Characterization of IS46, an insertion sequence found on two IncN plasmids

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• IncN plasmids R46 and N3 harbor two copies of an insertion sequence, termed IS46.
• IS46 is 0.81 kilobases long and possesses single PstI and SalI restriction sites.

Purpose of the Study:

• To investigate the transpositional capabilities and recombination activities of the IS46 insertion sequence.
• To elucidate the role of IS46 in plasmid evolution, cointegrate formation, and deletion events.

Main Methods:

• Analysis of IS46 restriction sites and size.
• Assessment of cointegrate formation and resolution in different cellular contexts (Rec+ and RecA-).
• Investigation of IS46-mediated recombination events, including inversions and deletions.

Main Results:

• All four IS46 copies facilitated cointegrate formation, but the intervening DNA (tetracycline resistance gene) was not transposable as a compound transposon.
• IS46-mediated cointegrates resolved efficiently in Rec+ cells but not in RecA- cells.
• Recombination between IS46 copies led to inversions, explaining distinct R46 forms, and IS46-mediated deletions likely formed plasmid pKM101 from R46.
• IS46 showed homology with IS15 but not with IS1.

Conclusions:

• IS46 is an active insertion sequence involved in plasmid recombination and evolution.
• IS46 plays a significant role in generating genetic diversity within plasmids through cointegrate formation, resolution, inversions, and deletions.
• The findings contribute to understanding the mechanisms of plasmid instability and adaptation.