Target choice and orientation preference of the insertion sequence IS903

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Bacterial insertion elements (transposons) are mobile genetic elements that can alter host genomes.
• IS903 is a well-studied insertion element with implications in genetic engineering and bacterial evolution.
• Understanding transposon insertion site specificity is crucial for predicting their genomic impact.

Purpose of the Study:

• To investigate the DNA targeting preferences of the bacterial insertion element IS903.
• To identify the sequence and structural determinants of IS903 insertion site selection.
• To elucidate the role of conjugal DNA transfer in IS903 transpositional polarity.

Main Methods:

• Transposition assays using the conjugative plasmid pOX38 and the insertion element IS903.
• Analysis of insertion sites through sequencing and alignment.
• Cloning of preferred target regions into a second conjugative plasmid (pUB307).
• Experimental manipulation of DNA replication (oriV) and DNA transfer (oriT) directions.

Main Results:

• IS903 insertions clustered in specific regions associated with conjugal DNA transfer on pOX38.
• No strict consensus sequence was found at insertion sites, but flanking sequences influenced target preference.
• A cloned preferred target region retained its preference in a different plasmid context.
• Transposition displayed a strong orientation preference in pUB307, dependent on the direction of conjugal transfer.

Conclusions:

• IS903 exhibits non-random targeting of conjugative plasmids, favoring regions involved in DNA transfer.
• Target selection involves sequence and/or structural interactions flanking the insertion site.
• Conjugal DNA transfer machinery imposes a polarity that influences IS903 insertion orientation.