon pyrosequencing of narG, nirK/nirS and nosZ (encoding nitrate, nitrite and N₂O reductases, respectively) yielded ≈49 000 quality-filtered sequences with an average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were detected per soil and gene, many of which were distantly related to cultured denitrifiers or environmental sequences. Denitrification-associated gene diversity in cryoturbated and in unturbated peat soils differed. Quantitative PCR (inhibition-corrected per DNA extract) revealed higher copy numbers of narG in cryoturbated than in unturbated peat soil. Copy numbers of nirS were up to 1000 × higher than those of nirK in both soils, and nirS nirK⁻¹ copy number ratios in cryoturbated and unturbated peat soils differed. The collective data indicate that the contrasting N₂O emission patterns of cryoturbated and unturbated peat soils are associated with contrasting denitrifier communities.