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Applied and Environmental Microbiology, February 2003, p. 1121-1128, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1121-1128.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

Courtney L. Frazier,1 Joseph San Filippo,2 Alan M. Lambowitz,2 and David A. Mills1*

Department of Viticulture and Enology, University of California at Davis, Davis, California 95616-8749,1 Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 787122

Received 3 October 2002/ Accepted 21 November 2002

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.

* Corresponding author. Mailing address: Department of Viticulture and Enology, University of California at Davis, 1 Shields Ave., Davis, CA 95616-8749. Phone: (530) 754-7821. Fax: (530) 752-0382. E-mail: damills{at}ucdavis.edu.

Applied and Environmental Microbiology, February 2003, p. 1121-1128, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1121-1128.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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