Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.
Microbiology (Reading)
; 147(Pt 9): 2529-2536, 2001 Sep.
Article
em En
| MEDLINE
| ID: mdl-11535792
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Rhodococcus
/
Elementos de DNA Transponíveis
Idioma:
En
Ano de publicação:
2001
Tipo de documento:
Article