Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.
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.
Fernandes
Paula J
PJ
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK1.
Powell
Justin A C
JAC
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK1.
Archer
John A C
JAC
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK1.
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018274:Electroporation; D016254:Mutagenesis, Insertional; D009154:Mutation; D016133:Polymerase Chain Reaction; D012240:Rhodococcus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012988:Soil Microbiology
D017931:DNA Primers; D004251:DNA Transposable Elements