Type IV secretion is used by pathogenic microorganisms to transfer effector macromolecules to eukaryotic target cells. The VirB͞D4 apparatus of Agrobacterium tumefaciens transfers DNA and proteins to plant cells. We postulated that the cell pole is the site of assembly of the A. tumefaciens type IV apparatus. Using immunofluorescence microscopy, we now demonstrate that 10 of the VirB proteins localized primarily to one cell pole and a macromolecular VirB complex is assembled at the pole. Neither the assembly of the complex nor polar localization of a VirB protein requires ATP utilization by the VirB ATPases. The requirement of other VirB proteins for the polar localization of at least six VirB proteins indicates an essential role of protein-protein interaction in polar targeting. Four proteins (VirB3, VirB4, VirB8, and VirB11) could target themselves to a cell pole independent of a VirB protein. We provide evidence that VirB6 -VirB10 are the structural components of the type IV apparatus. Using strains that express defined subsets of the virB genes, we demonstrate that VirB7-VirB10 are the minimum components sufficient for the assembly of a polar VirB complex. VirB6 associates with this complex to form the type IV secretion apparatus. VirB8 functions as the assembly factor and targets the apparatus to the cell pole.cell pole ͉ polar targeting protein ͉ VirB proteins ͉ scaffold protein

SummaryAgrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico , can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.

The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.