Fang, Ferric CNewman, Shannon L.2017-10-262017-10-262017-09Newman_washington_0250E_17716.pdfhttp://hdl.handle.net/1773/40651Thesis (Ph.D.)--University of Washington, 2017-09Incorporation of horizontally acquired genes into transcriptional networks is essential for regulated expression of virulence in the bacterial pathogen Salmonella enterica serovar Typhimurium. The nucleoid-associated protein H-NS is able to recognize and bind AT-rich horizontally acquired genes, such as the csg regulon required for curli synthesis, and silence their expression. While previous studies have implicated a role of the alternative sigma factor σS in directly disrupting H-NS silencing, our system reveals that σS plays a more indirect role, acting through transcriptional regulators such as CsgD, IHF, and MlrA. I have identified an essential role of CsgD as a direct counter-silencer to H-NS silencing. Using a suite of footprinting techniques in vitro, I was able to determine that CsgD causes a topological distortion to disrupt the H-NS nucleofilament and reinstate RNA polymerase open complex formation. While σS appears to be required for csgD expression in vivo, csgD is σS-independent in vitro. Analysis of regulators further upstream in the pathway suggest that both MlrA and IHF are required for csgD expression in vivo and are also dependent on σS. Initial studies in vitro suggest that MlrA may serve as a counter-silencer of H-NS, although the mechanism for this process awaits further characterization. Additionally, we identified a previously uncharacterized input for biofilm regulation. Vitamin B12 likely serves as an allosteric regulator of MlrA and may serve as a photosensor to trigger biofilm formation in response to harmful UV irradiation. Characterization of counter-silencers in Salmonella can help us understand mechanisms of gene regulation that can be extrapolated to other organisms and provide new insights into the evolution of regulatory networks in bacteria.application/pdfen-USCC BYCounter-SilencingcsgH-NSSalmonellaMolecular biologyMicrobiologyGeneticsMolecular and cellular biologyThesis