Genetic structure, function and regulation of the transposable element IS21

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The IncP plasmid R68.45 and related plasmids with tandem repeats of insertion sequence IS21 ((IS21)2) facilitate high-frequency replicon fusions in gram-negative bacteria.
• Plasmids with a single IS21 copy exhibit rare replicon fusion events.

Purpose of the Study:

• To investigate the mechanism of high-frequency replicon fusion mediated by tandem IS21 elements.
• To elucidate the role of IS21's nucleotide sequence, gene products, and flanking regions in transposition and fusion events.

Main Methods:

• Nucleotide sequencing of the IS21 insertion sequence.
• Analysis of polypeptide expression in E. coli maxicells using (IS21)2 and IS21 plasmids.
• Genetic analysis of (IS21)2 plasmids to identify promoter activity and functional elements.
• Fusion of a single IS21 element to an inducible tac promoter to assess its role in replicon fusion.

Main Results:

• The IS21 sequence contains inverted repeats at its ends and two open reading frames, istA and istB.
• The istA (46 kDa) and istB (30 kDa) gene products are expressed from (IS21)2 plasmids but not from single IS21 copy plasmids.
• IS21-IS21 junctions within (IS21)2 plasmids act as a promoter for the istAB operon.
• Replicon fusion requires both IS21-IS21 junctions and the istAB operon, with intervening sequences between IS21 elements being excluded from fusion products.
• IS21 exhibits specific, non-random insertion, creating 4 bp (occasionally 5 bp) target duplications.

Conclusions:

• Tandem IS21 elements, through their junctions and the istAB operon, are crucial for efficient replicon fusion.
• The IS21 transposase action is facilitated by the IS21-IS21 junction.
• IS21 transposition is a specific process with a preference for certain target sites.