Cointegrate-resolution of toluene-catabolic transposon Tn4651: determination of crossover site and the segment required for full resolution activity

Plasmid. 2013 Jan;69(1):24-35. doi: 10.1016/j.plasmid.2012.07.004. Epub 2012 Aug 1.


Tn3-family transposon Tn4651 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 Tn4651 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 Tn4651 cointegrate-resolution, we determined the extent of the functional resolution site (designated the rst site) of Tn4651 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 Tn4651 rst and a rst variant from the Tn4651-related transposon, Tn4661, 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 Tn4651-specified resolution system uses a different mechanism than other known resolution systems. Furthermore, comparative sequence analysis for Tn4651-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 Tn4651-related transposons.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • Computational Biology / methods
  • Crossing Over, Genetic*
  • DNA Transposable Elements*
  • DNA, Bacterial / genetics
  • Electrophoretic Mobility Shift Assay
  • Genetic Vectors / genetics
  • Inverted Repeat Sequences
  • Molecular Sequence Data
  • Plasmids / genetics
  • Promoter Regions, Genetic
  • Protein Binding
  • Pseudomonas putida / genetics*
  • Recombination, Genetic
  • Toluene / metabolism*


  • Bacterial Proteins
  • DNA Transposable Elements
  • DNA, Bacterial
  • Toluene