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dc.contributor.authorDavey, Lauren Elizabeth
dc.date.accessioned2017-04-03T17:22:39Z
dc.date.available2017-04-03T17:22:39Z
dc.identifier.urihttp://hdl.handle.net/10222/72781
dc.description.abstractDisulfide bonds are important for the stability of certain extracellular proteins, including bacterial virulence factors. Thiol disulfide oxidoreductases (TDORs) catalyze disulfide bond formation. Although these enzymes have been well characterized in some organisms, little is known about how disulfide bonds are formed in Gram-positive bacteria, particularly among facultative anaerobes such as streptococci. An analysis of the Streptococcus gordonii sequenced genome found five putative TDORs. These TDORs were systematically investigated for their affect on autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This revealed a single TDOR with a pleiotropic phenotype that we named Streptococcus disulfide bond protein A (SdbA). SdbA was demonstrated to catalyze disulfide bond formation, and using an in silico approach, we identified the major autolysin AtlS as a natural substrate. The low homology of SdbA to known TDORs prompted us to investigate its catalytic mechanism. TDORs catalyze disulfide bonds using a CXXC motif. Typically, the N-terminal cysteine interacts with substrates, while the C-terminal cysteine is buried, and both are essential for activity. We show that SdbA functions differently, and that mutants with a single, buried C-terminal cysteine of the CXXC motif can complement a ΔsdbA mutant, and restore disulfide bond formation to recombinant and natural substrates. These results distinguish SdbA from previously described TDORs. Finally, we found that mutation of SdbA generated an activating signal for the CiaRH two-component system. CiaRH is involved in stress response and affects virulence in pathogenic streptococci. Our results suggest that CiaRH can sense disulfide bond formation in S. gordonii. Activation of CiaRH repressed a second signaling system, ComDE, and eliminated bacteriocin production. Together, CiaRH and ComDE regulate >100 genes. By modulating the activity of CiaRH, SdbA can influence a broad spectrum of bacterial physiological processes that are not directly related to disulfide bond formation. In summary, this study establishes SdbA as a novel TDOR that catalyzes disulfide bond formation in S. gordonii and is important for the normal physiology of the cell.en_US
dc.language.isoenen_US
dc.subjectStreptococcusen_US
dc.subjectDisulfide bondsen_US
dc.titleIdentification and Characterization of SdbA, a Novel Thiol-Disulfide Oxidoreductase in Streptococcus gordoniien_US
dc.date.defence2016-03-07
dc.contributor.departmentDepartment of Microbiology & Immunologyen_US
dc.contributor.degreeDoctor of Philosophyen_US
dc.contributor.external-examinerDennis Cvitkovitchen_US
dc.contributor.graduate-coordinatorBrent Johnstonen_US
dc.contributor.thesis-readerNikhil Thomasen_US
dc.contributor.thesis-readerJohn Rohdeen_US
dc.contributor.thesis-readerRafael Gardunoen_US
dc.contributor.thesis-supervisorSong Leeen_US
dc.contributor.thesis-supervisorScott Halperinen_US
dc.contributor.ethics-approvalNot Applicableen_US
dc.contributor.manuscriptsYesen_US
dc.contributor.copyright-releaseYesen_US
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