Date of Award

May 2013

Degree Type

Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Charles F. Wimpee

Committee Members

Mark J. McBride, Sonia L. Bardy

Keywords

Bioluminescence, Lux Operon, Luxr Homolog, Quorum Sensing, Transcriptional Activation, Vibrio Harveyi

Abstract

Bacterial bioluminescence is observed in over twenty known species, primarily in the family Vibrionaceae. However, only Vibrio fischeri and Vibrio harveyi bioluminescence expression mechanisms are well studied. In V. harveyi, expression of the lux operon is activated by the transcription factor LuxR (LuxRVH), resulting in bioluminescence. Homologs of LuxRVH in other Vibrio species have been shown to regulate transcription of a variety of genes. Three parallel quorum sensing pathways co-regulate the expression of LuxRVH. The first objective was to assess possible quorum sensing regulation of lux operon expression in V. cholerae, V. chagasii, V. orientalis, and V. vulnificus using V. harveyi as the control. Secondly, cross-species induction of bioluminescence by LuxRVH was tested on the aforementioned Vibrio lux operons using an Escherichia coli dual vector expression system. This was accomplished by first generating a plasmid with the V. harveyi luxR gene (luxRVH) driven by an arabinose promoter. Secondly, individual E. coli systems had one of the five Vibrio species-specific lux operons cloned on a separate plasmid. Luminescence was assayed qualitatively on plates, and quantitatively using a luminometer. Relative light per cell was calculated by dividing measured light by OD600. Relative light assays showed quorum sensing regulation of the lux operon in all five Vibrio species. Quantitative V. harveyi lux operon expression assays in the E. coli dual vector systems showed significant increase in light production in samples provided with arabinose. This demonstrated that induction of luxRVH by arabinose resulted in upregulation of lux genes. The level of LuxRVH activation of other Vibrio lux operons reflects the distance of evolutionary relationships to V. harveyi. The most induction was seen with the V. vulnificus lux operon followed by V. orientalis, V. chagasii, and finally V. cholerae. This implies conservation of the lux operon regulatory mechanism between closely related species and suggests that the studied Vibrios utilize LuxRVH-type transcription factors. The known LuxRVH homologs, SmcR (V. vulnificus) and HapR (V. cholerae) were suspected lux activators. In addition, other LuxRVH-type regulators are now implicated in the other Vibrio species. Furthermore, mechanistic conservation of these transcription factors implies regulation by V. harveyi-type quorum sensing.

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