nirS Clones Research Articles

Enrichment of Type I Methanotrophs with <i>nirS</i> Genes of Three Emergent Macrophytes in a Eutrophic Wetland in China

The pmoA gene, encoding particulate methane monooxygenase in methanotrophs, and nirS and nirK genes, encoding bacterial nitrite reductases, were examined in the root and rhizosphere sediment of three common emergent macrophytes (Phragmites australis, Typha angustifolia, and Scirpus triqueter) and unvegetated sediment from eutrophic Wuliangsuhai Lake in China. Sequencing analyses indicated that 334 out of 351 cloned pmoA sequences were phylogenetically the most closely related to type I methanotrophs (Gammaproteobacteria), and Methylomonas denitrificans-like organisms accounted for 44.4% of the total community. In addition, 244 out of 250 cloned nirS gene sequences belonged to type I methanotrophs, and 31.2% of nirS genes were the most closely related to paddy rice soil clone SP-2-12 in Methylomonas of the total community. Three genera of type I methanotrophs, Methylomonas, Methylobacter, and Methylovulum, were common in both pmoA and nirS clone libraries in each sample. A quantitative PCR (qPCR) analysis demonstrated that the copy numbers of the nirS and nirK genes were significantly higher in rhizosphere sediments than in unvegetated sediments in P. australis and T. angustifolia plants. In the same sample, the nirS gene copy number was significantly higher than that of nirK. Furthermore, type I methanotrophs were localized in the root tissues according to catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Thus, nirS-carrying type I methanotrophs were enriched in macrophyte root and rhizosphere sediment and are expected to play important roles in carbon/nitrogen cycles in a eutrophic wetland.

Diversity of denitrifying bacteria in the greenwater system of coastal aquaculture

Greenwater technology is based on integrating finfishes inside pens for zero water exchange system of shrimp aquaculture. Nitrogen transformation could be improved by rearing euryhaline finfishes like grey mullet, milkfish which have a broad diet and tolerate poor water quality. The abundance of four denitrifying functional genes coding for nitrate reductase (narG and napA), nitrite reductase (nirS), nitricoxide reductase (qnorB) and nitrousoxide reductase (nosZ) has been examined in the greenwater system through a metagenomic approach. Phylogeny revealed homology of narG clones with uncultured environmental clones, whereas napA clone sequences were found to have homology with cultured (Stappia aggregata) and uncultured microorganisms. The nirS clones show uniqueness with Marinobacter hydrocarbonoclasticus, Aromatoleum aromaticum, and Ruegeria pomeroyi. The qnorB gene has been reported for the first time from culture systems along the Indian coast and clone exhibited 84–87 % identity with different uncultured bacteria. The nosZ clones are closely affiliated with S. aggregate and alpha bacterium. This study revealed denitrifying diversity from a greenwater system which could eventually be used in planning the future strategy for comprehending nitrogen fluxes, greenhouse gases and their mitigation in coastal aquaculture systems.

Diversity and abundance of Bacteria and nirS-encoding denitrifiers associated with the Juan de Fuca Ridge hydrothermal system

Denitrification, which results in the loss of bioavailable nitrogen—an essential macronutrient for all living organisms—may potentially affect chemosynthetic primary production in hydrothermal vent ecosystems where sub-oxic conditions favorable to denitrification are common. Here we describe the diversity and abundance of denitrifying bacteria in the subsurface biosphere at Axial Volcano and the Endeavour Segment on the Juan de Fuca Ridge using a combination of quantitative polymerase chain reaction assays, and small subunit ribosomal RNA (SSU or 16S rRNA) pyrotag and nitrite reductase (nirS) clone library sequencing methods. Bacterial communities were diverse and dominated by members of the e- and γ-proteobacteria, including taxonomic groups containing known denitrifiers. Assemblages of denitrifiers that could be evaluated by nirS gene sequence comparisons showed low diversity. The single nirS sequence shared by the two locations, affiliated with a γ-proteobacteria isolated from estuarine sediments (Pseudomonas sp. BA2), represented more than half of all sequences recovered when clustered at 97 % identity. All other nirS sequences clustered into different taxonomic groups, indicating important differences in denitrifier community membership between the two sites. Total nirS gene abundance was at least two orders of magnitude lower than 16S rRNA abundance. Overall, our results demonstrate that the diversity and abundance of the nirS gene-containing bacterial community are rather low, as might be expected under the extreme conditions encountered in the subsurface biosphere of hydrothermal vent systems, and do not correlate clearly with any environmental variables investigated (i.e., pH, temperature, and H2S, NO3 −, NH4 + concentrations).

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.

Denitrifying bacterial community in manure compost pellets applied to an Andosol upland field

A large amount of nitrous oxide (N2O), one of the most important greenhouse gases, was emitted after the application of manure compost pellets (MCPs) compared to treatments with ordinary manure composts or chemical fertilizers. Emission of N2O was found to be due to denitrification in the pellets. To study the relationship between N2O emission and denitrifiers, the denitrifying bacterial communities in the MCPs were investigated by comparing clone libraries of nitrite reductase genes (nirK and nirS) fragments. Pellet samples were obtained three days (P3) and 26 days (P26) after pellet application to the field. The P3 samples were emitting high levels of N2O, and the P26 samples had ceased N2O emission. Polymerase chain reaction (PCR) amplicons of both nirK and nirS were obtained from pellet samples. The diversity index (Shannon–Weiner index) of clone libraries was higher for the nirS genes than for the nirK genes for the same pellet samples and higher in P3 than in P26 for both nir genes. The clone libraries showed that the compositions of nirK denitrifiers in the pellets were relatively stable, but those of the nirS denitrifiers were variable within a month after pellet application; nirS clones that showed a lower similarity with nirS of known denitrifiers were dominant in P3 and those related to nirS of Cupriavidus spp. and Herbaspirillum spp. were dominant in P26. Most of the nirK clones in this study were related to the nirK genes of Rhizobiales denitrifying bacterial isolates. The nirS clones were divided into three groups; (1) those related to nirS of Bradyrhizobium spp., and Rhodanobacter sp. D206a, (2) those related to nirS of Cupriavidus spp., Herbaspirillum spp., and (3) those that had a lower similarity with nirS of known denitrifiers. This study showed a different response of nirK and nirS denitrifiers in the applied MCPs. In order to understand the relation between the denitrifying diversity and the N2O emissions, transcriptional and metabolic analysis will be also needed.

Transitions in nirS-type denitrifier diversity, community composition, and biogeochemical activity along the Chesapeake Bay estuary

Chesapeake Bay, the largest estuary in North America, can be characterized as having steep and opposing gradients in salinity and dissolved inorganic nitrogen along the main axis of the Bay. In this study, the diversity of nirS gene fragments (encoding cytochrome cd1-type nitrite reductase), physical/chemical parameters, and benthic N2-fluxes were analyzed in order to determine how denitrifier communities and biogeochemical activity vary along the estuary salinity gradient. The nirS gene fragments were PCR-amplified, cloned, and sequenced from sediment cores collected at five stations. Sequence analysis of 96–123 nirS clones from each station revealed extensive overall diversity in this estuary, as well as distinct spatial structure in the nirS sequence distributions. Both nirS-based richness and community composition varied among stations, with the most dramatic shifts occurring between low-salinity (oligohaline) and moderate-salinity (mesohaline) sites. For four samples collected in April, the nirS-based richness, nitrate concentrations, and N2-fluxes all decreased in parallel along the salinity gradient from the oligohaline northernmost station to the highest salinity (polyhaline) station near the mouth of the Bay. The vast majority of the 550 nirS sequences were distinct from cultivated denitrifiers, although many were closely related to environmental clones from other coastal and estuarine systems. Interestingly, 8 of the 172 OTUs identified accounted for 42% of the total nirS clones, implying the presence of a few dominant and many rare genotypes, which were distributed in a non-random manner along the salinity gradient of Chesapeake Bay. These data, comprising the largest dataset to investigate nirS clone sequence diversity from an estuarine environment, also provided information that was required for the development of nirS microarrays to investigate the interaction of microbial diversity, environmental gradients, and biogeochemical activity.

Evaluating wastewater stabilizing constructed wetland, through diversity and abundance of the nitrite reductase gene nirS, with regard to nitrogen control

The diversity and abundance of the nitrite reductase gene nirS were investigated for a free surface flow constructed, in both the summer and winter seasons. Nitrate was efficiently removed especially by the Typha wetland, under anoxic conditions, in both seasons. Clone library analysis, which used the distance based operational taxonomic unit richness program, suggested that the Typha wetland sediment samples, collected in September, provided the highest diversity and richness, in terms of corresponding indices (Simpson D, Shannon-Weiner ( H′ ), and Chao 1). The phylogenetic analysis based on deduced amino acids, divided 211 nirS clones into eight clusters. Although most of the clones were loosely related to the nirS gene of cultivated denitrifying bacteria, some clones were related to the nirS of Xanthomonadales, Burkholderiales, Rhodocyclales and Hydrogenophilales bacteria, over the sampling time, respectively. The real time polymerase chain reaction results showed that the copy numbers of nirS from both the Acorus and Typha wetlands in the summer (13.2 ± 2.0 and 1.8 ± 0.2 × 10 8copies/g soil) were significantly higher than those of the corresponding nirS genes in the winter (8.6 ± 1.1 and 0.7 ± 0.1 × 10 8copies/g soil). These results suggest that the diversity and abundance of the nirS can affect, in regard to nitrogen removal efficiency.

Construction of a Consortium Comprising Ammonia-oxidizing Bacteria and Denitrifying Bacteria Isolated from Marine Sediment

A unique consortium of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria was obtained via a long-term, 3-step cultivation of isolates from organically-enriched marine sediment. We developed this microbial consortium for possible applications in the remediation of degraded habitats in closed aquaculture or other aquatic environments via microbial degradation. Analysis of media components found definitive evidence of nitrogen removal via the coupling of ammonia-oxidation and denitrification. The phylogenetic diversity of the consortium was investigated by performing polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) that targeted the 16S rRNA gene, and the functional genes involved in ammonia-oxidation (amoA) and denitrification (nirS, nirK, norB and nosZ). Consequently, no significant divergence was observed, and thus it is suggested microbial populations were selected via a long-term, 3-step incubation process. All of the 16S rRNA clones fell into three phylogenetic groups, namely, gamma-proteobacteria, Actinobacteria and Flavobacteria. For almost half of the clones, the closest relatives in the database were identified as Alcanivorax spp. and these clones were present at all cultivation stages. The presence of these species as the dominant clones is significant since these bacterial species are known to reduce nitrate to nitrite. Accordingly, their abundance in our microbial consortium may have been responsible for the observed stepwise denitrification. All sequences of the amoA gene were identified to be Nitrosomonas lineage. Half of the nirS clones were identified to be from one major group of well-known denitrifying bacteria, Pseudomonas sp. Furthermore, 70% of the nirK clones were closely related to the nirK sequences of uncultured bacterial clones isolated from arable soil. The qnorB clones consisted of clusters exclusively, and formed a distinct cluster from the novel sequences of cultivated species. The nosZ clones also were not found in any of the closest relatives in the database including the uncultured bacterium from marine sediment. The unique clones obtained from the functional genes were related to each denitrification step.

Denitrification Activity and Relevant Bacteria Revealed by Nitrite Reductase Gene Fragments in Soil of Temperate Mixed Forest

Denitrification activity and bacterial community constituents were investigated in both well-drained and poorly drained soils of a temperate forest in central Japan by (15)N tracer experiments and a cloning-sequencing approach. Denitrification activity was much higher in wet soil than in dry soil, based on (15)N(15)N ((30)N(2)) and (15)N(15)NO ((46)N(2)O) production. Labeled nitrate ((15)NO(3)(-)) was immediately reduced to (30)N(2) in wet soil, whereas it was only reduced to (46)N(2)O in dry soil. Thus, the wet soil at the lower end of the catchment is a functional site for the scavenging for NO(3)(-) and N(2)O. Nitrite reductase gene (nirK and nirS) fragments from these soils were PCR amplified, cloned, and sequenced. Both nirK and nirS fragments were detected in the wet soil, whereas only nirK fragments were detected in the dry soil. All the nirK and nirS clones showed less than 90% similarity to known clones. Numerous operational taxonomic units for nirK and nirS were found in the wet soil. Considerable diversification within the largest clade on the nirK phylogenetic tree, which contained no known sequence, was observed in wet soil. Thus, a wet soil environment can provide both the habitat and conditions for the expression of denitrification activity.

Identification of Novel Betaproteobacteria in a Succinate-Assimilating Population in Denitrifying Rice Paddy Soil by Using Stable Isotope Probing

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification have not been well characterized. In this study, we employed Stable Isotope Probing (SIP) to study succinate-assimilating denitrifiers in soil microcosms amended with nitrate and (13)C-succinate. Microbial populations represented in (12)C- and (13)C-DNA fractions were different based on denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragment. A nearly full-length 16S rRNA gene was also amplified, cloned, and sequenced from (13)C-DNA fraction. Both PCR-DGGE and clone library analyses revealed that Burkholderiales and Rhodocyclales dominated the succinate-assimilating population in denitrifying soil after 24 h incubation. Among these, novel Betaproteobacteria, possibly within the order Rhodocyclales, represented 43% of the clones obtained. Nitrite reductase genes, nirS and nirK, were also amplified and cloned from the (13)C-DNA fraction. While most nirK clones in this study were similar to the nirK sequences from Rhizobiales, a majority of the nirS clones were similar to the nirS sequences from Burkholderiales and Rhodocyclales, consistent with the 16S rRNA gene analysis. These groups of bacteria, including the novel Betaproteobacteria, may play an important role in denitrification in rice paddy soil.

Diversity of the nitrite reductase gene nirS in the sediment of a free-water surface constructed wetland.

The diversity of the nitrite reductase gene nirS was studied in the bulk sediment of a free-water surface constructed wetland (FWS-CW) located next to the Empuriabrava wastewater treatment plant (WWTP), in Castelló d'Empúries (Girona, NE Spain). The study period extended from the inception of the treatment wetland, in June 1998, until March 1999 and comprised periods of relatively high nitrate and ammonium concentrations at the influent and low nitrate-removal efficiencies. To evaluate nirS diversity, partial gene sequences were obtained by cloning of the respective PCR products. Rarefaction curves based on DOTUR analyses of the deduced amino-acid sequences predicted a greater diversity of nirS genes in samples containing higher ammonium concentrations. Estimated Shannon-Weaver indices of the four cloned samples showed a positive relationship with the N-NH4 +/N-NO3 - ratios measured at the FWS-CW inlet. Identities between the deduced amino-acid sequences and those previously deposited in public databases ranged from 72 to 97%. Phylogenetic analysis based on these deduced sequences grouped 165 nirS clones in seven main clusters according to high similarity indices. Up to 60% of the clones clustered together in a highly homogeneous group with little homologies to any sequence retrieved from cultured representatives. Moreover, prevailing environmental conditions appeared to select for particular denitrifying populations, e.g., with respect to ammonium load and nitrogen removal efficiencies. This observation is of particular interest for the management of treatment wetlands, in which only slight variations in the theoretical denitrification potential of the system can occur.

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.

Vertical distribution of nitrite reductase genes (nir S) in continental margin sediments of the Gulf of Mexico

Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.

Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene

Nitrite reductase gene (nirS) fragments in the activated sludge obtained from a sequencing batch reactor (SBR) under anaerobic-aerobic condition were cloned and classified by restriction fragment length polymorphism (RFLP) analysis, and representative fragments were sequenced. One of the nirS clones was approximately 70% of all nirS clones in anaerobic/aerobic (existing oxygen and nitrate) cycle operation in which a large amount of anoxic phosphate uptake was observed. Although the activated sludge samples analyzed might contain bacteria that did not accumulate polyphosphate, it was likely that this nirS fragment sequence was that from denitrifying polyphosphate-accumulating organisms (DNPAOs) which can utilize both oxygen and nitrate as electron acceptors. The sequence was similar to the nirS sequences of Thauera mechernichensis (83% similarity) and Azoarcus tolulyticus (83% similarity) both of which belong to the Rhodocyclus group.

Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater.

Nitrate-contaminated groundwater samples were analysed for nirK and nirS gene diversity. The samples differed with respect to nitrate, uranium, heavy metals, organic carbon content, pH and dissolved oxygen levels. A total of 958 nirK and 1162 nirS clones were screened by restriction fragment length polymorphism (RFLP) analysis: 48 and 143 distinct nirK and nirS clones, respectively, were obtained. A single dominant nirK restriction pattern was observed for all six samples and was 83% identical to the Hyphomicrobium zavarzinii nirK gene. A dominant nirS pattern was observed for four of the samples, including the background sample, and was 95% identical to the nirS of Alcaligenes faecalis. Diversity indices for nirK and nirS sequences were not related to any single geochemical characteristic, but results suggested that the diversity of nirK genes was inversely proportional to the diversity of nirS. Principal component analysis (PCA) of the sites based on geochemistry grouped the samples by low, moderate and high nitrate but PCA of the unique operational taxonomic units (OTUs) distributions grouped the samples differently. Many of the sequences were not closely related to previously observed genes and some phylogenetically related sequences were obtained from similar samples. The results indicated that the contaminated groundwater contained novel nirK and nirS sequences, functional diversity of both genes changed in relation to the contaminant gradient, but the nirK and nirS functional diversity was affected differently.

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.