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.     

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 x 10(5) to 8.9 x 10(5) 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 x 10(3) to 2.6 x 10(4), 7.4 x 10(2) to 1.4 x 10(3), 2.5 x 10(2) to 6.4 x 10(3), and 1.2 x 10(3) to 5.5 x 10(3), 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.     

Genetic characterization of denitrifier communities with contrasting intrinsic functional traits

Microorganisms capable of denitrification are polyphyletic and exhibit distinct denitrification regulatory phenotypes (DRP), and thus, denitrification in soils could be controlled by community composition. In a companion study (D?rsch et al., 2012) and preceding work, ex situ denitrification assays of three organic soils demonstrated profoundly different functional traits including N(2) O/N(2) ratios. Here, we explored the composition of the underlying denitrifier communities by analyzing the abundance and structure of denitrification genes (nirK, nirS, and nosZ). The relative abundance of nosZ (vs. nirK + nirS) was similar for all communities, and hence, the low N(2) O reductase activity in one of the soils was not because of the lack of organisms with this gene. Similarity in community composition between the soils was generally low for nirK and nirS, but not for nosZ. The community with the most robust denitrification (consistently low N(2) O/N(2) ) had the highest diversity/richness of nosZ and nirK, but not of nirS. Contrary results found for a second soil agreed with impaired denitrification (low overall denitrification activity, high N(2) O/N(2) ). In conclusion, differences in community composition and in the absolute abundance of denitrification genes clearly reflected the functional differences observed in laboratory studies and may shed light on differences in in situ N(2) O emission of the soils.     

Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.     

Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes

In this study, we report the cloning and characterization of denitrifying gene clusters of Pseudomonas fluorescens C7R12 containing the narXLDKGHJI, nirPOQSM, norCB and nosRZDFYL genes. While consensus sequences for Fnr-like protein binding sites were identified in the promoter regions of the nar, nir, nor and nos genes, consensus sequences corresponding to the NarL binding sites were identified only upstream the nar genes. Monitoring by mRNA analysis the expression of the narG, nirS, norB and nosZ structural genes suggests a sequential induction of the denitrification system in P. fluorescens.     

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.     

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.     

NnrA is required for full virulence and regulates several Brucella melitensis denitrification genes

We identified two regulators of denitrification genes in Brucella melitensis 16M: NarR, which regulates the nitrate reductase (nar) operon, and NnrA, which is involved in the expression of the last three reductases of the denitrification pathway (nirK, norB, and nosZ). NnrA is required for virulence in mice and for intracellular resistance to nitric oxide.     

Characterization of the denitrifying bacterial community in a full-scale rockwool biofilter for compost waste-gas treatment

The potential denitrification activity and the composition of the denitrifying bacterial community in a full-scale rockwool biofilter used for treating livestock manure composting emissions were analyzed. Packing material sampled from the rockwool biofilter was anoxically batch-incubated with 15N-labeled nitrate in the presence of different electron donors (compost extract, ammonium, hydrogen sulfide, propionate, and acetate), and responses were compared with those of activated sludge from a livestock wastewater treatment facility. Overnight batch-incubation showed that potential denitrification activity for the rockwool samples was higher with added compost extract than with other potential electron donors. The number of 16S rRNA and nosZ genes in the rockwool samples were in the range of 1.64–3.27 × 109 and 0.28–2.27 × 108 copies/g dry, respectively. Denaturing gradient gel electrophoresis analysis targeting nirK, nirS, and nosZ genes indicated that the distribution of nir genes was spread in a vertical direction and the distribution of nosZ genes was spread horizontally within the biofilter. The corresponding denitrifying enzymes were mainly related to those from Phyllobacteriaceae, Bradyrhizobiaceae, and Alcaligenaceae bacteria and to environmental clones retrieved from agricultural soil, activated sludge, freshwater environments, and guts of earthworms or other invertebrates. A nosZ gene fragment having 99% nucleotide sequence identity with that of Oligotropha carboxidovorans was also detected. Some nirK fragments were related to NirK from micro-aerobic environments. Thus, denitrification in this full-scale rockwool biofilter might be achieved by a consortium of denitrifying bacteria adapted to the intensely aerated ecosystem and utilizing mainly organic matter supplied by the livestock manure composting waste-gas stream.     

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

Assessment of denitrifying bacterial composition in activated sludge

The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.     

Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47.

The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.     

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 (O(2) and NO(3)(-)) 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.     

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.     

Impacts of nitrogen application rates on the activity and diversity of denitrifying bacteria in the Broadbalk Wheat Experiment

Bacterial denitrification results in the loss of fertilizer nitrogen and greenhouse gas emissions as nitrous oxides, but ecological factors in soil influencing denitrifier communities are not well understood, impeding the potential for mitigation by land management. Communities vary in the relative abundance of the alternative dissimilatory nitrite reductase genes nirK and nirS, and the nitrous oxide reductase gene nosZ; however, the significance for nitrous oxide emissions is unclear. We assessed the influence of different long-term fertilization and cultivation treatments in a 160-year-old field experiment, comparing the potential for denitrification by soil samples with the size and diversity of their denitrifier communities. Denitrification potential was much higher in soil from an area left to develop from arable into woodland than from a farmyard manure-fertilized arable treatment, which in turn was significantly higher than inorganic nitrogen-fertilized and unfertilized arable plots. This correlated with abundance of nirK but not nirS, the least abundant of the genes tested in all soils, showing an inverse relationship with nirK. Most genetic variation was seen in nirK, where sequences resolved into separate groups according to soil treatment. We conclude that bacteria containing nirK are most probably responsible for the increased denitrification potential associated with nitrogen and organic carbon in this soil.