Transposition of Tn4451 and Tn4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX

Abstract

SummaryTn4451 is the paradigm element of a family of mobilizable chloramphenicol resistance transposons from Clostridium perfringens and Clostridium difficile. The unique feature of these 6.3 kb elements is that their excision to form a circular molecule is mediated by TnpX, a member of the large resolvase family of site-specific recombinases. By optimizing the transposition assay system in Escherichia coli, we showed that Tn4453a from C. difficile transposed at a higher frequency than the C. perfringens element, Tn4451, and that transposition of both Tn4451 and Tn4453a was significantly enhanced by the provision of a multicopy tnpX gene in trans. The complete nucleotide sequence of Tn4453a was determined, but its comparison with Tn4451 did not reveal why it transposed at a higher frequency. Using experiments involving a chromosomal derivative of Tn4453a, we have confirmed that the circular form is the transposition intermediate. As the tnpX gene is located very close to one end of these elements, primer extension analysis was used to determine the transcription start point. The results showed that the formation of the circular intermediate creates a strong tnpX promoter, which consists of a 210 box originally located at the left end of the transposon and a 235 box originally located at the right end. The data provide strong evidence that transcription of tnpX is likely to occur from the non-replicating circular intermediate, which would facilitate the subsequent insertion of the transient circular molecule. It is postulated that, when the transposon is in an integrated state, transcription of tnpX would depend on the presence of an appropriately spaced 235 sequence in the DNA flanking the insertion site or the presence of an alternative upstream promoter.

Identification of the Structural and Functional Domains of the Large Serine Recombinase TnpX from Clostridium perfringens

“…The final steps in recombination involve strand cleavage, strand trans- fer, and religation. In the excision reaction, TnpX binds with equal affinity to the attL and attR ends of the transposon and strand exchange leads to the formation of a non-replicating circular intermediate (5,17). By contrast, integration involves the binding of TnpX to the attCI site at the joint of the circular intermediate, significantly weaker binding to an insertional target site, attT, and subsequent strand exchange (17).…”

“…The N-terminal domain, domain I, was found to extend to a region around residue 170 and encompassed the catalytic resolvase region (aa 1-104), which is highly conserved among serine recombinases (7) and has been shown to be essential for TnpX function (5,16). Domain II extended from aa 170 to 266 and contained two regions that were conserved between members of the large serine recombinase group (7).…”

“…Antibiotic resistance transposons identified in the clostridia include the integrative mobilizable elements Tn4451 and Tn4453a, which confer chloramphenicol resistance. Integration and excision of these elements is mediated by the large serine recombinase TnpX (3)(4)(5).…”

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function

“…To determine the effect of these mutations on the function of TnpX, the 19 mutated plasmids were introduced individually into suitable reporter strains and assayed for excision, insertion and transposition ability as previously described (Crellin and Rood, 1997;Lyras and Rood, 2000;Lyras et al ., 2004). Note that the actual frequencies shown graphically in Fig.…”

“…The 82 kDa TnpX protein catalyses both insertion and excision of the Tn 4451 -family of elements in the absence of other element-or host-encoded proteins (Lyras and Rood, 2000;Lyras et al ., 2004). These two site-specific recombination events, once thought to be biochemically the reverse of each other (Crellin and Rood, 1997), have been shown to be distinct due to the demonstration of differences in target site affinity between TnpX and its separate insertion and excision substrates .…”