%0 Journal Article %A Fernandes, Paula J %A Powell, Justin A. C %A Archer, John A. C %T Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes %D 2001 %J Microbiology, %V 147 %N 9 %P 2529-2536 %@ 1465-2080 %R https://doi.org/10.1099/00221287-147-9-2529 %K iPCR, inverse PCR %K kanamycin %K Gram-positive %K Mycobacterium %K transposon %K Nocardia %I Microbiology Society, %X 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. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-147-9-2529