An improved temperature-sensitive shuttle vector system for scarless gene deletion in human-gut-associated species.

is a prevalent bacterial taxon in the human gut that comprises over 10 (sub)species. Previous studies suggest that these species use evolutionarily distinct strategies for symbiosis with their hosts. However, the underlying species-specific mechanisms remain unclear due to the lack of efficient gene knockout systems applicable across different species. Here, we developed improved temperature-sensitive shuttle vectors by introducing Ser139Trp into the replication protein RepB. We then used temperature-sensitive plasmids to construct a double-crossover-mediated scarless gene deletion system. The system was employed for targeted gene deletion in subsp. , subsp. , , , , and . Deletion of genes involved in capsular polysaccharide biosynthesis, aromatic lactic acid production, and sugar utilization resulted in the expected phenotypic changes in the respective (sub)species. The temperature-sensitive plasmids developed in this study will aid in deciphering the evolutionary traits of the human-gut-associated species.