title: Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.
abstract: In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1. Yoshida, Yamaguchi 753-8515, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishida
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1. Yoshida, Yamaguchi 753-8515, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1. Yoshida, Yamaguchi 753-8515, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Tachibana
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1. Yoshida, Yamaguchi 753-8515, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1. Yoshida, Yamaguchi 753-8515, Japan. Electronic address: watarai@yamaguchi-u.ac.jp.
authors.forename: Masahisa
authors.initials: M
authors.lastname: Watarai
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D058947:Bacterial Secretion Systems / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007612:Kanamycin / Q000494:pharmacology; D016952:Legionella pneumophila / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D058947:Bacterial Secretion Systems; D004269:DNA, Bacterial; D049452:Green Fluorescent Proteins; D007612:Kanamycin
keywords: Intracellular bacteria; Legionella pneumophila; Plasmid; Shuttle vector