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Nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica and construction of a conjugative shuttle vector
Author(s) -
Malhotra Shweta,
Majumdar Swati,
Kumar Mukesh,
Bhasin V. K.,
Gartemann KarlHeinz,
Lal Rup
Publication year - 2008
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200700326
Subject(s) - replicon , shuttle vector , plasmid , open reading frame , biology , origin of replication , nucleic acid sequence , bacterial conjugation , escherichia coli , genetics , homology (biology) , dna , peptide sequence , microbiology and biotechnology , recombinant dna , vector (molecular biology) , gene
The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self‐replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli ‐ Amycolatopsis shuttle‐cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)