Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.