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"Structural Biology of Bacterial Transcription Factors Regulated by Pheromones or Second Messengers"

by
Atul Ashok Khataokar
Interdisciplinary Program
B.S., Shivaji University, 2006, Kolhapur, Maharashtra, India
M.S., New Jersey Institute of Technology, 2009, Newark, NJ



Thesis Advisor: Matthew Neiditch, Ph.D.
Associate Professor
Department of Microbiology, Biochemistry and Molecular Genetics

Wednesday, April 19, 2017
12:00 PM, ICPH Auditorium


Abstract

Quorum sensing is density-dependent bacterial cell-cell signaling mediated by secreted pheromones that coordinate a wide range of phenotypic responses including, among others, virulence factor expression and biofilm formation. The Streptococcus species employ peptide pheromones called small hydrophobic peptides, or SHPs, for cell-cell communication. SHPs bind to and regulate cytosolic transcription factors known as Rgg proteins. In previous structure-function studies of Rgg proteins we showed that they possess an N-terminal DNA-binding domain and a C-terminal pheromone binding domain. Each of these domains contributes to the Rgg-Rgg dimer interface. Buried at the dimer interface formed by the DNA binding domains, is an intermolecular disulphide bond. This disulphide is formed by a cysteine residue that is conserved in each of the greater-than 120 identified Rgg proteins. To further explore the mechanism of Rgg function, we have determined X-ray crystal structures of Rgg mutants that cannot form the disulphide bond. These structures, among others solved in our parallel studies of wild-type Rgg proteins, were determined in the presence and absence of target promoter DNA. These X-ray crystal structures reveal the molecular basis of Rgg-DNA interaction, and begin to explain how the Rgg-Rgg disulphide bond contributes to receptor function.

In addition to my crystallographic studies of bacterial intercellular (cell-cell) communication, I will also present the results of my studies of bacterial intracellular communication, i.e., second messenger signal transduction. Bacteria can exist in a motile state and form non-motile aggregates called biofilms. Vibrio cholerae transitions between the motile lifestyle to a biofilm-based infectious lifestyle in the human gastrointestinal tract. This transition between the motile and biofilm based lifestyles is controlled by the intracellular concentration of the bacterial second messenger, c-di-GMP. High levels of c-di-GMP induce biofilm formation and repress flagellar gene expression, thus c-di-GMP functions antithetical to motility. Low levels of c-di-GMP have the opposite effect, triggering motility and repressing biofilm formation. C-di-GMP can modulate this transition by directly regulating transcription factors and thus transcription initiation. I have worked in collaboration with the Waters lab (Michigan State University) to show how c-di-GMP regulates the activity of two V. cholerae bacterial enhancer binding proteins (bEBPs) FlrA and VpsR. C-diGMP inhibits the transcriptional activity of FlrA, the master regulator of flagellar biosynthesis. Conversely, c-di-GMP activates the transcriptional activity of VpsR, the master regulator of biofilm gene expression. Our biochemical, structural, genetic, and computational studies seek to explain how c-di-GMP functions to activate VpsR and repress FlrA.


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