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Elucidating the Role of Iron and Cis-acting Regulatory Regions on the Expression of Aerobactin, an Iron Acquisition System Detected in Salmonella enterica IncFIB Plasmids.

Authors:
Poster Author(s)
Bushman, Summer, FDA/NCTR (Student); Gudeta, Dereje, FDA/NCTR (Mentor); Khajanchi, BIjay, FDA/NCTR (Mentor); Foley, Steven, FDA/NCTR (Mentor); Henry, Savannah, FDA/NCTR (Student)
Center:
Contributing Office
National Center for Toxicological Research

Abstract

Poster Abstract

Salmonella enterica is a leading cause of foodborne illness in the United States and around the globe. The Centers for Disease Control and Prevention (CDC) estimates that Salmonella infects over 1.3 million people annually in the US, with over 26,000 cases requiring hospitalization. This bacterium is known to harbor chromosomal and plasmid-encoded host immune evading virulence factors. Siderophores are iron acquisition virulence factors that can be encoded from a plasmid or the chromosome. Aerobactin is a plasmid encoded siderophore that we detected on an IncFIB plasmid of Salmonella enterica strain SE163. Aerobactin is encoded from the iucABCD-iutA operon, and the expression of genes in this operon is likely driven by a single promoter, which is located upstream of the iucA gene. The promoter contains a cis-acting Fur binding region often called Fur box. Fur, a transcriptional regulator, requires iron as a binding cofactor. Thus, under iron poor conditions, Fur loses its binding affinity for the Fur-box clearing the promoter for RNA polymerase activity, resulting in expression of the genes in the operon. The aims of this project are: i) to mutate the Fur box detected on the iucA promoter from GATAATGAGAATCATTATT to GATAgagtcgcatcctagg, which will create “Fur binding mutant” (fbm), ii) to contrast IutA-GFP translational fusion and iucA promoter GFP transcriptional fusion in fbm and wild type background iii) to determine the effect Fur-box mutation on iucA promoter activity and iutA gene expression using GFP as a reporter under iron replete and iron poor conditions. The mutants are generated using our recently developed allelic replacement vector. Our PCR results confirmed that we have tagged GFP at the 3' end of the iutA gene and construction of the remaining mutants is currently in progress. The GFP assay will be performed in spectrophotometer in triplicates using an excitation wavelength of 485nm.

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