Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker

Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.