title: The dimer interface of Agrobacterium tumefaciens VirB8 is important for type IV secretion system function, stability, and association of VirB2 with the core complex.
abstract: Type IV secretion systems are virulence factors used by many gram-negative bacteria to translocate macromolecules across the cell envelope. VirB8 is an essential inner membrane component of type IV secretion systems, and it is believed to form a homodimer. In the absence of VirB8, the levels of several other VirB proteins were reduced (VirB1, VirB3, VirB4, VirB5, VirB6, VirB7, and VirB11) in Agrobacterium tumefaciens, underlining its importance for complex stability. To assess the importance of dimerization, we changed residues at the predicted dimer interface (V97, A100, Q93, and E94) in order to strengthen or to abolish dimerization. We verified the impact of the changes on dimerization in vitro with purified V97 variants, followed by analysis of the in vivo consequences in a complemented virB8 deletion strain. Dimer formation was observed in vivo after the introduction of a cysteine residue at the predicted interface (V97C), and this variant supported DNA transfer, but the formation of elongated T pili was not detected by the standard pilus isolation technique. Variants with changes at V97 and A100 that weaken dimerization did not support type IV secretion system functions. The T-pilus component VirB2 cofractionated with high-molecular-mass core protein complexes extracted from the membranes, and the presence of VirB8 as well as its dimer interface were important for this association. We conclude that the VirB8 dimer interface is required for T4SS function, for the stabilization of many VirB proteins, and for targeting of VirB2 to the T-pilus assembly site.
authors.affiliation: McMaster University, Department of Biology, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Durga
authors.initials: D
authors.lastname: Sivanesan
authors.forename: Christian
authors.initials: C
authors.lastname: Baron
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005816:Genetic Complementation Test; D014644:Genetic Variation; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors