Dual regulatory role of IS91-encoded Orf121 in IS91 transposition

Insertion sequences (IS) are key players in bacterial genome plasticity and horizontal gene transfer. IS 91 , belonging to the HUH superfamily of single-strand nucleases, is often linked with antibiotic resistance genes. Besides the transposase tnpA gene, IS 91 also carry the sequence called orf121 , whose stop codon overlaps with the start codon of tnpA , a highly conserved feature in IS 91 isoforms. Here, we demonstrate the role of this overlapping in the regulation of tnpA expression. We also showed that Orf121 serves as a dual regulator of IS 91 transposition: Orf121 inhibits transposition activity of TnpA while facilitating accurate excision of IS 91 single-strand circular intermediates, reducing one-ended transposition events. These findings highlight a sophisticated regulatory strategy balancing IS 91 mobility and genetic stability.