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. The focus of study in this laboratory is a type IV secretion (T4S) system of Agrobacterium tumefaciens. This system is ancestrally related and functionally similar to bacterial conjugation systems, as well as recently described protein translocation systems used by bacterial pathogens during the course of infection. This T4S system mediates transfer of diverse substrates across the A. tumefaciens cell envelope. The substrates include nucleoprotein particle such as the VirD2 relaxase covalently linked to a single strand of DNA, and protein monomers such as the VirE2 effector protein. The T4S system translocates these substrates to a variety of phylogenetically-diverse bacterial and eukaryotic cell targets through a process dependent on direct cell-to-cell contact. This T4S system, assembled from VirD4 and 11 VirB subunits, is an especially attractive system for detailed mechanistic studies of macromolecular transport because of the ease of manipulation of A. tumefaciens, the large numbers strains, constructs, and other molecular tools at hand, and the wealth of information available about it and closely related transfer systems. The overall goal of work in this laboratory is to describe in complete molecular terms the dynamic processes required for: i) biogenesis of the VirB/D4 secretion channel, ii) recognition of secretion substrates, and iii) translocation of substrates across the Gram-negative envelope. The studies will use state-of-the-art biochemical and structure-based approaches in combination with powerful in vivo technologies developed in this laboratory during the current grant cycle to define how model DNA and protein substrates are recruited to and through the secretion channel. First, contributions of two ATPases, the VirD4 substrate receptor and VirB11, to early- and late-stage substrate transfer reactions will be determined. Second, a newly-discovered phenomenon of DNA substrate- and ATP-mediated activation of a conformational switch of the VirB10 subunit required for channel assembly or gating will be fully characterized. Finally, the contributions of functionally interacting pairs of inner membrane subunits, VirB6 and VirB8, and outer membrane-associated subunits, VirB2 and VirB9, to substrate translocation across the cell envelope will be characterized. The proposed studies will develop a mechanistic understanding of type IV secretion in unprecedented detail.