Date of Award

January 2018

Degree Type

Open Access Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

First Advisor

Marcia M. Pierce

Second Advisor

Rebekah L. Waikel

Third Advisor

William J. Staddon

Abstract

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen. This species is a common cause of food-borne illness outbreaks with high fatality rates. L. monocytogenes is known to produce a number of virulence factors, including enzymes and toxins, which are involved in the disease process. The arginine deiminase pathway has the ability to regulate acidic environments by converting arginine to ammonia, ornithine, and ATP. With the help of a membrane-bound antiporter, the ornithine is transported out of the cell while the ammonia is converted to ammonium. The ATP by-product can also be used with the F0F1-ATPase system to help maintain homeostasis. The prfA gene, which codes for the PrfA protein, is considered to be a crucial virulence regulator in L. monocytogenes. The PrfA protein has the ability to bind to palindromic promoter boxes within the chromosome in order to recruit necessary components for transcription of ten crucial virulence factors. This study attempted to produce genetic knockouts of the prfA gene and ADI pathway. The splicing overlap extension polymerase chain reaction method was used for genetic manipulation in this study. The 6.9 kb shuttle vector pKSV7 was used to introduce the spliced genetic insert into the wild type strain. The pKSV7-AD vector for the Δarc was successfully introduced into L. monocytogenes. The integration of the plasmid into the host chromosome was also successful; however the plasmid did not integrate with the gene of interest. The attempted Δarc was unsuccessful due to the inability of the spliced SoeAD-pKSV7 insert to bind to the gene of interest. The pKSV7-AD vector for the attempted ΔprfA successfully incorporated into the L. monocytogenes chromosome. The prfA SoeAD-pKSV7 inserted in the correct gene of interest, resulting in a successful mutant strain.

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