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.

FIG. 1

Phylogenetic relationship of nirS gene products (partial, 112 amino acids; accession numbers in brackets). The dendrogram was generated by phylogenetic distance analysis with a neighbor-joining algorithm with Roseobacter denitrificans [AJ224911] as the outgroup. Values indicate the percentage of 100 replicate trees supporting the branching order. Bootstrap values below 50 are omitted. Scale bar, 10 mutations per 100 sequence positions. Clones obtained from Puget Sound and Washington coast samples are labeled p and w, respectively. Phylogenetic positions of pure cultures based on 16S rDNA genes are indicated by α, β, and γ for the subgroups of the Proteobacteria. General taxonomic positions of marine isolates based on 16S rDNA genes in percent similarity determined by BLAST search is given in parenthesis after isolate designations. Roman numbers indicate the clusters of nirS sequences from isolates and environmental clones.

FIG. 2

Rarefraction curves indicating the diversity of denitrifying bacteria as detected by RFLP analysis of cloned nitrite reductase genes (nirK and nirS) from Puget Sound and Washington coast samples. nirK clones were digested with the tetrameric restriction enzymes MspI and HaeIII, and nirS clones were digested with MspI and HhaI in a single reaction. PS and WC stand for Puget Sound and Washington coast, respectively.

FIG. 3

Dot blot hybridization of nirS and nirK clones obtained from Puget Sound and Washington coast sediment samples. (A) Cloned nirK fragments were hybridized to four different probes: nirK PCR products from Pseudomonas sp. strain G-179 (1), from B. denitrificans (2), from R. sphaeroides f. sp. denitrificans (3), and from Alcaligenes sp. (4). Strength of hybridization signals was assigned as 0 (no hybridization signal) to 6 (strong hybridization signal). Frequency (%) expresses the total number of clones hybridizing with each probe. (B) Cloned nirS fragments were hybridized to four different probes: nirS PCR products from P. stutzeri/P. aeruginosa (1), from P. denitrificans (2), from marine isolate B9-12 (3), and from marine isolate D4-14 (4). Strength of hybridization signals was assigned as for panel A. (C) Strength of nirS hybridization signals was correlated to homology values of the probes to 37 sequenced nirS clones. Bars show standard deviation.

FIG. 4

Phylogenetic relationship of nirK gene products (partial, 111 amino acids; accession numbers in brackets). The dendrogram was generated by phylogenetic distance analysis with a neighbor-joining algorithm with B. denitrificans [AJ224906] as the outgroup. Values indicate the percentage of 100 replicate trees supporting the branching order. Bootstrap values below 50 are omitted. Scale bar, 10 mutations per 100 sequence positions. Phylogenetic positions of pure cultures based on 16S rDNA genes are indicated by α, β, and γ for the subgroups of the Proteobacteria.