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Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

Helen Rawsthorne, Kevin N. Turner, and David A. Mills^*

University of California at Davis, Department of Viticulture and Enology, One Shields Ave., Davis, California 95616

Received 19 December 2005/ Accepted 9 July 2006

Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

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Present address: Department of Food Science, Box 7624, North Carolina State University, Raleigh, NC 27695-7624.

Presnet address: University of California at Irvine, Irvine, CA 92697.

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