Architecture of the Agrobacterium tumefaciens vir -encoded Type IV secretion system

• Main Author: Draper, Olga
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2002
• Subjects: Microbiology; Molecular biology
• ISBN: 9780496300181; 0496300180

Agrobacterium tumefaciens is a soil borne pathogen that infects a broad range of plants including fruit and nut trees and causes crown gall disease, known for its tumorous phenotype. A. tumefaciens genetically modifies its host when a fragment of its tumor inducing plasmid is transferred and integrated into the host genome. The transferred DNA (T-DNA) is nicked, displaced, and covalently bound to a “pilot” protein. The nucleoprotein (T-complex) is then exported through the bacterial membranes by a Type IV secretion system (T4SS) composed of twelve proteins, VirBI-VirB11 (the transporter) and VirD4 (a “coupling” protein that delivers the appropriate substrate to the transporter). The T4SS is an evolutionarily conserved mechanism used for secretion of virulence factors or for distribution of antibiotic resistance by pathogenic bacteria. To understand the network of interactions that define the architecture of the VirB transporter, I began my analysis with the yeast two-hybrid system. I helped create plasmids to ease peptide linkage mapping with complex libraries and screened a library of VirB fragments to map potential domains of interaction among the VirB proteins. Interacting domains show several subsets of previously unknown interactors: VirB1-VirB4-VirB8-VirB11-VirB10, VirB7-VirB9-VirB1-VirB11, and either dimers or self-interacting domains of VirB1, VirB4, and VirB8. The interaction domains of disulfide-linked proteins, VirB7 and VirB9, were further delimited by the two-hybrid results. The subsets of interactors support a model of physical interactions that span the bacterial envelope. To test the biological relevance of the yeast two-hybrid interactions, I modified an in vivo T-DNA transfer assay. Merodiploid expression of a VirB4 fragment missing the WalkerA box, a domain critical for ATP hydrolysis, leads to a significant decrease of transporter function. Interference with transporter function implies the domain is intimately associated with the transporter structure. I also created a number of plasmids that can express epitope-tagged fragments of VirB proteins at near-wildtype levels that will be useful for biochemical approaches to dissecting the architecture of the transporter. Understanding the transporter architecture will enable predictions of individual protein functions and facilitate further research, perhaps providing targets for disruption of T4SSs that may combat disease or hinder the spread of antibiotic resistance in bacterial populations.