In Vivo Evidence of Single (13)C and (15)N Isotope–Labeled Methanotrophic Nitrogen-Fixing Bacterial Cells in Rice Roots

Methane-oxidizing bacteria (methanotrophs) play an ecological role in methane and nitrogen fluxes because they are capable of nitrogen fixation and methane oxidation, as indicated by genomic and cultivation-dependent studies. However, the chemical relationships between methanotrophy and diazotrophy and aerobic and anaerobic reactions, respectively, in methanotrophs remain unclear. No study has demonstrated the cooccurrence of both bioactivities in a single methanotroph bacterium in its natural environment. Here, we demonstrate that both bioactivities in type II methanotrophs occur at the single-cell level in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). We first verified that difluoromethane, an inhibitor of methane monooxygenase, affected methane oxidation in rice roots. The results indicated that methane assimilation in the roots mostly occurred due to oxygen-dependent processes. Moreover, the results indicated that methane oxidation-dependent and methane oxidation-independent nitrogen fixation concurrently occurred in bulk root tissues. Subsequently, we performed fluorescence in situ hybridization and NanoSIMS analyses, which revealed that single cells of type II methanotrophs (involving six amplicon sequence variants) in paddy rice roots simultaneously and logarithmically fixed stable isotope gases (15)N(2) and (13)CH(4) during incubation periods of 0, 23, and 42 h, providing in vivo functional evidence of nitrogen fixation in methanotrophic cells. Furthermore, (15)N enrichment in type II methanotrophs at 42 h varied among cells with an increase in (13)C accumulation, suggesting that either the release of fixed nitrogen into root systems or methanotroph metabolic specialization is dependent on different microenvironmental niches in the root.