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Applied and Environmental Microbiology, September 2003, p. 5574-5584, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5574-5584.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

Carl-Alfred Alpert,{dagger} Anne-Marie Crutz-Le Coq, Christine Malleret, and Monique Zagorec*

Flore Lactique et Environnement Carné, Institut National de la Recherche Agronomique, 78350 Jouy en Josas, France

Received 31 March 2003/ Accepted 16 June 2003

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.

* Corresponding author. Mailing address: UFLEC, INRA-CRJ, Domaine de Vilvert, F-78350 Jouy-en-Josas, France. Phone: 33 (0)1 34-65-22-89. Fax: 33 (0)1 34-65-21-05. E-mail: zagorec{at}jouy.inra.fr.

{dagger} Present address: Deutsches Institut für Ernährungsforschung, Abteilung Gastrointestinale Mikrobiologie, 14558 Bergholz-Rehbrücke, Germany.

Applied and Environmental Microbiology, September 2003, p. 5574-5584, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5574-5584.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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