The absence of pmoA sequence in surface soil suggested a preferred habitat in deep soil for n-damo bacteria. The 14 sequences retrieved from the other three depths together with the published pmoA, pxmA and amoA nucleic acid sequences were phylogenetically analyzed (Fig. 3). Most of the sequences in this study showed high identity to each other and were closely related (difference up to 90–92% nucleotide and up to 94–95% protein identity) to the pmoA gene of M. oxyfera (FP565575 or CBE69519). The sequences obtained from the paddy soil formed

a unique clade in the tree along with other pmoA sequences from ditch, aquifer environments, and WWTPs reported previously (Luesken et al., 2011a,c). The low diversity CAL-101 nmr of pmoA sequences obtained from the paddy soil was consistent with previous studies (Deutzmann & Schink, 2011; Luesken et al., 2011c; Kojima et al., 2012). The fact that the sequences obtained were not highly divergent from each other was probably caused by the functional conservation of pmoA gene reflected by the unique oxygenic pathway of n-damo bacteria (Luesken et al., 2011c). In addition, the primers used in this

study were designed based IWR-1 supplier on the limited references available. It cannot be ruled out that they were too narrow to cover all the pmoA gene of the n-damo bacteria (Deutzmann & Schink, 2011). Therefore, further improvement in specific primers was needed to analyze the diversity of the n-damo at a functional level (Kojima et al., 2012). Because there was no suitable primer pair targeting the pmoA gene for qPCR so far, the abundance of n-damo bacteria was estimated by quantifying their 16S rRNA gene. The copy numbers Ketotifen ranged from 1.0 ± 0.1 × 105 (0–10 cm) to 7.5 ± 0.4 × 104 copies g−1

dry soil (30–40 cm; Fig. 2b). Below 40 cm depth, the abundance decreased gradually from 4.9 ± 0.1 × 104 (40–50 cm) to 6.5 ± 0.4 × 103 (60–70 cm) copies g−1 dry soil. Below 70 cm depth, the abundance decreased beyond the limit of detection. As the primers used were designed based on enrichment samples and have not been previously applied on environmental samples. Therefore, the clones of 16S rRNA gene were also sequenced for a comparison with the known n-damo bacteria (Fig. S10). The phylogenetic analysis showed that sequences from 40 to 50 and 60 to 70 cm depths clustered within group a, which comprises sequences closely related to the enrichment n-damo bacteria (DQ369742) (Ettwig et al., 2009), whereas sequences from 0 to 10 and 20 to 30 cm depths were distantly related to the known n-damo bacteria. This means the quantification based on the 16S rRNA gene probably overestimated the abundance in the upper soils because of the less specificity of the primer set.