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J. Biol. Chem., Vol. 280, Issue 28, 26349-26359, July 15, 2005

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Identification of the VirB4-VirB8-VirB5-VirB2 Pilus Assembly Sequence of Type IV Secretion Systems^*

Qing Yuan, Anna Carle, Chan Gao, Durga Sivanesan, Khaled Ahmed Aly, Christoph Höppner, Lilian Krall, Natalie Domke, and Christian Baron¶

From the Department of Biology, McMaster University, Hamilton, Ontario LS8 4K1, Canada and Department Biologie I, Bereich Mikrobiologie, Ludwig-Maximilians-Universität, D-80638 München, Germany

Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.

Received for publication, March 2, 2005 , and in revised form, May 5, 2005.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-64300, Natural Sciences and Engineering Research Council of Canada Grant 262104, and the European Union Frame Programme 5 Contract QLK2-CT-2001-01200 (to C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario LS8 4K1, Canada. Tel.: 905-525-9140 (ext. 26692); Fax: 905-522-6066; E-mail: baronc{at}mcmaster.ca.

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