Localization and topology of VirB proteins of Agrobacterium tumefaciens

Agrobacterium tumefaciens causes tumors on plants by transferring part of its Ti (tumor inducing) plasmid, the T-DNA or transferred DNA, to plant cells. The virB operon, the largest operon of the virulence (Vir) region located on the Ti plasmid, is necessary for tumor induction on all plant species. Previously, the complete nucleotide sequences of the virB operons of several Ti plasmids of A. tumefaciens were determined. The 11 predicted proteins mostly have signal sequences and/or hydrophobic domains. Hence, these proteins are thought to be located in or transported over the agrobacterial inner membrane. The VirB proteins are suggested to form a pore structure in the membrane through which the T-DNA-protein complex is transported. To obtain direct evidence for transport of these proteins over the inner membrane, we made fusions between genes of the virB operon and the gene for the enzyme alkaline phosphatase. Here we show the localization of several VirB proteins in the bacterial membrane and predict the topology of the membrane-localized VirB proteins. The finding of a fusion between the VirB7 protein and the enzyme alkaline phosphatase provides the first evidence for the expression of the small virB7 gene. The VirB2 protein shows similarity with the TraA propilin of the Escherichia coli F plasmid. Here we show that the predicted topology of the VirB2 protein in the inner membrane is identical to that of the TraA protein. Therefore, we hypothesize that the VirB2 protein is part of an agrobacterial pilus-like structure.