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Applied and Environmental Microbiology, January 2006, p. 869-879, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.869-879.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Gene Targeting in the Gram-Positive Bacterium Lactococcus lactis, Using Various Delta Ribozymes

Karine Fiola,1,2 Jean-Pierre Perreault,2* and Benoit Cousineau1*

Department of Microbiology and Immunology, McGill University, Montréal, Québec H3A 2B4, Canada,1 RNA Group/Groupe ARN, Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada2

Received 1 June 2005/ Accepted 20 October 2005

The trans-acting antigenomic delta ribozyme, isolated from the human hepatitis delta virus, was shown to be highly stable and active in vitro, as well as in mammalian cell lines. However, the stability and gene-targeting competence of this small ribozyme have not been studied previously in bacterial cells. In this paper we describe the use of two variants of the trans-acting antigenomic delta ribozyme targeting the abundant EF-Tu mRNA in the industrially important gram-positive bacterium Lactococcus lactis. These two delta ribozyme variants were expressed at significant levels and were shown to be highly stable in vivo. The half-life of the EF-Tu mRNA was slightly but consistently reduced in the presence of the classical delta ribozymes (7 to 13%). In contrast, delta ribozymes harboring a specific on/off riboswitch (SOFA-delta ribozymes) targeting the same sites on the EF-Tu mRNA considerably reduced the half-life of this mRNA (22 to 47%). The rates of catalysis of the SOFA-delta ribozymes in L. lactis were similar to the rates determined in vitro, showing that this new generation of delta ribozymes was highly efficient in these bacterial cells. Clearly, SOFA-delta ribozymes appear to be an ideal means for development of gene inactivation systems in bacteria.

* Corresponding author. Mailing address for Benoit Cousineau: McGill University, Lyman Duff Medical Building, 3775 University Street, Montréal, Québec H3A 2B4, Canada. Phone: (514) 398-8929. Fax: (514) 398-7052. E-mail: benoit.cousineau{at}mcgill.ca. Mailing address for Jean-Pierre Perreault: Université de Sherbrooke, Centre Hospitalier de l'Université de Sherbrooke, 3001 12th Avenue, Sherbrooke, Québec J1H 5N4, Canada. Phone: (819) 564-5310. Fax: (819) 564-5340. E-mail: Jean-Pierre.Perreault{at}USherbrooke.ca

Applied and Environmental Microbiology, January 2006, p. 869-879, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.869-879.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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