Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.