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Simon Jakubowski

Advisor: Peter J. Christie, Ph.D.

Accepted post-doctoral position at University of West Florida after receiving PhD
Now a Principal Science Technology Consultant at AlphaFix LLC, Houston, TX

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plants cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies using two complementing techniques, reporter fusions of VirB6 to periplasmically active alkaline phosphatase phoA’ and cytoplasmically active b-galactosidase and cysteine accessibility support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop. five transmembrane segments, and a cytoplasmic C terminus. A combination of Transfer DNA immunoprecipitation (TrIP) assays and topology studies have been used to identify several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the exiting T-strand, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. These findings support a model in which the large periplasmic loop compromises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate translocation to the cell exterior. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 Interactions with VirB7 and VirB10 were localized to the C- and N-terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier.


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Research Info

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB/D4 Type IV Secretion System (T4SS)