Allelic Exchange in Pseudomonas Aeruginosa Using Novel ColE1-type Vectors and a Family of Cassettes Containing a Portable oriT and the Counter-Selectable Bacillus Subtilis sacB Marker

Mol Microbiol. 1992 May;6(9):1195-204. doi: 10.1111/j.1365-2958.1992.tb01558.x.

Abstract

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Bacillus subtilis / genetics*
  • Base Sequence
  • DNA, Recombinant
  • Escherichia coli / genetics
  • Genetic Markers
  • Genetic Vectors*
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Plasmids
  • Pseudomonas aeruginosa / genetics*
  • Transduction, Genetic*

Substances

  • DNA, Recombinant
  • Genetic Markers