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Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK¹

Author for correspondence: John A. C. Archer. Tel: +44 1223 333 999. Fax: +44 1223 333 992. e-mail: jaca1{at}mole.bio.cam.ac.uk

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.

Keywords: transposon, kanamycin, Gram-positive, Mycobacterium, Nocardia

Abbreviations: iPCR, inverse PCR

^a Present address: Paradigm Therapeutics, Department of Physiology, Downing Street, Cambridge, CB2 3EG, UK.

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