Functional Characterization of a Novel Nucleobase Transporter from Erwinia amylovora, the Bacterial Causative Agent of Fire Blight Disease in Apples and Pears

Amanda J Stoffer, Purdue University

Abstract

Erwinia amylovora is the bacterial causative agent of fire blight, a devastating disease that affects members of the Rosaceae family (e.g. apples and pears). Upon infection, E. amylovora enters the plant through surface wounds and travels in the vascular system resulting in blockage of water and nutrients to distal portions of the plant. This ultimately leads to necrosis of plant tissue with a blackened or “scorched” appearance. To establish infection, E. amylovora must uptake/transport nitrogen-rich compounds from the plant’s extracellular spaces into its own cell. This transport necessitates integral membrane protein transporters responsible for the movement of nitrogen-rich compounds and are ideal drug targets for protection against fire blight. The objective of this research was to identify the substrate specificity of E. amylovora EaUraA. To achieve that, the gene encoding EaUraA was isolated from the pathogen E. amylovora, sequenced, and expressed in E. coli deficient in its native UraA. EaUraA shares 84%/95% identity/similarity with E. coli UraA. E. coli expressing EaUraA was then tested for its ability to grow in the presence of a panel of different toxic nucleobase structural analogs. To elucidate EaUraA’s substrate specificity, E. coli expressing EaUraA were tested for their ability to transport several different radiolabeled nucleobases from the growth medium into the cells. Additionally, kinetic parameters (Km and Vmax) were determined for the substrate.

Degree

M.S.

Advisors

Mourad, Purdue University.

Subject Area

Molecular biology|Genetics|Biochemistry

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