An integrated microbial genome-wide association studies-based characterization of nitric oxide homeostasis by probiotic Bacillus subtilis on aging and neurodegeneration using Caenorhabditis elegans and mouse models.

Microbial nitric oxide (NO) metabolism plays a critical role in regulating host redox homeostasis, yet its probiotic relevance remains largely unexplored. In this study, we performed a genome-wide phenotypic screen of 3984 Bacillus subtilis 168 (B. subtilis 168) gene deletion strains in Caenorhabditis elegans (C. elegans), identifying 12 core genetic factors linked to aging modulation. Among these, nosA (nitric oxide synthase) and yojO (putative nitric oxide reductase) were prioritized for validation based on their consistent host phenotypic effects and defined roles in NO regulation. Treatment with wild-type B. subtilis 168 extended C. elegans lifespan by 20 %, enhanced locomotor activity and chemotaxis index by 30-40 %, and reduced amyloid-beta accumulation by over 35 %. In contrast, ΔnosA and ΔyojO strains abolished these benefits, resulting in lifespan and behavior indices comparable to Escherichia coli OP50. In a BSO-induced oxidative stress mouse model, B. subtilis 168 administration significantly improved performance in Y-maze, open field, and novel object recognition tasks, whereas ΔnosA and ΔyojO groups exhibited diminished behavioral recovery and no improvement in cognitive outcomes. These findings demonstrate that microbial NO synthesis and detoxification are critical for mediating the anti-aging and neuroprotective functions of B. subtilis. Our study highlights a genetically tractable framework for dissecting host-microbe interactions relevant to redox signaling and age-associated neurodegeneration.