title: Characterization of a small cryptic plasmid pK50-2 isolated from Lactobacillus reuteri K50.
abstract: The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866 bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (-) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.
authors.affiliation: Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, ROC.
authors.forename: Yi-Chih
authors.initials: YC
authors.lastname: Chang
authors.forename: Jun-Yuan
authors.initials: JY
authors.lastname: Huang
authors.forename: Ming-Tang
authors.initials: MT
authors.lastname: Chiou
authors.forename: Tung-Ching
authors.initials: TC
authors.lastname: Chung
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Hsu
authors.forename: Chuen-Fu
authors.initials: CF
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D052200:Limosilactobacillus reuteri / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D017385:Sequence Homology
chemical_list: D004269:DNA, Bacterial