title: Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems.
abstract: 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.
authors.affiliation: Department of Biology, McMaster University, Hamilton, Ontario LS8 4K1, Canada.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
authors.forename: Anna
authors.initials: A
authors.lastname: Carle
authors.forename: Chan
authors.initials: C
authors.lastname: Gao
authors.forename: Durga
authors.initials: D
authors.lastname: Sivanesan
authors.forename: Khaled Ahmed
authors.initials: KA
authors.lastname: Aly
authors.forename: Christoph
authors.initials: C
authors.lastname: Höppner
authors.forename: Lilian
authors.initials: L
authors.lastname: Krall
authors.forename: Natalie
authors.initials: N
authors.lastname: Domke
authors.forename: Christian
authors.initials: C
authors.lastname: Baron
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000502:physiology*; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D014774:Virulence; D037521:Virulence Factors / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors