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

Abstract

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.Denitrification is a dissimilatory process where oxidized nitrogen compounds (NO 3 Ϫ and NO 2 Ϫ ) are used as alternative electron acceptors for energy production. Nitrogen oxides are reduced stepwise to gaseous end products (NO, N 2 O, and N 2 ) which are concomitantly released, leading to a loss of fixed nitrogen to the environment. In marine coastal sediments, 40 to 50% of external inputs of dissolved inorganic nitrogen are removed by denitrification (23), resulting in an unbalanced nitrogen budget in the ocean (5, 7). Accumulation of the intermediates NO and N 2 O contributes to global warming and the destruction of the ozone layer (6). Thus, the underlying communities of denitrifying bacteria and their responses to environmental conditions need to be understood. Commonly, 16S rDNA molecules are tools to investigate microbial communities which avoid limitations of culturability (9,11,16). Lately, several functional genes were shown to be us...

Effect of pyrene on denitrification activity and abundance and composition of denitrifying community in an agricultural soil

“…Most denitrifiers belong to a wide range of subclasses of Proteobacteria, while there are still some closely related to Archaea, halophilic and hyperthermophilic branches as well as mitochondria of certain fungi (Zumft, 1997). The common difference between true denitrifiers and other microorganisms with nitrate-reducing ability is that the true denitrifiers have either a copper-containing enzyme encoded by nirK or a cytochrome cd1 enzyme encoded by nirS (Braker et al, 2000;Zumft, 1997). Therefore, the nirK and nirS genes have frequently been used as gene markers to analyze denitrifying community.…”

“…Since not all denitrifiers have the complete suite of denitrification enzymes (Zumft, 1997), two or more functional genes are often used as molecular markers for this microbial group. In most studies, the analyses of the molecular ecology of the denitrifying community were based on nirK, nirS and nosZ genes (Braker et al, 2000;Throbäck et al, 2004). The activity of reductases and the expression of functional genes involved in denitrification process varies among individual strains (Zumft, 1997), and community composition is thus considered to be able to influence functionality at both community and ecosystem levels (Braker et al, 2010).…”

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

“…However, a group-specific 16S rDNA approach is not suitable for community analysis of denitrifying bacteria, as this functional group is widely distributed over the phylogenetic tree (31,38). The genetic diversity of denitrifiers in marine sediments was explored by cloning nirK and nirS genes, which encode copper-and cytochrome cd 1 -containing nitrite reductases, respectively, key enzymes in the denitrification process (4). The PCR method to detect nir genes was highly specific for nirS and evaluated novel and diverse denitrifier communities in selected marine sediment samples (4).…”

“…In a computer simulation, nirS sequences from 34 marine sediment clones obtained in a previous study (4) were cleaved with the chosen restriction endonucleases for nirS. The lengths of these theoretical T-RFs were calculated, and clones were assigned to peaks found in the chromatograms from two sediment samples (C/1 and 304/1) (Fig.…”

“…The genetic diversity of denitrifiers in marine sediments was explored by cloning nirK and nirS genes, which encode copper-and cytochrome cd 1 -containing nitrite reductases, respectively, key enzymes in the denitrification process (4). The PCR method to detect nir genes was highly specific for nirS and evaluated novel and diverse denitrifier communities in selected marine sediment samples (4). To more systematically explore bacterial communities on a functional level, PCR-amplified functional genes such as nirS genes can be used with subsequent T-RFLP analysis.…”