Date of Award
© 2004 Wonjae Benjamin Rhee. All rights reserved. Access to this work is restricted to users within the Swarthmore College network and may only be used for non-commercial, educational, and research purposes. Sharing with users outside of the Swarthmore College network is expressly prohibited. For all other uses, including reproduction and distribution, please contact the copyright holder.
Bachelor of Arts
Amy Cheng Vollmer
Periplasmic deletions of the homodimeric Agrobacterium tumefaciens sensor histidine kinase VirA result in a loss of interaction with the wild type periplasmic domain in a yeast two-hybrid assay, suggesting that this domain may contribute to dimerization. Modifications were made to the wild type periplasmic domain using mutagenic PCR. Two of the mutations, peri5 and peri6, were found to have enhanced and weak interactions respectively, relative to wild-type peri versus itself in the yeast two-hybrid assay. Further characterization of these mutants was done after substituting the wild type periplasmic domain of a 2.6 kb virA fragment with these modified domains and cloning into a high copy vector, pYW15b. These mutant strains were assayed for vir gene expression and virulence in the virA null strain, A348-3, carrying a P_(virB):lacZ reporter fusion using a β-galactosidase assay. We found that in comparison to wild type VirA, the VirA^(peri5) mutant has an elevated level of vir gene expression in the absence of arabinose, while vir gene expression is decreased in the presence of arabinose. This is a deviation from normal VirA behavior where the primary phenolic inducer signal is amplified in the presence of monosaccharides. Similar behavior was observed in a quantitative tobacco virulence assay. Clearly, there is a need to analyze these mutants further. Assuming that VirA requires dimer formation to function, our study implies that neither the peri5 nor the peri6 mutants seem to contribute to VirA dimerization, as evidenced by the ability of these mutant VirAs to function in vir gene expression and virulence assays. We also provide evidence that yeast-2-hybrid interactions do not necessarily correlate with biological interactions.
Rhee, Wonjae Benjamin , '04, "Structure-Function Analysis of the Periplasmic Domain of the VirA Sensor Kinase from Agrobacterium tumefaciens" (2004). Senior Theses, Projects, and Awards. 51.