Dual regulatory role of IS91-encoded Orf121 in IS91 transposition

Insertion sequences (IS) are key players in bacterial genome plasticity and horizontal gene transfer. IS91 family members, belonging to the HUH superfamily of single-strand nucleases, are often linked with antibiotic resistance genes. Among these, the element IS91 is unique as it also carries a sequence called orf121, whose stop codon overlaps with the start codon of tnpA, a highly conserved feature of IS91 isoforms. We show that Orf121 serves as a dual regulator of IS91 transposition: Orf121 inhibits transposition activity of TnpA while facilitating accurate excision of IS91 single-strand circular intermediates. This accurate excision reduces one-ended transposition events, i.e., events arising when proper termination fails, leading to the co-mobilization of adjacent DNA. We also provide evidence that the bottom-stranded ssDNA circular intermediate is the functional substrate IS91. These findings highlight a sophisticated regulatory strategy balancing IS91 mobility and genetic stability. This study identifies Orf121 as a dual regulator that limits IS91 transposition while ensuring accurate excision of mobile DNA. The findings reveal a built-in mechanism that enables bacteria to balance genome plasticity with stability.