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Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
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Recombination Between Two Adjacent ICESan95 Generated a New Transferable Integrative and Conjugative Element in
1College of Applied Engineering, Henan University of Science and Technology, Sanmenxia, China.
None:
ICESan95 is a serine integrase integrative and conjugative element (ICE) that targets methyltransferase genes for integration. The mosaic ICE ICESag084 contains ICESan95 and partial fragments from two distinct genes (snf2 and hsdM), suggesting a recombination event. This study aimed to experimentally investigate the recombination of multiple ICESan95, leading to the formation of a mosaic ICE resembling ICESag084. 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 ICESag084 comprises an ICESan95-like ICE, a tyrosine integrase element, and a partial ICESpy009 sequence. The ICESan95-like ICE was flanked by truncated snf2 and hsdM genes, indicating ancestral cointegration of two ICESan95-like ICEs followed by excision and sequence capture. Experiments confirmed that tandem integration of ICESan95 into adjacent snf2 (from an ICESpy009-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 ICESag084. 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 ICESpy009, ICESan95, 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.
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