Natronobacterium gregoryi Argonaute inhibits class 1 integron integrase-mediated excision and integration

Argonaute (Ago) proteins, ubiquitous in all domains of life, serve as key components in defense against foreign nucleic acids. While eukaryotic Agos (eAgos) are well characterized for guide RNA-mediated RNA targeting, prokaryotic Agos (pAgos) exhibit diverse functions, particularly in protecting bacteria from invasive DNA. The previous study identified Class 1 integron integrase (IntI-1), a tyrosine site-specific recombinase involved in horizontal transfer of antibiotic resistance genes, as a potential interaction partner of Natronobacterium gregoryi Argonaute (NgAgo), a member of pAgos. Here, we demonstrated that this interaction was direct, depended on the PIWI domain, and was independent of the catalytic activity of NgAgo. Notably, no interaction occurred between NgAgo and Cre (another tyrosine site-specific recombinase), highlighting the specificity of NgAgo-IntI-1 interaction. Furthermore, NgAgo could inhibit binding of IntI-1 to its target DNA, and then impede IntI-1-mediated integration and excision. Consistent with the above finding, few pAgos could be found in prokaryotic genomes containing IntI, whereas IntI showed significant co-occurrence with another bacterial defense system, CRISPR-Cas. In summary, our study elucidated a novel defense mechanism of pAgos through interaction with IntI-1 for inhibiting IntI-1-mediated gene excision/integration process.