THE ESSENTIAL VIRULENCE PROTEIN VIRB8 LOCALIZES TO THE INNER MEMBRANEOF AGROBACTERIUM-TUMEFACIENS

Agrobacterium tumefaciens genetically transforms plant cells by transf erring a specific DNA fragment from the bacterium through several biol ogical membranes to the plant nucleus where the DNA is integrated. Thi s complex DNA transport process likely involves membrane-localized pro teins in both the plant and the bacterium. The 11 hydrophobic or membr ane-localized proteins of the virB operon are excellent candidates to have a role in DNA export from agrobacteria. Here, we show by TnphoA m utagenesis and immunogold electron microscopy that one of the VirB pro teins, VirB8, is located at the inner membrane. The observation that a virB8::TnphoA fusion restores export of alkaline phosphatase to the p eriplasm suggests that VirB8 spans the inner membrane. Immunogold labe ling of VirB8 was detected on the inner membrane of dr-induced A. tume faciens by transmission electron microscopy. Compared with that of the controls, VirB8 labeling was significantly greater on the inner membr ane than on the other cell compartments. These results confirm the inn er membrane localization of VirB8 and strengthen the hypothesis that V irB proteins help form a transfer DNA export channel or gate.