Analysis of nucleotide sequence of a novel plasmid, pILR091, from Lactobacillus reuteri L09 isolated from pig

Lee, Deog-Yong; Kang, Sang-Gyun; Rayamajhi, Nabin; Kang, Milan; Yoo, Han Sang

Korean Journal of Veterinary Research 2008;48(4):441-449.

Deog-Yong Lee, Sang-Gyun Kang, Nabin Rayamajhi, Milan Kang, Han Sang Yoo

College of Veterinary Medicine, KRF Zoonotic Diseases Institute and BK 21 Program for Veterinary Science, Seoul National University

Abstract

The genus Lactobacillus is the largest of the genera included in lactic acid bacteria and is associated with mucosal membranes of human and animal. Only a few Lactobacillus plasmid-encoded functions have been discovered and used. In this study, a novel plasmid (pILR091) was isolated from a wild L. reuteri isolated from pig and described the characteristics of its replicons, genetic organization, and relationship with other plasmids. After digestion of the plasmid, pILR091, with SalI, plasmid DNA was cloned into the pQE-30Xa vector and sequenced. The complete sequence was confirmed by the sequencing of PCR products and analyzed with the Genbank database. The isolate copy number and stability were determined by quantitative-PCR. The complete sequence of L. reuteri contained 7,185 nucleotides with 39% G-C content and one cut site by two enzymes, SalI and HindIII. The similar ori sequence of the pC194- rolling circle replication family (TTTATATTGAT) was located 63 bp upstream of the protein replication sequence, ORF 1. Total of five ORFs was identified and the coding sequence represented 4,966 nucleotides (70.4%). ORF1 of pILR091 had a low similarity with the sequence of pTE44. Other ORFs also showed low homology and E-values. The average G-C content of pILR091 was 39%, similar with that of genomic DNA. The copy number of pILR091 was determined at approximately 24 to 25 molecules per genomic DNA. These results suggested that pILR091 might be a good candidate to construct a new vector, which could be used for cloning and expression of foreign genes in lactobacilli.

Key Words: G-C content, Lactobacillus reuteri, ORF, plasmid DNA, replication