Translocation of DNA across the Agrobacterium Envelope

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DESCRIPTION (provided by applicant): Translocation of macromolecules between cells is a major area of biomedical interest. In bacteria, the intercellular transfer of macromolecules is commonly orchestrated by surface organelles termed type IV secretion systems. Many bacterial species use type IV systems to deliver DNA substrates within or across species, genus, or even kingdom boundaries through a process termed conjugation. Conjugative DNA transfer contributes to genome plasticity over evolutionary time, and the spread of antibiotic resistance genes and other virulence traits on a more immediate time scale. Many medically-important bacterial pathogens also use type IV systems to deliver effector proteins to eukaryotic target cells during infection processes. The VirB/VirD4 system of Agrobacterium tumefaciens serves as important model for detailed mechanistic studies of type IV machine function. This system, assembled from subunits VirB1 - VirB11 and VirD4, translocates DNA and proteins to phylogenetically-diverse bacterial and eukaryotic target cells by a mechanism dependent on direct cell-to-cell contact. The overall goal of work in this laboratory is to describe in complete molecular terms the VirB/VirD4 machine biogenesis pathway, the basis for substrate recognition and transfer across the cell envelope, and the nature of the VirB/VirD4 machine - target cell contact. Recent studies identified three novel features of VirB10 that focus our attention on this channel subunit: i) it spans the entire Gram-negative cell envelope, a novel property among all described bacterial proteins, ii) it forms an ?-helical pore at the outer membrane, only the second described ?-helical outer membrane pore protein, and iii) it undergoes an ATP-mediated structural transition required for substrate translocation across the outer membrane. We hypothesize that VirB10 forms a structural scaffold across the entire cell envelope for assembly of two structures, the VirB/VirD4 translocation channel and the extracellular T pilus, the latter being important for establishment of target cell contact. We propose to: (A) define the architecture, assembly dynamics, and function of the ?-helical outer membrane pore with respect to substrate transfer and pilus biogenesis, (B) define how VirB10 contributes to channel function through biochemical and structural characterization of VirB10-containing machine subassemblies, and (C) elucidate how the VirB10 transmembrane domain and the VirB4 ATPase located at the inner membrane contribute to pilus biogenesis. Studies of type IV secretion are essential for at least two reasons. First, these are widely used machines among most if not all prokaryotic cells and yet their mechanisms of action remain poorly understood. Second, type IV secretion is a major contributor to the proliferation of antibiotic resistance as well as successful infection by many bacterial pathogens; these systems are therefore excellent targets for therapies aimed at suppressing disease progression.