TY - JOUR
T1 - Cointegrate-resolution of toluene-catabolic transposon Tn4651
T2 - Determination of crossover site and the segment required for full resolution activity
AU - Yano, Hirokazu
AU - Genka, Hiroyuki
AU - Ohtsubo, Yoshiyuki
AU - Nagata, Yuji
AU - Top, Eva M.
AU - Tsuda, Masataka
N1 - Funding Information:
We thank Dr. Christopher M .Thomas at University of Birmingham for providing us unpublished sequence information of plasmid Rms148. We thank Shiho Wakase and Dr. Yukari Sato at Tohoku University for technical advice and meaningful discussion. We also thank Gail E. Deckert at University of Idaho for improving the manuscript. This research was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
PY - 2013/1
Y1 - 2013/1
N2 - 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.
AB - 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.
KW - Cointegrate-resolution
KW - Pseudomonas
KW - Tn3
KW - Tn4652
KW - Transposon
KW - Tyrosine recombinase
UR - http://www.scopus.com/inward/record.url?scp=84871952203&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84871952203&partnerID=8YFLogxK
U2 - 10.1016/j.plasmid.2012.07.004
DO - 10.1016/j.plasmid.2012.07.004
M3 - Article
C2 - 22878084
AN - SCOPUS:84871952203
SN - 0147-619X
VL - 69
SP - 24
EP - 35
JO - Plasmid
JF - Plasmid
IS - 1
ER -