Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• IS91 is a mobile genetic element known to transpose within bacterial genomes.
• Understanding the mechanisms of IS91 transposition is crucial for comprehending genome dynamics and gene regulation.

Purpose of the Study:

• To investigate the transposition mechanism of IS91.
• To identify factors influencing IS91 transposition frequency and target selection.
• To analyze the role of ORF121 in IS91 transposition.

Main Methods:

• Construction of plasmids containing IS91 transposable cassettes (IRL-kan-IRR).
• Analysis of transposition products using molecular techniques.
• Site-directed mutagenesis of ORF121 and assessment of transposition specificity.

Main Results:

• IS91 transposition efficiency is similar whether transposase is provided in cis or trans.
• Transposition frequency increases significantly (approx. 100-fold) with induced transposase expression.
• 87% of transposition events resulted in simple insertions, while 13% formed plasmid fusions/co-integrates.
• Mutations in ORF121 altered the insertion specificity of IS91.
• Intramolecular transposition led to the insertion of additional copies of the cassette within the cat gene, causing plasmid instability.

Conclusions:

• IS91 transposition can occur via a rolling-circle mechanism.
• ORF121 plays a role in target selection during IS91 transposition.
• IS91 transposition can lead to both extrachromosomal and intramolecular rearrangements, contributing to genome plasticity.