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Related Experiment Videos

New thermosensitive plasmid for gram-positive bacteria.

E Maguin1, P Duwat, T Hege

  • 1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique-Domaine de Vilvert, Jouy en Josas, France.

Journal of Bacteriology
|September 1, 1992
PubMed
Summary

Researchers developed a thermosensitive plasmid, pVE6002, useful as a suicide vector for genetic engineering in diverse bacteria. This tool facilitates gene transfer and transposition studies, achieving approximately 1% transposition frequency in Bacillus subtilis.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Bacterial Genetics

Background:

  • Broad-host-range plasmids are essential tools in molecular biology for genetic manipulation across various bacterial species.
  • Thermosensitive plasmids offer conditional replication control, enabling their use as suicide vectors for targeted gene delivery and elimination.
  • The pWV01 replicon is a well-characterized plasmid system utilized for genetic studies in bacteria.

Purpose of the Study:

  • To isolate and characterize a replication-thermosensitive mutant of the broad-host-range plasmid pWV01.
  • To evaluate the utility of the thermosensitive mutant, pVE6002, as a suicide vector for bacterial genetic engineering.
  • To investigate the potential of pVE6002 for facilitating transposition studies in bacteria.

Main Methods:

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  • Isolation and characterization of a thermosensitive mutant (pVE6002) from the pWV01 replicon.
  • Determination of the thermosensitive phenotype in both gram-negative and gram-positive bacteria.
  • Genetic analysis to identify mutations within the replication protein gene.
  • Construction of cloning-convenient derivatives of the thermosensitive plasmid.
  • Application of pVE6002 as a delivery vector for a transposon Tn10 derivative in Bacillus subtilis.

Main Results:

  • A replication-thermosensitive mutant, pVE6002, was successfully isolated from pWV01.
  • pVE6002 exhibited thermosensitivity above 35°C in both gram-negative and gram-positive bacteria.
  • Four clustered mutations were identified in the replication protein gene of pVE6002.
  • A related plasmid, pE194, showed a thermosensitive mutation in an analogous region.
  • Derivatives of pVE6002 suitable for cloning were constructed.
  • pVE6002 demonstrated utility as a suicide vector for bacteria with limited temperature growth ranges.
  • Transposition frequencies of approximately 1% were achieved using pVE6002 for Tn10 delivery in Bacillus subtilis.

Conclusions:

  • The thermosensitive plasmid pVE6002 is a valuable tool for conditional replication and suicide vector applications in diverse bacterial hosts.
  • The identified mutations provide insights into the replication mechanism of pWV01-type plasmids.
  • pVE6002 facilitates efficient transposition studies, particularly in bacterial species with restricted temperature growth ranges.