Open AccessComparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector
Author(s) -
Ju-Hoon Lee,
Jamie S. Halgerson,
Jeong Hwan Kim,
Daniel J. O’Sullivan
Publication year - 2007
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00099-07
Subject(s) - plasmid , biology , orfs , replicon , shuttle vector , genetics , lactobacillus , streptococcus thermophilus , cloning vector , lactobacillus delbrueckii subsp. bulgaricus , open reading frame , escherichia coli , gene , microbiology and biotechnology , peptide sequence , bacteria , molecular cloning , vector (molecular biology) , recombinant dna
While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated withLactobacillus delbrueckii , with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain ofLactobacillus 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 characteristicori 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 availableL. 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 forL. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids fromStreptococcus thermophilus . This observation and the ability of the replicon to function inS. thermophilus support the idea that the origin of plasmids inL. delbrueckii was likely fromS. thermophilus . This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons fromL. delbrueckii has previously been constructed, anEscherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15Aori , the chloramphenicol resistance gene of pCI372, and thelacZ polylinker from pUC18. This cloning vector was successfully introduced intoE. coli ,L. delbrueckii subsp.bulgaricus ,S. thermophilus , andLactococcus lactis . This shuttle cloning vector provides a new tool for molecular analysis ofLactobacillus delbrueckii and other lactic acid bacteria.