Translocation of nucleic acids across biological membranes is of fundamental importance for diverse cellular processes in prokaryotes and eukaryotes. A striking example of DNA translocation with profound implications for genome evolution occurs during Agrobacterium tumefaciens infection of plant cells. In response to specific plant signal molecules, this prokaryotic pathogen synthesizes translocation-competent DNA/protein complex that cross not only the bacterial envelope but also plant membranes enroute to the nucleus. Expression of the transferred genes ultimately disrupts endogenous plant hormone balances, resulting in loss of cell division control and the formation of tumors. Because of this unique ability to incite plant tumor formation through DNA transfer, the A. tumefaciens plant transformation system offers an ideal model for examining fundamental processes related to host-pathogen signal exchange, macromolecular transport, and eukaryotic cell division control and tumorigenesis. The focus of this research program is to elucidate the structural and functional features of the apparatus at the A. tumefaciens membrane required for exporting macromolecules to plant cells. By several genetic and biochemical criteria, the about 9.5 kilobase (kb) virB operon codes for some or all of the components of this interkingdom transport system. The proposed research will evaluate the contributions of two putative ATP-binding/hydrolysis proteins, VirB4 and VirB11, to this DNA transport process. Both proteins contain conserved domains found in a superfamily of prokaryotic and eukaryotic mononucleotide binding/hydrolysis proteins. An initial study showed that purified VirB11 protein binds ATP, possesses ATPase activity, and autophosphorylates in vitro. A combination of classical and molecular genetic techniques, and protein biochemistry, will be used to examine the structures and functions of these proteins. VirB11 is required for DNA transport, but this remains to be definitively established for VirB4. The importance of VirB4 protein for DNA transport will be examined by constructing a nonpolar virB4 null mutation and assessing the ability of the corresponding mutant to transport DNA. Biochemical activities of both proteins will be characterized by protein purification and in vitro assays for ATP binding, ATP hydrolysis, and phosphorylation. Specific residues important for VirB4 and VirB11 biochemical activities will be identified and mutated by site-directed mutagenesis. Random mutations will be introduced to identify other regions of the proteins important for structure and/or function. Mutant proteins will be examined for altered enzymatic activity and effects on A. tumefaciens virulence and DNA transport. VirB4 and VirB11 membrane topologies, subcellular localization, and the potential for interacting with other cellular constituents will be evaluated. Corresponding analyses of mutant proteins will facilitate identification of domains or residues that are critical for protein configuration. A novel DNA transfer assay has been developed in this laboratory based on the ability of A. tumefaciens to transfer intron-containing reporter genes to plant cells and protoplasts. The sensitivity of the assay will be tested throughout the proposed studies by evaluating DNA-transfer efficiencies of wild-type strains and virB4 and virB11 mutants. These studies will test the utility of the assay as a genetic screen for identifying DNA-transfer deficient mutants. This assay will form the basis of future genetic studies of the A. tumefaciens infection process.
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