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Identification of cis- and trans-acting elements in pHW126, a representative of a novel group of rolling circle

Wilfried Rozhon1, Mamoona Khan, Elena Petutschnig

  • 1Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria. wilfried.rozhon@univie.ac.at

Plasmid
|September 22, 2010
PubMed

Insights

This study identifies the replication protein (Rep) and origin of replication for the novel rolling circle plasmid pHW126. The Rep protein acts in trans, and specific elements within the origin are essential for replication and maintenance in Enterobacteriaceae.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Rolling circle plasmids represent a diverse group of extrachromosomal DNA elements.
  • The pHW126 plasmid belongs to a novel, uncharacterized family of rolling circle plasmids.
  • Understanding plasmid replication mechanisms is crucial for genetic engineering and microbial studies.

Purpose of the Study:

  • To characterize the replication mechanism of the novel rolling circle plasmid pHW126.
  • To identify the gene encoding the replication protein (Rep) and the origin of replication.
  • To determine the host range and maintenance factors of pHW126.

Main Methods:

  • Site-directed mutagenesis and deletion analysis of open reading frames and cis-acting regions.
  • Complementation experiments using compatible vectors to assess Rep function.
  • Plasmid replication and stability assays in various bacterial hosts.

Main Results:

  • The second open reading frame of pHW126 encodes a trans-acting replication protein (Rep).
  • A cis-acting origin of replication, approximately 300 bp, comprises an accessory region, a conserved stretch, and four tandem repeats, with the latter two being essential.
  • pHW126 exhibits replication in Enterobacteriaceae but not in Agrobacterium tumefaciens or Pseudomonas syringae, indicating a limited host range.

Conclusions:

  • The identified Rep protein and origin of replication are key determinants of pHW126 replication.
  • The accessory region of the origin is vital for stable plasmid maintenance under non-selective conditions.
  • pHW126 possesses a restricted host range, primarily within the Enterobacteriaceae family.

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