Transposome insertional mutagenesis and direct sequencing of microbial genomes

Abstract

Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.