Analysis of the virulence protein VirB6 and the assembly of the type IV secretion apparatus of Agrobacterium tumefaciens

Type IV secretion (T4S) is used by pathogenic microorganisms to transfer effector macromolecules to eukaryotic cells. The VirB/D4 apparatus of Agrobacterium tumefaciens transfers DNA and proteins to plant cells and forms a conduit spanning the bacterial membranes, allowing substrate export. This study investigated the role of VirB6 in T4S. VirB6 is a polytopic inner-membrane protein. Computer models predict up to nine hydrophobic regions that can function as transmembrane (TM) segments. Using alkaline phosphatase fusions, I observed that only four of these participate in membrane-spanning. Both the N and C-terminii of the protein lie in the cytoplasm. Mutagenesis studies suggested the periplasmic loop P1 and TM3 are important for VirB6 function. Deletion of virB6 showed a loss of VirB7 homodimer formation, but did not affect VirB3 and VirB5 accumulation. Using immuno-fluorescence microscopy, VirB6 was found to localize to the cell pole. Polar localization required five VirB proteins, VirB7-VirB11, suggesting that a complex of proteins is necessary to target VirB6 to the cell pole. A conserved tryptophan at position 197, as well as the C-terminus of the protein are essential for its polar targeting. A similar analysis demonstrated that ten VirB proteins localized to the cell pole and assemble a macromolecular VirB complex at the pole. Neither the assembly of the complex nor polar localization of a VirB protein requires ATP. Instead, other VirB proteins are required for the polar localization of at least six VirB proteins suggesting the importance of protein-protein interactions in the targeting of VirB proteins to the pole. Analysis of mutants and strains expressing a specific virB gene indicated that four proteins, VirB3, VirB4, VirB8 and VirB11, could target themselves to a cell pole. Colocalization and other experiments indicated that VirB6-VirB10 are the structural components of the T4S apparatus. Analysis of strains that express virB6-virB11 or its subsets indicated that VirB7-VirB10 are the minimum components necessary for the assembly of a polar VirB complex. In the presence of VirB11, VirB6 associates with this complex to form the T4S apparatus. The VirB8 protein functions as the assembly factor, targeting the apparatus to a cell pole.