A genetic analysis system of Burkholderia cepacia: Construction of mobilizable transposons and a cloning vector

Mitsuko Abe, Masataka Tsuda, Mitsuaki Kimoto, Sachiye Inouye, Atsushi Nakazawa, Teruko Nakazawa

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest. To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying Km(R) and Tp(R) genes. The plasmids have the origin of ColE1 replication and the mobilization gene of RP4. Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec). Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1. Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome. Thirteen protease-deficient mutants were also lipase deficient. An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, Ap(R) and Cm(R) genes, and several unique restriction sites for cloning. Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production.

Original languageEnglish
Pages (from-to)191-194
Number of pages4
JournalGene
Volume174
Issue number2
DOIs
Publication statusPublished - 1996
Externally publishedYes

Keywords

  • Insertion mutation
  • Plasmid pSa
  • Protease production
  • Recombinant DNA
  • Tn5

ASJC Scopus subject areas

  • Genetics

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