Construction, transfer and properties of a novel temperature‐sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus

J. B. Luchansky, A. K. Benson, A. G. Atherly

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram‐positive bacteria. The use of a restriction‐deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325‐4. Plasmid pPQ126 (13.6 kb) is a novel, temperature‐sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30°C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39°C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination‐deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30°C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.

Original languageEnglish (US)
Pages (from-to)65-78
Number of pages14
JournalMolecular Microbiology
Volume3
Issue number1
DOIs
StatePublished - Jan 1989

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Plasmids
Chloramphenicol
Erythromycin
Gentamicins
Temperature
Staphylococcus aureus
Organism Cloning
Replicon
Protoplasts
Insertional Mutagenesis
Genes
Chromosomes
Bacteria
DNA
Population

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Construction, transfer and properties of a novel temperature‐sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus. / Luchansky, J. B.; Benson, A. K.; Atherly, A. G.

In: Molecular Microbiology, Vol. 3, No. 1, 01.1989, p. 65-78.

Research output: Contribution to journalArticle

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