Access Type

Open Access Dissertation

Date of Award

January 2010

Degree Type


Degree Name



Immunology and Microbiology

First Advisor

Jeffrey H. Withey


Vibrio cholera, the causative agent of the severe diarreal illness cholera, uses a complex array of gene regulation to induce its virulence determinants. During the early stage of infection, and upon response to unknown signals, virulence genes are turned on. ToxT protein is the primary virulence gene transcription activator. Once ToxT is produced, it amplifies its own expression through an auto-regulatory loop and directly binds and activates expression of various virulence factors including the toxin-coregulated pilus (TCP) and cholera toxin (CT). During the late stage of infection, virulence genes are turned off and the bacteria escape the host to resume their lifestyle in the aquatic environment. While posttranscriptional regulation of ToxT has been observerd, most studies were focused on revealing how toxT expression is initiated. Here, the regulation of ToxT protein at the posttranscriptional level has been explored.

In chapter one, experiments are presented that indicate bicarbonate is a positive effector molecule that enhances ToxT activity. Culturing the bacteria in the presence of bicarbonate increases the ToxT activity without inreasing the protein level. Bicarbonate is found in the small intestine where V. cholerae colonize humans and could be the natural signal that the bacteria sense during the course of infection to maximally induce its virulence determinents.

In chapter two, the mechanism of virulence down-regulation as V.cholerae terminates its virulence expression during the late stage of infection preparing to enter back into the environment is assessed. The data suggest that virulence expression could terminate through ToxT proteolytic degradation. ToxT proteolysis was observed when culturing the bacteria at high temperature and pH, condition that has been found to repress virulence induction. Further analysis revealed that the unstructured motif which is located between amino acids 100-110 of ToxT is important for this degradation.

In the last chapter, a method of utilizing fluorescence-activated cell sorting (FACS) technology in conjuction with transposon mutagenesis is described. This method was used to isolate bacterial mutants that produce different gene expression profiles in response to environmental cues while cultured in vitro in liquid growth media.This technique should be applicable for isolating bacterial mutants that respond differently to chemical and physical inducers or repressors that are present in the liquid growth conditions.