The ISApl1 ₂ Dimer Circular Intermediate Participates in mcr-1 Transposition

Area of Science:

• Molecular Biology
• Genetics
• Antimicrobial Resistance

Background:

• The mobile colistin resistance gene, mcr-1, poses a significant global health threat.
• The composite transposon Tn6330 and its circular intermediates are implicated in mcr-1 spread, but their precise roles are unclear.

Purpose of the Study:

• To investigate the intermediates involved in the transposition of the Tn6330 composite transposon.
• To elucidate the mechanistic role of Tn6330 in the spread of the mcr-1 gene.

Main Methods:

• Engineered Escherichia coli strains with intact or deleted Tn6330 derivatives.
• Utilized PCR assays to detect IR-IR junctions and circular intermediates.
• Conducted transposition experiments, whole genome sequencing, and ISMapper analyses to determine frequency and integration sites.

Main Results:

• An intact Tn6330 is essential for mcr-1 transposition, though circular intermediates can form with or without the IR regions.
• The tandem repeat ISApl1 dimer was observed only in constructs with an intact Tn6330.
• mcr-1 transposition occurred at a frequency of approximately 10^-6 per cell, with integration sites showing 2 bp target duplications in AT-rich regions.

Conclusions:

• mcr-1 transposition is dependent on the presence of an intact Tn6330.
• The formation of the ISApl1 tandem repeat is identified as a critical intermediate in the transposition process.
• These findings provide essential mechanistic insights into how mcr-1 is mobilized and spreads.