Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector

Takashi Nishida, Kenta Watanabe, Masato Tachibana, Takashi Shimizu, Masahisa Watarai

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584 bp 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 (5058 bp, 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 × 101 to 1.0 × 105 CFU/ng. The relative number of pNT562 was estimated at 5.7 ± 1.0 copies and 73.6% of cells maintained the plasmid after 1 week 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.

Original languageEnglish
Pages (from-to)30-37
Number of pages8
JournalPlasmid
Volume90
DOIs
Publication statusPublished - Mar 1 2017
Externally publishedYes

Fingerprint

Legionella pneumophila
Genetic Vectors
Green Fluorescent Proteins
Plasmids
Proteins
Amino Acids
Organism Cloning
Kanamycin Resistance
Escherichia coli
Genes
Replication Origin
Kanamycin
Base Composition
Genetic Promoter Regions
Open Reading Frames

Keywords

  • Intracellular bacteria
  • Legionella pneumophila
  • Plasmid
  • Shuttle vector

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector. / Nishida, Takashi; Watanabe, Kenta; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa.

In: Plasmid, Vol. 90, 01.03.2017, p. 30-37.

Research output: Contribution to journalArticle

Nishida, Takashi ; Watanabe, Kenta ; Tachibana, Masato ; Shimizu, Takashi ; Watarai, Masahisa. / Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector. In: Plasmid. 2017 ; Vol. 90. pp. 30-37.
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AB - In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584 bp 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 (5058 bp, 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 × 101 to 1.0 × 105 CFU/ng. The relative number of pNT562 was estimated at 5.7 ± 1.0 copies and 73.6% of cells maintained the plasmid after 1 week 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.

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