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.     

PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd₁- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd₁ primers were designed to amplify a 778 to 799-bp region of cd₁-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd₁-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd₁-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

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.     

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.     

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.     

Presence of Two Different Active nirS Nitrite Reductase Genes in a Denitrifying Thauera sp. from a High-Nitrate-Removal-Rate Reactor

The nirS nitrite reductase genes were studied in two strains (strains 27 and 28) isolated from two denitrifying reactors and characterized as Thauera according to their 16S rRNA gene sequences. Strain 28 contains a single nirS sequence, which is related to the nirS of Thauera mechernichensis, and strain 27 contains two nirS sequences; one is similar to the nirS sequence from Thauera mechernichensis (gene 2), but the second one (gene 8) is from a separate clade with nirS from Pseudomonas stutzeri, Azoarcus species, Alcaligenes faecalis, and other Thauera species. Both genes were expressed, but gene 8 was constitutively expressed while gene 2 was positively regulated by nitrate.     

Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples

The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.     

Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes

Nitrite reduction is the key step in the denitrification reaction with two predominant types of nitrite reductase genes: nirS and nirK. The diversity of denitrifying bacteria in a municipal wastewater treatment plant is described by using both these genes. Of the cultured colonies, 22.5% contained the NirS gene and 12.5% the nirK gene. These nitrite reductase-containing colonies could be further divided into five different types by using both restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis. Phylogenetic analysis showed that these five types of denitrifying bacteria were phylogenetically diverse. Finally, one nirS gene was obtained and compared with the published sequences.     

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.     

Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10⁶ gene copies (r² = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.     

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.     

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.Nitrogen (N) is a limiting nutrient in most Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest ecosystems. Understanding the links between forest management and forest ecosystem function, including the cycling of N, is of paramount importance to researchers and forest managers. Management practices such as thinning and clear-cutting can alter the soil microbial community, potentially altering the rate and amount of net N addition or loss to the forest floor. Clear-cutting alters the functional diversity of soil microorganisms and alters soil characteristics (temperature, pH, moisture, and nutrient status). Thinning and clear-cutting can increase nitrification, denitrification, and leaching of N in soil, all of which can reduce the available N (2, 13, 22, 41, 47). Clear-cutting in Douglas-fir forests can also remove associated gene pools of diazotrophic microorganisms (46). It is not yet well understood how clear-cutting or thinning affects the abundance of N-cycling microorganisms. We focus on two populations of N-cycling microorganisms: diazotrophs, which biologically fix N₂ gas to ammonia, and denitrifiers, which reduce N oxides and result in the release of N-containing gasses.Fixation of N by diazotrophic microorganisms is the primary source of N addition to undisturbed, unfertilized forest soil ecosystems (9, 39). The diazotrophic community is most often studied in situ using the marker gene for nitrogenase reductase (nifH); the diversity and abundance of diazotrophic microorganisms as determined by nifH characterization may be used as an indicator of overall soil ecological health. Diazotrophs can be symbiotic, associated (e.g., with a specific plant or fungal biomass), or free-living in the soil. Endophytic diazotrophs fix ∼100 times more N than free-living strains (9). Free-living diazotrophs such as Azotobacter vinelandii and A. chroococcum may fix between 0 and 60 kg of N ha⁻¹ year⁻¹ (9) and, because of a relative dearth of endophytic interactions in coniferous forests, free-living diazotrophs can be an important source of N in these soils. Cultural studies have shown that free-living diazotrophs improve the establishment of mycorrhizae and conifer seedlings, with relative activity fluctuating according to season, site aspect, and moisture conditions (11). Fixed-N inputs act as a catalyst for interlinked N-cycling events, e.g., fungal decomposition of woody debris and organic material (28). Nitrogen fixation in temperate forest soil is directly related to the amounts of soil organic matter (17). However, it is unclear how nifH gene abundance relates to the amount of total carbon (C) and organic matter (OM) and N in forest soil. It is also unknown how common silvicultural practices (e.g., clear-cutting and thinning) affect diazotrophic abundance or how diazotrophic abundance may in turn affect cycling of soil nutrients.The reduction of inorganic N oxides by denitrifying microorganisms can cause N loss from forest soil ecosystems, as well as the release of greenhouse gases into the atmosphere. The loss of N from temperate forest soil as N₂O has been reported as ranging from 0.2 to 7.0 kg ha⁻¹ year⁻¹, depending largely on soil nitrogen status, soil moisture, and temperature (57). Robertson and Tiedje (44) state that soil N loss in coniferous ecosystems due to denitrification is regulated by nitrification potential (e.g., nitrate levels) in the soil, and while not considered a major N loss component following clear-cutting, this loss is generally of the same magnitude as the N loss due to leaching. Denitrification is primarily studied using molecular approaches by monitoring several genes in the denitrification pathway: cytochrome cd₁-containing nitrite reductase (nirS), Cu-containing nitrite reductase (nirK), nitrous oxide reductase (nosZ), and membrane-bound nitrate reductase A (narG). The nirS and nirK genes were the denitrification genes used in the present study. Studies demonstrating (i) that the nirS gene is more diverse than nirK in soil and (ii) the domination of the nirK population by a relatively reduced number of clones have been published (42, 45). However, recent meta-analysis of studies involving nirK and nirS has shown that both communities tend to be phylogenetically clustered in undisturbed soils (23).To compare the effects of silvicultural practices on the abundance of diazotrophs and denitrifiers, we used quantitative real-time PCR (qPCR) assays to quantify nifH, nirS, and nirK genes in soil. This method can be used to quantify target sequences in environmental samples. Several qPCR protocols for the analysis of functional gene abundance in soil have been developed for N-cycling genes, including nifH, ammonia monooxygenase (amoA), nirK, nirS, nosZ, and narG (21, 24, 31, 38, 43, 54, 55). The objectives of the present study were (i) to quantify nifH, nirS, and nirK; (ii) to compare the effects of thinning and clear-cutting in Douglas-fir stands on the abundance of total diazotrophs, free-living diazotrophs, and denitrifiers; and (iii) to elucidate the relationships between N-cycling genes and nutrient abundance in forest soils.     

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Steep vertical gradients of oxidants (O₂ and NO₃⁻) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.     

Simultaneous PCR Detection of Multiple Classes of Integron Integrase Genes for Determining the Presence of Multidrug-Resistant Bacteria in Environmental Samples

Dissemination of multidrug-resistant bacteria, particularly in hospitals, has become a serious public health problem. Integrons impart antibiotic multidrug resistance in gram-negative and some gram-positive bacteria by capturing and then disseminating antibiotic resistance genes. This mechanism plays a major role in contributing to the alarmingly high prevalence of bacterial drug resistance. A universal polymerase chain reaction (PCR) primer set was attempted to design to more sensitively and specifically detect integrons in environmental samples. One set, designated intCiF3_a, intCiF3_b, intCiiiR3_a, and intCiiiR3_b, simultaneously amplifies the conserved region of the tyrosine recombinase gene family between box I and box II. This primer set generates PCR products derived from classes 1, 2, and 3 integron integrases from environmental samples such as wastewater. An unexpected finding of this study was the detection of new putative integron integrase gene sequences. This is the subject of ongoing research, which aims to provide a clear understanding of the risk to human health posed by these genetic elements.     

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.