The research focus of the Withey lab is the regulation of gene expression with an emphasis on genes involved in bacterial pathogenesis.  Our current model system is Vibrio cholerae, the causative agent of the severe diarrheal disease cholera.  The two major virulence factors of V. cholerae are the cholera toxin (CT) and the toxin co-regulated pilus (TCP).  V. cholerae uses a complex network of transcription regulators to control expression of the genes involved in virulence, including those that encode CT and TCP, together with a collection of other genes whose exact roles in pathogenesis are unclear.  The direct transcriptional activator of the majority of these virulence genes is ToxT protein, which is a member of the large AraC/XylS family of transcription regulators.  Understanding the function of ToxT at the molecular level is our primary goal at present.

            We have identified and characterized the DNA sequence to which ToxT binds upstream of all of the genes it is known to activate.  This sequence, the toxbox, is a 13 base-pair, degenerate, A-T rich sequence.  ToxT is able to activate transcription of different virulence genes using toxboxes in a variety of configurations and locations relative to the core promoter elements.  Currently we are investigating further how ToxT interacts with toxboxes in different configurations, and determining the interactions that occur between ToxT and RNA polymerase during transcription activation at different promoters.  We are also investigating how ToxT activity is regulated post-transcriptionally and are interested in the functions of genes activated by ToxT in pathogenesis or colonization of the host intestine. 

            Our research uses a combination of bacterial genetics and in vitro biochemical techniques.  The power of bacterial genetics allows us to identify genes and mutants of interest, which we then characterize using in vivo techniques such as promoter-reporter fusions and in vitro techniques such as electrophoretic mobility shift assays (EMSA) and DNA footprinting.