Tn. 3-family transposon Tn. 4651 from Pseudomonas putida mt-2 plasmid pWW0 carries two divergently transcribed genes, tnpS and tnpT, for cointegrate-resolution. While tnpS encodes a tyrosine recombinase, tnpT encodes a protein that shows no homology to any other characterized protein. The Tn. 4651 resolution site was previously mapped within the 203-bp fragment that covered the tnpS and tnpT promoter region. To better understand the molecular mechanisms underlying the Tn. 4651 cointegrate-resolution, we determined the extent of the functional resolution site (designated the rst site) of Tn. 4651 and the location of the crossover site for the cointegrate-resolution. Deletion analysis of the rst region localized the fully functional rst site to a 136-bp segment. The analysis of the site-specific recombination between Tn. 4651 rst and a rst variant from the Tn. 4651-related transposon, Tn. 4661, indicated that the crossover occurs in the 33-bp inverted repeat region, which separates the 136-bp functional rst site into the tnpS- and tnpT-proximal segments. Electrophoretic mobility shift assays demonstrated specific binding of TnpT to the 20-bp inverted repeat region in the tnpT-proximal segment. The requirement for accessory sequences on both sides of the crossover site and the involvement of the unique DNA-binding protein TnpT suggest that the Tn. 4651-specified resolution system uses a different mechanism than other known resolution systems. Furthermore, comparative sequence analysis for Tn. 4651-related transposons revealed the occurrence of DNA exchange at the rst site among different transposons, suggesting an additional role of the TnpS-TnpT-. rst system in the evolution of Tn. 4651-related transposons.
- Tyrosine recombinase