Natural transformation drives large-scale genome mosaicism in human gut bifidobacteria

Although horizontal gene transfer drives bacterial diversification, its contribution to chromosome-scale variation in human gut commensals remains unclear. This study demonstrated that human-associated bifidobacteria undergo extensive chromosomal transfer through natural transformation. Comparative genomics of coexisting Bifidobacterium pseudocatenulatum isolates from a single individual revealed extensive recombination signatures between the lineages. We experimentally reproduced this recombination by co-culturing strains, resulting in the transfer of multiple chromosomal regions and generation of mosaic genomes. Individual recombination tracts reached up to 247 kb per site, with cumulative replacements accounting for up to 28.9% of the recipient chromosome. These transfers occurred with heat-killed donors or purified DNA and were abolished by DNase, thereby identifying natural transformation as the underlying mechanism. Furthermore, we observed that environmental factors strongly influenced transformation frequency, suggesting that gut environmental conditions play a role regulating this process. Using natural transformation, we established a simple markerless genome-editing method that enables efficient gene deletions. Deletions of the Tad pili, ComEA-ComEC, or DprA-ComM-YraN gene clusters abolished transformation, defining the core machinery. The conservation of these genes across the genus Bifidobacterium and experimental demonstration of natural transformation in Bifidobacterium longum and Bifidobacterium breve indicate that natural transformation capacity is widespread within the genus. Our findings establish natural transformation as a key mechanism that promotes genome plasticity and contributes to adaptive evolution in bifidobacteria, thereby expanding our current understanding of horizontal gene transfer in the human gut microbiota.