The ISPpu9 insertion sequence of <i>Pseudomonas putida</i> 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, inserted into repetitive extragenic palindromic sequences. In five of these copies, a gene specifying the Ssr9 small RNA is located downstream of the transposase gene. Three additional ssr9 copies are present elsewhere in the genome. When transferred into a different P. putida strain, ISPpu9 inserted at sites closely resembling those in KT2440. Ssr9 was not required for transposition. Circular DNA intermediates were detected containing the transposase gene alone, the transposase gene along with ssr9, or ssr9 alone. This finding explains the presence of these distinct modules in KT2440 chromosome and suggests that ssr9 acts as a selfish genetic element that uses ISPpu9 transposase for its own propagation. Minicircle formation required the transposase and short sequences at the IS ends. Unlike other IS110 elements, ISPpu9 minicircles did not generate a hybrid promoter to enhance transcription of the ISPpu9 transposase gene. Instead, transcription occurred efficiently from its native promoter. In contrast, minicircles from ISPpu10, another IS110 element in strain KT2440, did form a strong hybrid promoter that significantly enhanced transposase transcription compared to its native promoter.