title: Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.
abstract: To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis.
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Moon-Sun
authors.initials: MS
authors.lastname: Jang
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Fujita
authors.affiliation: Division of Applied Biological Sciences, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Ikawa
authors.affiliation: Division of Applied Biological Sciences, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Hanawa
authors.affiliation: Division of Applied Biological Sciences, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Yamamura
authors.affiliation: Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Division of Applied Biological Sciences, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hayakawa
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takeaki
authors.initials: T
authors.lastname: Tezuka
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: ayasuo@mail.ecc.u-tokyo.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
mesh_terms: D039903:Actinobacteria / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded
keywords: Actinoplanes missouriensis; Couchioplanes caeruleus; Plasmid; Rare actinomycetes; Rolling-circle replication; Shuttle vector