Nitric oxide reductase (norB) genes from pure cultures and environmental samples

Abstract

A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.

FIG. 1.

Diversity of nitric oxide-reducing bacteria in environmental samples as evaluated by RFLP analysis of cloned nitric oxide (norB) genes from marine and freshwater sediments. qnorB clones were hydrolyzed with restriction enzymes MspI and RsaI, and cnorB clones were hydrolyzed with HhaI and MspI.

FIG.2.

Phylogenetic analysis of norB genes. Neighbor joining tree (Jones-Taylor-Thornton model of amino acid exchange) based on partial norB amplicons (100 amino acids; accession numbers in parentheses). A consensus tree was constructed from distance methods (neighbor joining and FITCH), parsimony, and maximum likelihood by introducing multifurcations (dashed lines) where tree topology was not consistently resolved. Bootstrap values were generated from 1,000 replicates of neighbor joining and parsimony analysis. •, bootstrap values >90%; ○, bootstrap values 50 to 90%; bootstrap values of <50% are omitted. Clones obtained from the Washington margin and Red Cedar River are designated WM and RCR, respectively, plus q and c for qnorB and cnorB, respectively. The phylogenetic positions of isolates are indicated by α, β, and γ for the subgroups of the Proteobacteria. Roman numbers indicate clusters of norB genes.