THE RESOLVASE INVERTASE DOMAIN OF THE SITE-SPECIFIC RECOMBINASE TNPX IS FUNCTIONAL AND RECOGNIZES A TARGET SEQUENCE THAT RESEMBLES THE JUNCTION OF THE CIRCULAR FORM OF THE CLOSTRIDIUM-PERFRINGENS TRANSPOSON TN4451/

Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostrid ium perfringens and is found on the conjugative plasmid pIP401. The el ement undergoes spontaneous excision from multicopy plasmids in Escher ichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is prob ably the transposition intermediate. Excision of Tn4451 is dependent u pon the site-specific recombinase TnpX, which contains potential motif s associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid res idues within these domains was used to show that the resolvase/inverta se domain was essential for TnpX-mediated excision of Tn4451 from mult icopy plasmids in E. coli, An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity, The Tn 4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanke d hv directly repeated GA dinucleotides, and there was also a GA at th e junction of the circular form, where the left and right termini of T n4451 were fused, We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp stagg ered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives wi th altered CA dinucleotides provided experimental evidence to support the model.