Identification of Novel Betaproteobacteria in a Succinate-Assimilating Population in Denitrifying Rice Paddy Soil by Using Stable Isotope Probing

Abstract

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification have not been well characterized. In this study, we employed Stable Isotope Probing (SIP) to study succinate-assimilating denitrifiers in soil microcosms amended with nitrate and (13)C-succinate. Microbial populations represented in (12)C- and (13)C-DNA fractions were different based on denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragment. A nearly full-length 16S rRNA gene was also amplified, cloned, and sequenced from (13)C-DNA fraction. Both PCR-DGGE and clone library analyses revealed that Burkholderiales and Rhodocyclales dominated the succinate-assimilating population in denitrifying soil after 24 h incubation. Among these, novel Betaproteobacteria, possibly within the order Rhodocyclales, represented 43% of the clones obtained. Nitrite reductase genes, nirS and nirK, were also amplified and cloned from the (13)C-DNA fraction. While most nirK clones in this study were similar to the nirK sequences from Rhizobiales, a majority of the nirS clones were similar to the nirS sequences from Burkholderiales and Rhodocyclales, consistent with the 16S rRNA gene analysis. These groups of bacteria, including the novel Betaproteobacteria, may play an important role in denitrification in rice paddy soil.