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

Area of Science:

• Microbial genetics
• Molecular biology
• Bacterial genomics

Background:

• Pseudomonas putida KT2440 harbors multiple copies of the IS110 family insertion sequence, ISPpu9.
• ISPpu9 contains a transposase gene and, in many copies, a downstream Ssr9 small RNA gene.

Purpose of the Study:

• To investigate the relationship between ISPpu9, its transposase, and the Ssr9 small RNA.
• To elucidate the mechanism of ISPpu9 transposition and minicircle formation.
• To understand the role of Ssr9 in the context of ISPpu9's genomic organization.

Main Methods:

• Genomic analysis of Pseudomonas putida KT2440 to identify ISPpu9 and Ssr9 locations.
• Transposition experiments in a different P. putida strain to assess ISPpu9 insertion patterns.
• Detection and characterization of circular DNA intermediates (minicircles) formed during transposition.

Main Results:

• ISPpu9 insertion sites in a new strain mimicked those in KT2440, indicating target site specificity.
• Ssr9 was found to be non-essential for ISPpu9 transposition.
• Circular DNA intermediates containing transposase alone, Ssr9 alone, or both were detected, suggesting modular propagation.
• ISPpu9 minicircles did not enhance transposase transcription via hybrid promoters, unlike ISPpu10 minicircles.

Conclusions:

• Ssr9 functions as a selfish genetic element, leveraging the ISPpu9 transposase for its own replication and dissemination.
• The formation of distinct minicircles explains the presence of separate transposase and Ssr9 modules in the genome.
• ISPpu9 exhibits a unique transposition mechanism compared to other IS110 elements, relying on its native promoter for efficient transcription.