Dual regulatory role of IS91-encoded Orf121 in IS91 transposition

Prokaryotic insertion sequences (IS) are pivotal in the propagation of bacterial multidrug resistance, with IS91 notably linked to virulence and antibiotic resistance genes. However, the precise mechanism by which IS91 contributes to gene dissemination remains elusive. Unique among its family, IS91 features a small open reading frame (orf) upstream of the tnpA transposase gene, potentially encoding a 121-amino acid protein, orf121, which may be translationally coupled with tnpA. Using a genetic system based on the mating-out assay in Escherichia coli, we explored the role of orf121 in the in vivo transposition of IS91. Our findings indicate that the overlap between orf121 and tnpA is crucial for tnpA transcription, with both being primarily transcribed as bicistronic mRNAs from the Porf121 promoter. Additionally, the expression of orf121 (whether in cis or trans) significantly reduces the frequency of IS91 transposition and the rate of one-ended transposition. Furthermore, only the single-stranded DNA circles of IS91 intermediates can integrate into new target sequences, and Orf121 negatively influences this insertion step. In summary, orf121 acts as a negative regulator of transposition while ensuring the expression of tnpA, thus providing insights into the complex mechanisms underlying IS91-mediated gene dissemination and its potential role in antibiotic resistance propagation.