Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis

The L1.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. L1.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of L1.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for L1.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (10(7)-fold detection range), we demonstrate that L1.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for L1.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented L1.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.