The ISPpu9 insertion sequence of Pseudomonas putida KT2440 generates various circular intermediates enabling modular transposition

Pseudomonas putida KT2440 contains seven copies of an insertion sequence (IS) belonging to the IS110 family, designated ISPpu9, which are inserted into repetitive extragenic palindromic sequences. Five of these copies include a small RNA, ssr9, located downstream of the transposase gene. Additionally, three separate copies of ssr9 are also present. When transferred to a different P. putida strain, ISPpu9 inserted at a specific target site highly similar to that of the KT2440 strain. Ssr9 was not needed for transposition. Circular intermediates were detected containing either the transposase, the transposase along with ssr9, or ssr9 alone. This finding explains the presence of these distinct modules in KT2440 chromosome. Minicircle formation required the transposase and short sequences located within the IS ends, which were identified. Unlike other ISs of this family, minicircle formation in ISPpu9 did not produce a hybrid promoter to enhance transcription of the ISPpu9 transposase. Instead, transcription occurred efficiently from its native promoter. In contrast, minicircles formed by ISPpu10, another IS of the same family also present in KT2440, generated a hybrid promoter significantly stronger than its weak native transposase promoter. This suggests that hybrid promoters might only emerge when the native transposase promoter is inherently weak.