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Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence
K M Stephens1, C Roush, E Nester
1Department of Microbiology, University of Washington, Seattle 98195.
Journal of Bacteriology
|January 1, 1995
Summary
VirB11 protein is essential for Agrobacterium tumefaciens T-DNA transport. Mutations in its nucleotide-binding site impair virulence, indicating the importance of this site for bacterial conjugation and plant infection.
Area of Science:
- Microbiology
- Molecular Biology
- Plant Pathology
Background:
- The virB11 gene is crucial for Agrobacterium tumefaciens T-DNA transfer into plant cells.
- VirB11 is a putative ATPase localized to the inner membrane, despite lacking a typical export signal.
Purpose of the Study:
- To investigate the localization and function of VirB11.
- To characterize the role of the Walker nucleotide-binding site in VirB11 activity and virulence.
Main Methods:
- Purification of VirB11 using a maltose-binding protein fusion in E. coli.
- Complementation assays with a virB11 deletion mutant.
- Site-directed mutagenesis of the Walker nucleotide-binding site (K175A, K175R).
- Analysis of protein localization, ATPase activity, and virulence phenotypes.
Main Results:
- Purified VirB11 lacked ATPase and autophosphorylation activity.
- Mutations in the Walker site (K175A, K175R) resulted in avirulent or attenuated virulence phenotypes.
- Mutant proteins localized to the inner membrane but were present at higher levels.
- VirB11K175R protein showed instability after cell lysis.
Conclusions:
- VirB11's ATPase and nucleotide-binding activities are critical for its function in T-DNA transport.
- The conserved lysine in the Walker site is essential for VirB11's role in Agrobacterium virulence.
- VirB11 localization to the inner membrane is independent of other Ti plasmid proteins.