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Journal of Bacteriology, November 2009, p. 6877-6887, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00918-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface{triangledown}

Sanja Mihajlovic,1 Silvia Lang,1 Marta V. Sut,1 Heimo Strohmaier,1 Christian J. Gruber,1 Günther Koraimann,1 Elena Cabezón,2 Gabriel Moncalián,2 Fernando de la Cruz,2 and Ellen L. Zechner1*

University of Graz, Institute of Molecular Biosciences, Humboldtstrasse 50, A-8010 Graz, Austria,1 Departamento de Biología Molecular and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC-IDICAN, Cardenal Herrera Oria s/n, 39011 Santander, Spain2

Received 14 July 2009/ Accepted 9 September 2009

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraD{Delta}N130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo ({Delta}Lk = –4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraD{Delta}N130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.

* Corresponding author. Mailing address: University of Graz, Institute of Molecular Biosciences, Humboldtstrasse 50, A-8010 Graz, Austria. Phone: 43 316 380 5624. Fax: 43 316 380 9019. E-mail: ellen.zechner{at}uni-graz.at

{triangledown} Published ahead of print on 18 September 2009.

Journal of Bacteriology, November 2009, p. 6877-6887, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00918-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sut, M. V., Mihajlovic, S., Lang, S., Gruber, C. J., Zechner, E. L. (2009). Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1. J. Bacteriol. 191: 6888-6899 [Abstract] [Full Text]  


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