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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM048746-03
Application #
2186282
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1993-01-01
Project End
1997-12-31
Budget Start
1995-01-01
Budget End
1995-12-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Miscellaneous
Type
Schools of Public Health
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Gordon, Jay E; Christie, Peter J (2014) The Agrobacterium Ti Plasmids. Microbiol Spectr 2:
Garza, Isaac; Christie, Peter J (2013) A putative transmembrane leucine zipper of agrobacterium VirB10 is essential for t-pilus biogenesis but not type IV secretion. J Bacteriol 195:3022-34
Sarkar, Mayukh K; Husnain, Seyyed I; Jakubowski, Simon J et al. (2013) Isolation of bacterial type IV machine subassemblies. Methods Mol Biol 966:187-204
Thanassi, David G; Bliska, James B; Christie, Peter J (2012) Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function. FEMS Microbiol Rev 36:1046-82
Berry, Trista M; Christie, Peter J (2011) Caught in the act: the dialogue between bacteriophage R17 and the type IV secretion machine of plasmid R1. Mol Microbiol 82:1039-43
Banta, Lois M; Kerr, Jennifer E; Cascales, Eric et al. (2011) An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect. J Bacteriol 193:2566-74
Kerr, Jennifer E; Christie, Peter J (2010) Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system. J Bacteriol 192:4923-34
Jakubowski, Simon J; Kerr, Jennifer E; Garza, Isaac et al. (2009) Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis. Mol Microbiol 71:779-94
Alvarez-Martinez, Cristina E; Christie, Peter J (2009) Biological diversity of prokaryotic type IV secretion systems. Microbiol Mol Biol Rev 73:775-808
Fronzes, RĂ©mi; Christie, Peter J; Waksman, Gabriel (2009) The structural biology of type IV secretion systems. Nat Rev Microbiol 7:703-14

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