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Nucleic Acids Research Advance Access published online on March 27, 2007
Nucleic Acids Research, doi:10.1093/nar/gkl1146
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

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

Kamila Belhocine, Anthony B. Mak and Benoit Cousineau*

Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada H3A 2B4

*To whom correspondence should be addressed. Tel: +1 514 398 8929; Fax: +1 514 398 7052; E-mail: benoit.cousineau{at}mcgill.ca

Received August 29, 2006. Revised December 15, 2006. Accepted December 15, 2006.

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.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 Ll.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 Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (107-fold detection range), we demonstrate that Ll.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 Ll.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 Ll.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.


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