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Applied and Environmental Microbiology, July 2007, p. 4417-4424, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.00099-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector{triangledown}

Ju-Hoon Lee, Jamie S. Halgerson, Jeong-Hwan Kim,{dagger} and Daniel J. O'Sullivan*

Department of Food Science and Nutrition and Center for Microbial and Plant Genomics, University of Minnesota, Cargill Building for Microbial and Plant Genomics, 1500 Gortner Ave., St. Paul, Minnesota 55108

Received 15 January 2007/ Accepted 21 May 2007

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.

* Corresponding author. Mailing address: Cargill Building for Microbial and Plant Genomics, 1500 Gortner Ave., St. Paul, MN 55108. Phone: (612) 624-5335. Fax: (612) 625-5272. E-mail: dosulliv{at}umn.edu

{triangledown} Published ahead of print on 25 May 2007.

{dagger} Present address. Department of Food Science and Technology, Gyeongsang National University, Jinju 660-701, South Korea.

Applied and Environmental Microbiology, July 2007, p. 4417-4424, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.00099-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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