Novel mechanisms of selenite reduction in Bacillus subtilis 168：Confirmation of multiple-pathway mediated remediation based on transcriptome analysis

Selenite biotransformation by microorganisms is an effective detoxification and assimilation process. Bacillus subtilis is a probiotic bacterium that can reduce Se(IV) to SeNPs under aerobic conditions. However, current knowledge on the molecular mechanisms of selenite reduction by B. subtilis remains limited. Here, the reduction of Se(IV) by probiotic bacterium Bacillus subtilis 168 was systematically analysed, and the molecular mechanisms of selenium nanoparticle (SeNPs) formation were characterised in detail. B. subtilis 168 reduced 5.0 mM selenite by nearly 40% in 24 h, and the produced SeNPs were spherical and localised intracellularly or extracellularly. FTIR (Fourier transform infrared) spectroscopy suggested the presence of proteins, lipids, and carbohydrates on the surface of the isolated SeNPs. Transcriptome data analysis revealed that the expression of genes associated with the proline metabolism, glutamate metabolism, and sulfite metabolism pathways was significantly up-regulated. Gene mutation and complementation revealed the ability of PutC, GabD, and CysJI to reduce selenite in vivo. In vitro experiments demonstrated that PutC, GabD, and CysJI could catalyse the reduction of Se(IV) under optimal conditions using NADPH as a cofactor. To the best of our knowledge, our study is the first to demonstrate the involvement of PutC and GabD in selenite reduction. Particularly, our findings demonstrated that the reduction of Se(IV) was mediated by multiple pathways both in vivo and in vitro. Our findings thus provide novel insights into the molecular mechanisms of Se(VI) reduction in aerobic bacteria.