Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes

P. J. Fernandes, J. A.C. Powell, John Archer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

The ability to generate tagged mutants of Rhodococcus spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn5-based DNA-protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A Rhodococcus rhodochrous CW25 Tn5 insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn5 was confirmed by the presence of 9 bp direct repeats of Rhodococcus DNA flanking the transposon insertion site. To further test this system, a Tn5 insertion library was constructed in a wild-type soil isolate of Rhodococcus spp. This is the first viable transposon knockout system reported for Rhodococcus.

Original languageEnglish (US)
Pages (from-to)2529-2536
Number of pages8
JournalMicrobiology
Volume147
Issue number9
DOIs
StatePublished - Jan 1 2001

Keywords

  • Gram-positive
  • Kanamycin
  • Mycobacterium
  • Nocardia
  • Transposon

ASJC Scopus subject areas

  • Microbiology

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