Development of the First Shuttle Vector System and Optimization of Transformation in <i>Selenomonas sputigena</i>

<i>Selenomonas sputigena</i>, an anaerobic bacterium found in the human respiratory tract, has recently gained significant attention due to its dual role in human health - as both a periodontal pathogen and a protective agent against severe asthma. However, the absence of genetic tools for this organism has severely limited our understanding of its molecular mechanisms and therapeutic potential. Here, we report the first successful development of a genetic modification system for <i>S. sputigena</i> ATCC 35185. We constructed a shuttle vector carrying the Rep191 replication protein from <i>Selenomonas ruminantium</i> and systematically optimized transformation conditions. Through careful optimization of key parameters including DNA quantity, bacterial growth phase, membrane permeabilization, and post-pulse recovery time, we achieved a 22.5-fold improvement in transformation efficiency (from ~2,000 to ~45,000 CFU/μg DNA). The broad applicability of our system was demonstrated through successful transformation of multiple <i>Selenomonas</i> species, establishing the first standardized genetic modification system for this genus. We further validated the system's functionality by achieving stable GFP expression in <i>S. sputigena</i>, representing the first demonstration of ectopic protein expression in this organism. This work provides essential tools for investigating the molecular basis of <i>S. sputigena</i>'s therapeutic effects and pathogenic capabilities, potentially accelerating the development of novel microbiome-based treatments for both respiratory and oral diseases.