Metatranscriptomic analysis to reveal the coupling between nitrogen fixation and CH4 oxidation in root tissues of Phragmites australis

The root-associated type II methanotrophs significantly contribute to CH₄ oxidation-dependent N₂ fixation. However, it is unclear whether type I methanotrophs are involved in CH₄ oxidation and N₂ fixation, especially in natural wetlands. So far, limited attention has given to root-associated active microorganisms. Here, metatranscriptomic analysis of root-associated microbes has been proposed to reveal the aerobic methanotrophs contributing to CH₄ and nitrogen cycles in the roots of Phragmites australis grown in a natural wetland. Results showed Methylocystaceae (type II methanotrophs) and Methylococcaceae (type I methanotrophs) as major taxa (relative abundance, 14%) at transcription level. However, based on 16S rRNA gene sequencing, contribution of these taxa was < 1% at DNA level. Genes encoding methane monooxygenase (enzyme responsible for the first step of CH₄ oxidation) were detected in Methylomonas (pmoCBA) and Methylosinus (mmoXYZCB). Furthermore, genes related to methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase were also detected in Methyosinus and Methylomonas, while mcrA gene was observed in Methanospirillum and Methanofollis. Moreover, nitrogenase structural genes, such as nifHDK, were found in Methylosinus (Methylocystaceae) and Methylomonas (Methylococcaceae). Minor nitrogenase genes were detected in Cyanothece, Lyngbya, Pelobacter and Smithella of Cyanobacteriaceae family. In addition, N₂ fixing activity of P. australis was determined by analyzing the natural abundance of δ¹⁵N from June to August. The N₂ fixing activity of P. australis increased in presence of CH₄ in root system under ¹⁵N-N₂ feeding. Metatranscriptomic analysis revealed that not only type II methanotrophs, but also type I methanotrophs oxidize CH₄ and fix N₂.