Higher diversity and abundance of denitrifying microorganisms in environments than
considered previously
Wei Wei, Kazuo Isobe, Tomoyasu Nishizawa, Lin Zhu, Yutaka Shiratori, Nobuhito Ohte,
Keisuke Koba, Shigeto Otsuka, Keishi Senoo
Supplementary Figure legends
Supplementary Fig. S1. The core and clamp position of the forward (a) and reverse (b)
primers in the amino acid sequence alignment of nirK from 97 reference genomes and the
forward (c) and reverse (d) primers in the amino acid sequence alignment of the nirS from 75
reference genomes. The amino acid codons with different colored backgrounds denote the
primer-designed region of each cluster, and the black frames denote the region of the
currently used primer F1aCu/R3Cu and cd3aF/R3cd. The black and gray arrows in (a) and (b)
denote type I and type II copper ligands of NirK, and the black arrows in (c) denote active
sites of the nirS gene.
Supplementary Fig. S2. Maximum likelihood phylogeny of amino acid sequences of the nirK
copper center type I (a) and nirS cytochrome d1 heme (b). The sequences at the forward and
reverse primer sites are shown as seq-logo in the upper right and corresponding sequences
below. Bootstrap values (500 replicates) greater than 70% are denoted by dots above the
branches, and the branch lengths correspond to sequence differences indicated by the scale
bar. Symbols on tree tips indicate the taxonomic affiliations of reference sequences.
Supplementary Fig. S3. N2O fluxes in the soil microcosm. The error bars represent standard
deviations (n=3).
Supplementary Fig. S4. Maximum likelihood phylogeny, including the sequences of the
clones, denitrifying isolates and reference strains based on the partial nirK (a) and nirS (b)
gene. Bootstrap values (500 replicates) greater than 65% are indicated above the branches.
Branch lengths correspond to sequence differences indicated by the scale bar. The strains with
pink backgrounds exhibited the denitrifying isolates, which correspond to the values listed in
Supplementary Table S2. The clones with gray background exhibited the OTU sequences
from each nirK and nirS cluster.