Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

Abstract

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.