Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

The genus Vibrio includes serious human pathogens, such as V. vulnificus (Vv) and V. cholerae (Vc). Some species infect shellfish, such as Vibrio nigripulchritudo (Vn), which is a shrimp pathogen. Vibrio species encode PecS, a member of the multiple antibiotic resistance regulator (MarR) family of transcription factors. PecS is encoded by the pecS gene and expression is auto regulatory. The pecS gene is divergently oriented to pecM, which encodes an efflux pump. Vibrio species feature frequent duplication of pecS-pecM genes, which may facilitate how these species adapt from being free living motile bacteria in water columns to colonizing host tissues as biofilms to evade distinct environmental pressures.

Vibrio vulnificus, commonly associated with consumption of undercooked oyster encodes a single pecS-pecM gene pair. Vv PecS was shown to bind two sites within the pecS-pecM (iSM) intergenic region with Kd = 0.3 ± 0.1 nM, a binding that is attenuated by purine ligands xanthine and urate. A unique DNA-binding target for Vv PecS was found in the intergenic region between genes encoding the nitric oxide sensing transcription factor, NsrR, and nod; the nod-encoded nitric oxide dioxygenase is important for preventing nitric oxide stress in V. vulnificus. In vivo reporter gene assays indicate that Vv PecS-mediated repression of gene expression can be relieved in presence of ligands. Because xanthine and urate are produced as part of the oxidative burst during host defenses or under molluscan hypoxia, I hypothesize that these intermediates in the host purine degradation pathway function to promote bacterial survival during hypoxia and oxidative stress by targeting Vv PecS.

PecS homologs from V. cholerae and V. nigripulchritudo were also characterized by protein-DNA binding assays to determine their binding affinity for target gene promoters. Vn PecS bound the pecS-pecM intergenic region as well as the promoter for an indigoidine biosynthetic gene. The Vc PecS homolog bound the promoter for a gene encoding diguanylate cyclase, which is responsible for the synthesis of 3’ 5’ cyclic di-GMP. These in vitro assays lay the foundations for in vivo assays to determine the roles of these PecS proteins in biofilm formation and combating oxidation stress.

Date

3-18-2019

Committee Chair

Grove, Anne

Available for download on Thursday, December 24, 2020

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