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Journal of Bacteriology, October 2004, p. 6999-7006, Vol. 186, No. 20
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.20.6999-7006.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM

Andreas Beranek,1 Markus Zettl,2 Klaus Lorenzoni,3 Alexandra Schauer,1 Michael Manhart,1 and Günther Koraimann*

Institut für Molekulare Biowissenschaften, Karl-Franzens-Universität Graz,1 JSW Research, Graz, Austria,3 ICRF, London, United Kingdom2

Received 15 April 2004/ Accepted 23 July 2004

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 ({Delta}N155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 x 107 liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.

* Corresponding author. Mailing address: Institut für Molekulare Biowissenschaften, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. Phone: 43 (316) 380 5620. Fax: 43 (316) 380 9898. E-mail: guenther.koraimann{at}uni-graz.at.

Journal of Bacteriology, October 2004, p. 6999-7006, Vol. 186, No. 20
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.20.6999-7006.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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