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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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