Directed mutagenesis is an important approach in molecular microbiology, but has historically proven difficult in many bacteria, including clostridial species. This led to the development of the ClosTron system, which allowed the directed construction of stable mutants in Clostridium species using the Ll.LtrB intron of Lactococcus lactis. The intron is directed to insert into the desired DNA target by altering the sequence of the intron RNA-encoding ClosTron plasmid.
The procedure for ClosTron mutagenesis has been optimised to exploit key characteristics of group II introns, facilitating mutant generation in Clostridia:
As intron target specificity is determined by such a small region, it is cost-effective to out-source the synthesis and sub-cloning of the small DNA fragment. ClosTron plasmids are delivered ready-targeted, typically within 2 weeks of ordering.
Once the ClosTron plasmid is transferred into the target host, the constitutive expression of the intron leads to its spontaneous insertion into a proportion of the population. Thanks to the incorporation of a RAM (Retrotransposition-Activated Marker) into the intron the clones with an intron insertion become resistant to a second antibiotic (erythromycin), allowing their easy isolation / selection.
The LtrB protein is absolutely required for the retro-transposition of the intron to its target site, but its encoding gene (ltrB) does not accompany the intron when inserting into a new target. Rather, ltrB remains behind on the ClosTron plasmid. Subsequent loss of the plasmid from those cells in which the intron has inserted ensures that it is stably maintained at its new location.