ISPpu10 is a structure-gated bridge RNA recombinase that drives safe, large-scale genomic plasticity

Environmental stress drives massive genomic rearrangements in bacteria, yet how mobile elements mediate these changes while preserving host integrity remains obscure. Here, following adaptive laboratory evolution, we identified ISPpu10, an IS110-family recombinase driving >200 kb segmental amplifications in Pseudomonas putida KT2440. We show that the ISPpu10 bridge RNA (bRNA) possesses a distinct architecture lacking the repressive 5' stem-loop found in related systems. Uniquely, it utilized a dual-match logic requiring both bRNA complementarity and a target DNA 5' stem-loop. This genomic hairpin acts as a critical structural determinant, directing integration into intergenic safe zones and structurally stabilizing the recombinase-RNA complex. Harnessing this machinery, we achieved cross-species genomic integration, mobilized massive 22.9 kb DNA payloads, and captured split DNA substrates in trans. Furthermore, we systematically deciphered the hierarchical constraints governing bRNA reprogramming. These findings identify ISPpu10 as a structure-gated architect of genome evolution, offering a high-stringency paradigm for safe, large-scale synthetic biology.