MOLECULAR-GENETICS OF THE CHLORAMPHENICOL-RESISTANCE TRANSPOSON TN4451 FROM CLOSTRIDIUM-PERFRINGENS - THE TNPX SITE-SPECIFIC RECOMBINASE EXCISES A CIRCULAR TRANSPOSON MOLECULE

The chloramphenicol-resistance transposon Tn4451 undergoes precise con jugative deletion from its parent plasmid plP401 in Clostridium perfri ngens and precise spontaneous excision from multicopy plasmids in Esch erichia coli. The complete nucleotide sequence of the 6338 bp transpos on was determined and it was found to encode six genes. Genetic analys is demonstrated that the largest Tn4451-encoded gene, tnpX, was requir ed for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnp X gene was completely stable in both organisms, Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans- acting site-specific recombinase. Allelic exchange was used to introdu ce the tnpX Delta 1 allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens . It was also shown by hybridization and polymerase chain reaction (PC R) studies that TnpX-mediated transposon excision resulted in the form ation of a circular form of the transposon. The TnpX recombinase was u nique because it potentially contained the motifs of two independent s ite-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/inv ertase domain of TnpX was likely to be involved in the excision proces s by catalysing the formation of a 2 bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at th e junction of the circular form. The other Tn4451-encoded genes includ e tnpZ, which appears to encode a second potential site-specific recom binase. This protein has similarity to plasmid-encoded Mob/Pre protein s, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.