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Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.

ABSTRACT: 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.

SUBMITTER: Lee JH

PROVIDER: S-EPMC1932812 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.

Lee Ju-Hoon JH Halgerson Jamie S JS Kim Jeong-Hwan JH O'Sullivan Daniel J DJ

Applied and environmental microbiology 20070525 14

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 fu ...[more]

PMID: 17526779

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Project description:Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

Project description:BackgroundLactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria, which are widely distributed in various diverse natural habitats. These are used in a variety of industrial food fermentations and carry numerous traits with utmost relevance to the food industry. Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions.ResultsThe developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial "pediocin" produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts.ConclusionThe present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.

Dataset Information

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.

Publications

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.

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