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Journal of Bacteriology, August 2008, p. 5472-5479, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00321-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

ATPase Activity and Oligomeric State of TrwK, the VirB4 Homologue of the Plasmid R388 Type IV Secretion System{triangledown} ,{dagger}

Ignacio Arechaga,1* Alejandro Peña,1 Sandra Zunzunegui,1 María del Carmen Fernández-Alonso,2 Germán Rivas,2 and Fernando de la Cruz1

Departamento de Biología Molecular, Universidad de Cantabria (UC) and Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), c/Herrera Oria s/n, 39011 Santander, Spain,1 Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain2

Received 4 March 2008/ Accepted 23 May 2008

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.

* Corresponding author. Mailing address: Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, 39011 Santander, Spain. Phone: 34 942 202033. Fax: 34 942 201945. E-mail:arechagai{at}unican.es

{triangledown} Published ahead of print on 6 June 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, August 2008, p. 5472-5479, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00321-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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