Open AccessRandom insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes
Author(s) -
Mangan Michael W.,
Meijer Wim G.
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10955.x
Subject(s) - insertion sequence , transposition (logic) , genetics , insertional mutagenesis , biology , tn10 , mutagenesis , rhodococcus equi , gene duplication , gene , insertion , kanamycin , plasmid , transposable element , nucleic acid sequence , genome , microbiology and biotechnology , virulence , mutation , linguistics , philosophy
The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis. This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R. equi ATCC 33701. Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random. This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA. The presence of a 9 bp duplication of the target sequence showed that random integration of the transposome was due to a bona fide Tn5 transposition event.