Date of Award

Spring 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Christian Baron

Committee Member

Turlough Finan, Justin Nodwell

Abstract

Type IV Secretion Systems (T4SS) are machineries required for the virulence of many Gram-negative pathogens. They contribute to bacterial competence, conjugation and the translocation of toxins from bacteria into eukaryotic hosts. In the plant pathogen Agrobacterium tumefaciens, the T4SS complex is composed of 11 VirB proteins (VirB1-VirB11) and VirD4. The VirB/D4 complex spans the bacterial envelope and assembles filamentous T-pili, which extend into the extracellular environment and mediate inter-bacterial conjugation as well as the formation of Crown Gall tumors on plants.

In my Ph.D. project, the role of VirB5 during the T4SS process occupied the major focus. VirB5 is required for inter-bacterial conjugation and to incite tumors on plant surfaces. Previous research has demonstrated that VirB5 is indispensable for the assembly of the outermost T4SS section, the T-pilus. In addition, detailed analysis of the VirB5 ortholog, TraC, form the Escherichia coli plasmid system pKM101 revealed that this putative pilus protein might be exploited during the Type IV-dependent phage binding prior to entrance into bacterial cells. Collectively, previous research findings led to the hypothesis that VirB5 is a minor T4SS component with a T-pilus associated form, which contributes to agrobacterial pathogenesis (Schmidt-Eisenlohr, Domke et al. 1999). Towards addressing this hypothesis, several approaches were undertaken such as forced localization of VirB5 to various sub-cellular compartments, PCR mutagenesis, C-terminal truncation and alanine replacements. An optimized immuno-electron microscopy (immuno-EM) procedure was also applied during the course of my studies.

Forced localization of VirB5 to the outer membrane of the periplasmic space using an inner trans-membrane domain fused to its amino- or carboxyl-terminus led to a nonfunctional T4SS and abolished T-pilus assembly. Using immuno-EM, VirB5 was found to associate with the T-pilus tips, bacterial surface and the ends of the detached pili. Characterization of VirB5 C-terminal deletion and alanine replacement variants revealed that VirB5 may be assigned an important role in host cell specificity.

As result of other investigation of the necessity of VirB5 for agrobacterial pathogenesis, a former Ph.D. candidate in Dr. Baron’s laboratory in Munich (Dr. Lilian Krall) found that VirB5 interacts with the agrobacterial host range factor trans-zeatin synthesizing enzyme, Tzs. My follow-up work showed that, similar to VirB5, Tzs localizes to the bacterial surface. Unlike the T4SS transglyosylase VirB1, Tzs is not secreted into the culture supernatant. Using Blue Native Electrophoresis (BNE), it was shown that the association of Tzs with agrobacterial protein complexes of diverse molecular masses was significantly altered in case of the virB5 deletion mutant CB1005. Taken together, these results highlighted that VirB5 is a key protein required for the cell surface localization of the Tzs.

The result of my Ph.D. study have significantly contributed towards better understanding the role of VirB5 during the T4SS process and have opened numerous research avenues to better understand the method(s) by which T4SS machineries from the source pathogen can recognize the recipient.

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