Recombination Between Two Adjacent ICE San 95 Generated a New Transferable Integrative and Conjugative Element in Streptococcus agalactiae

ICE San 95 is a serine integrase integrative and conjugative element (ICE) that targets methyltransferase genes for integration. The mosaic ICE ICE Sag 084 contains ICE San 95 and partial fragments from two distinct genes ( snf2 and hsdM ), suggesting a recombination event. This study aimed to experimentally investigate the recombination of multiple ICE San 95, leading to the formation of a mosaic ICE resembling ICE Sag 084. Using conjugation assays, we tracked this evolutionary process, confirmed the structures via whole-genome sequencing, and assessed genomic stability through serial passages. Analysis showed that ICE Sag 084 comprises an ICE San 95-like ICE, a tyrosine integrase element, and a partial ICE Spy 009 sequence. The ICE San 95-like ICE was flanked by truncated snf2 and hsdM genes, indicating ancestral cointegration of two ICE San 95-like ICEs followed by excision and sequence capture. Experiments confirmed that tandem integration of ICE San 95 into adjacent snf2 (from an ICE Spy 009-like element) and hsdM (from the tyrosine integrase element) sites facilitated recombinational excision. This event deleted an ∼36.6-kb intervening fragment, yielding an ∼86.5-kb circularized intermediate and generating an ∼81.0-kb mosaic ICE highly similar to ICE Sag 084. The mosaic ICE was conjugatively transferable and remained stable for ≥60 generations, while the circularized ICE was lost within 20 generations. Thus, tandem integration and recombinational excision drive the emergence and spread of mosaic ICEs. This mechanism facilitates the modular assembly of multiple mobile genetic elements, including ICE Spy 009, ICE San 95, and a tyrosine integrase element. This process creates novel resistance islands with expanded traits, providing a key pathway for long-term adaptive evolution in bacterial pathogens.