Self-Assembly of the Agrobacterium tumefaciens VirB11 Traffic ATPase

The T-DNA transfer system of Agrobacterium tumefaciens is a member of the recently named type IV, or adapted conjugation, secretion systems in gram-negative bacteria (2, 5, 28, 40). These transporters share the property of having evolved from ancestral conjugation systems for novel purposes during the course of infection. Two subfamilies of the type IV transporters can be distinguished on the basis of the exported substrate(s). One subfamily is dedicated to the conjugal transfer of DNA as a nucleoprotein particle. This is a cell contact-dependent event, and transfer can occur across kingdom boundaries, as exemplified by A. tumefaciens-mediated delivery of T-DNA to plant cells and of other DNA to yeast, Neurospora, and other fungi (4, 8, 11). The second subfamily exports effector proteins into the extracellular milieu or directly into the cytosols of eukaryotic target cells. Identified in a rapidly expanding number of bacterial pathogens, these transporters are proposed to play diverse roles associated with virulence. The type IV Ptl system of Bordetella pertussis directs the six-subunit pertussis toxin, assembled in the periplasm, across the bacterial outer membrane to the extracellular milieu (2). Very recently, Helicobacter pylori was reported to use a type IV transporter encoded by the cag pathogenicity island to deliver the 145-kDa Cag protein directly to the mammalian host cell (31, 33). CagA undergoes tyrosine phosphorylation, forms a cylindrical structure, and appears to induce cytoskeletal rearrangements upon transfer to the host cell (31). Facultative intracellular pathogens including Legionella pneumophila, Rickettsia prowazekii, Brucella spp., and Bartonella spp. are thought to export factors that promote survival within macrophages, contribute to phagolysosome fusion, and/or contribute to macrophage killing (23, 32, 35). Significant progress has been made toward definition of the structure and function of the A. tumefaciens T-DNA transfer system (4, 8, 40). Composed of the VirB proteins, the T-DNA transporter likely assembles as a transenvelope channel through which T-DNA is translocated and a pilus for establishing productive interactions with target cells. A subset of VirB protein interactions, key early steps in transporter biogenesis, major and minor pilin subunits, and the pilus structure have been identified (4, 8, 13, 21, 29). Our laboratory has been investigating the roles of the cytoplasmic membrane ATPases VirB4 and VirB11 in T-DNA transfer (1, 4, 6, 7, 26, 38). VirB11 is related throughout its length to proposed subunits of other type IV transporters and to a number of archaeal proteins of unknown function. In addition, a region of VirB11 that spans the conserved Walker A and B nucleotide binding motifs and an adjacent His box is closely related to corresponding regions of the type II secretion pathway GspE family of proteins (27). Mutations of the conserved Walker A motifs of the VirB11 and GspE family members invariably abolish activities of the cognate systems, and ATP binding and hydrolysis has been confirmed for purified VirB11 (6) and VirB11-like proteins associated with conjugation including TrwD (20a, 27), TrbB, and H. pylori HP0525 (20a). Therefore, these proteins most probably share the general property of supplying energy from ATP hydrolysis to drive organellar assembly or substrate translocation at the gram-negative cell envelope. Mutations of the VirB11 Walker A motif are recessive (26). However, a screen of virB11 alleles generated by PCR mutagenesis resulted in the isolation of 11 dominant alleles (38). Of considerable interest, these alleles fell into two classes, one that coded for nonfunctional VirB11 proteins and a second that coded for fully functional proteins when synthesized in the absence of native VirB11. Isolation of this latter mutant class, which exhibit a phenotype only when synthesized in the presence of native VirB11, prompted a model that a functional T-complex transport system is composed of more than one VirB11 subunit (38). In the present report, we supply further biochemical and molecular genetic evidence for VirB11 self-association in vivo.