Agrobacterium tumefaciens uses DNA transmission in pathogenesis. The transfer of part of its genetic material to plants results in the formation of tumors. Novel DNA transfer from bacterial cells to cells of a higher organism is an unique, interesting and important phenomenon in microbiology and biotechnology. Genes required for this promiscuous DNA transmission are organized in a 28.6 kb regulon located on a 200 kb Ti plasmid that also contains the 25 kb transferred DNA (T-DNA). The regulon is comprised of seven operons (vfrA, vfrB, vfrG, virC, vfrD, vfrH and vfrE), of which the virB operon is the largest containing eleven genes; whose protein products are primarily associated with the A. tumefaciens membrane, are required for virulence, but whose precise functions remain unknown. From our recent studies, the VirB proteins show striking similarities to Tra proteins of conjugative plasmids such as IncW plasmid R388 and IncF plasmid F. These Tra proteins are involved in the synthesis and assembly of the conjugative sex pilus. Thus, one part of this three part research program focuses on the VirB proteins, which will include analysis for pilin or pilin-like structures, molecular escorting by the VirB4 protein as well as other VirB proteins for identifying their topology on the bacterial membranes. The second part is on the negative regulation of vir genes by the Ros protein encoded by the ros chromosomal gene, which includes further analysis of the putative zinc finger in the Ros protein, the identification of specific residues involved in binding to the ros box, the binding of VirG to the vir box upstream of ros, and further defining the ros box in binding Ros. The third part of the research will be on the nature of the suppression of oncogenicity by the Osa protein encoded by the osa gene on IncW plasmid pSa. The work will include determining function and identifying potential competition sites at the inner membrane, where Osa resides. The research will therefore employ a combination of genetic, immunochemical, biochemical as well as high resolution microscopical techniques to gain further insights on the T-DNA transfer apparatus, vir gene regulation and oncogenic suppression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM045550-25
Application #
2183234
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1979-09-01
Project End
1999-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
25
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California Davis
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618
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Archdeacon, J; Bouhouche, N; O'Connell, F et al. (2000) A single amino acid substitution beyond the C2H2-zinc finger in Ros derepresses virulence and T-DNA genes in Agrobacterium tumefaciens. FEMS Microbiol Lett 187:175-8
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Chou, A Y; Archdeacon, J; Kado, C I (1998) Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt. Proc Natl Acad Sci U S A 95:5293-8
Shirasu, K; Koukolikova-Nicola, Z; Hohn, B et al. (1994) An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol 11:581-8
Kado, C I (1994) Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. Mol Microbiol 12:17-22
Jones, A L; Shirasu, K; Kado, C I (1994) The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol 176:5255-61
Chen, C Y; Kado, C I (1994) Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa. J Bacteriol 176:5697-703
D'Souza-Ault, M R; Cooley, M B; Kado, C I (1993) Analysis of the Ros repressor of Agrobacterium virC and virD operons: molecular intercommunication between plasmid and chromosomal genes. J Bacteriol 175:3486-90
Shirasu, K; Kado, C I (1993) Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol Lett 111:287-94

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