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Journal of Bacteriology, August 2000, p. 4137-4145, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Self-Assembly of the Agrobacterium tumefaciens VirB11 Traffic ATPase

Svetlana Rashkova,dagger Xue-Rong Zhou,Dagger Jun Chen, and Peter J. Christie*

Department of Microbiology and Molecular Genetics, The University of Texas--- Houston Health Sciences Center, Houston, Texas 77030

Received 28 January 2000/Accepted 8 May 2000

The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of lambda  cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda  superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A. tumefaciens cells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A. tumefaciens cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5' and 3' halves of virB11. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (Delta GKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, The University of Texas---Houston Health Sciences Center, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-5440. Fax: (713) 500-5499. E-mail: christie{at}utmmg.med.uth.tmc.edu.

dagger Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Dagger Present address: CSIRO Plant Industry, Canberra, ACT 2601, Australia.

Journal of Bacteriology, August 2000, p. 4137-4145, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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