Coupling of functional gene diversity and geochemical data from environmental samples

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirS and nirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1 and dsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusual dsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of the nirS, nirK, and dsrAB1 genes did not generalize as well as the models for dsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.     

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.     

Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods.     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

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.     

Phylogenetic Diversity and Distribution of Dissimilatory Sulfite Reductase Genes from Deep-Sea Sediment Cores

The diversity and distribution of sulfate-reducing prokaryotes (SRP) was investigated in the Nankai Trough sediments of off-central Japan by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Bulk DNAs were extracted from five piston-cored samples (up to 4.5 m long) with 41 vertical sections, and full-length dsrABgene sequences (ca. 1.9 kb) were PCR amplified and cloned. A total of 382 dsrAB clones yielded eight phylogenetic groups with an indigenous group forming a unique dsrAB lineage. The deltaproteobacterial dsrAB genes were found in almost all sediment samples, especially in the surface layer. One unique dsrAB clone group was also widespread in the dsrAB profiles of the studied sediments, and the percentage of its clones was generally shown gradual increase with sediment depth.     

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.     

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.     

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.     

Diversity,abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface sediments of the South China Sea

In marine ecosystems, both nitrite‐reducing bacteria and anaerobic ammonium‐oxidizing (anammox) bacteria, containing different types of NO‐forming nitrite reductase–encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua‐nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO‐forming nitrite‐reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua‐nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua‐nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua‐nirS genes share similar niche in deep‐sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite‐reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).     

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.     

Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

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.     

Genetic and Functional Variation in Denitrifier Populations along a Short-Term Restoration Chronosequence

Complete removal of plants and soil to exposed bedrock, in order to eradicate the Hole-in-the-Donut (HID) region of the Everglades National Park, FL, of exotic invasive plants, presented the opportunity to monitor the redevelopment of soil and the associated microbial communities along a short-term restoration chronosequence. Sampling plots were established for sites restored in 1989, 1997, 2000, 2001, and 2003. The goal of this study was to characterize the activity and diversity of denitrifying bacterial populations in developing HID soils in an effort to understand changes in nitrogen (N) cycling during short-term primary succession. Denitrifying enzyme activity (DEA) was detected in soils from all sites, indicating a potential for N loss via denitrification. However, no correlation between DEA and time since disturbance was observed. Diversity of bacterial denitrifiers in soils was characterized by sequence analysis of nitrite reductase genes (nirK and nirS) in DNA extracts from soils ranging in nitrate concentrations from 1.8 to 7.8 mg kg⁻¹. High levels of diversity were observed in both nirK and nirS clone libraries. Statistical analyses of clone libraries suggest a different response of nirS- and nirK-type denitrifiers to factors associated with soil redevelopment. nirS populations demonstrated a linear pattern of succession, with individual lineages represented at each site, while multiple levels of analysis suggest nirK populations respond in a grouped pattern. These findings suggest that nirK communities are more sensitive than nirS communities to environmental gradients in these soils.     

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.     

The physiological potential of anammox bacteria as revealed by their core genome structure

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2− reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH₂OH by self-production for anammox metabolism.     

Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions

Nitrous oxide (N₂O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N₂O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N₂O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N₂O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N₂O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N₂O emissions than nirK type denitrifiers under favorable environmental conditions_. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N₂O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N₂O emissions.