Identification of active denitrifiers in rice paddy soil by DNA- and RNA-based analyses

Abstract

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.

Fig. 1

Denitrifying activities in the soil microcosm system as measured by the C₂H₂ block method.

Fig. 2

Changes in the amounts of (A) 16S rRNA gene, (B) nirS, (C) nirK from DNA samples and (D) 16S rRNA, (E) nirS transcripts from cDNA samples. X axes show incubation time. Y axes show numbers of gene copies or gene transcripts.

Fig. 3

Changes in the distribution of (A) 16S rRNA, (B) nirS, (C) nirK and (D) nosZ clones in response to denitrification-inducing conditions. Clones were grouped into clusters based on the phylogenetic trees shown in Supplementary Materials (Fig. S1, 2, 3, and 4).