A Two-Step SeCys Oxidation Pathway Bridging Soil Selenium Bioavailability and Rice Selenium Biofortification

Addressing global selenium (Se) deficiency requires enhancing soil Se bioavailability, a process largely governed by microbial oxidation whose molecular mechanisms remain elusive. Here, we systematically elucidated the SeCys₂ oxidation mechanism of Comamonas testosteroni JL40 and its role in promoting rice Se biofortification. Strain JL40 employed a novel two-step oxidative pathway, converting SeCys₂ sequentially to Se nanoparticles (SeNPs) and then to selenite (Se(IV)). SeoA-D were identified to catalyse Se oxidation by integrated proteomic, genetic and enzymatic analyses. Of these, SeoA and SeoB catalysed the complete two-step oxidation; SeoD exclusively mediates the first step of SeCys₂ to SeNPs, while SeoC drives the second step of Se(0) to Se(IV). This system demonstrates broad host diversity and environment distribution and represents a prevalent mechanism for Se activation in the environment. Acting in concert with sulphur-oxidising metabolic gene clusters, they facilitated Se-S cross-metabolism, assembling into Se-oxidising activity as a previously unrecognised functional trait. Crucially, strain JL40 stably colonised the rice rhizosphere, where it enhanced soil Se bioavailability by 150%, increased grain Se content by 87.5%, and concurrently improved plant physiological traits and reshaped rhizosphere microbial networks. This work reveals a key Se-cycle pathway and directly enables sustainable biofortification, bridging microbial ecology and crop nutrition.