Molecular and Biochemical Characterization Reveals the Solute Transport and Binding Profiles of an Adenine Permease from Erwinia amylovora, the Bacterial Causative Agent of Fire Blight in Apples

Candace R Alexander, Purdue University

Abstract

Fire blight is a devastating disease afflicting members of the family Rosaceae, including apples and pears. It’s caused by the bacterial pathogen, Erwinia amylovora. Fire blight is characterized by a black or “scorched” appearance to plant tissues that can ultimately lead to loss of the entire tree. This issue is of high economic importance, as fire blight affects crop yields across the globe. Current treatment involves the blanket use of antibiotics, such as streptomycin, and in areas where its use is permitted, antibiotic-resistant strains are arising. To establish infection, E. amylovora relies on the efflux of nitrogen-rich compounds from the plant’s tissue, and their transport into the bacterial cell is relied upon by nucleobase transporter proteins, which are possible drug targets. The goal of this research was to characterize the solute transport profile of EaAdeP of E. amylovora. EaAdeP exhibited an amino acid sequence identity/similarity of 81.35%/95.06% to PurP of E. coli. It was isolated from E. amylovora’s genome, sequenced, and expressed in a plasmid vector to be transformed into E. coli lacking their native PurP. This bacterial strain expressing EaAdeP was tested for its ability to grow in the presence of toxic nucleobase structural analog 8-azaadenine. To derive its substrate specificity, E. coli expressing EaAdeP were tested for their ability to transport a panel of radiolabeled nucleobases. Lastly, kinetic parameters were produced for positively transported substrates.

Degree

M.S.

Advisors

Mourad, Purdue University.

Subject Area

Molecular biology

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