Title: Transcriptional repression, anti-repression, and glycosylation in the regulation of flagellar motility in Listeria monocytogenes
Description: Flagellar motility is a critical environmental adaptation that allows bacteria to escape adverse conditions and populate new environments. In the facultative intracellular pathogen Listeria monocytogenes, flagellar motility is regulated in a temperature-dependent manner. Prior to the work presented in this thesis, two factors had been identified as regulating flagellar gene expression in L. monocytogenes. The MogR transcriptional repressor was shown to down-regulate the transcription of three flagellar genes in response to physiological temperature (37°C) and to be required for virulence in a mouse model of infection. The DegU response regulator was demonstrated to be required for the expression of all flagellar genes during growth at low temperatures (30°C and below). In this thesis, I characterized the roles of MogR and DegU in mediating temperature-dependent regulation of flagellar gene expression and identified a new component of this thermosensitive switch. MogR-mediated repression of flagellin (FlaA) was determined to specifically be required for L. monocytogenes virulence. MogR was shown to directly repress the expression of all known flagellar motility genes at 37°C by occluding RNA polymerase binding. The repression activity of MogR was determined to be directly antagonized by the MogR anti-repressor, GmaR, which occluded the DNA binding domain of MogR. Since GmaR anti-repression activity was temperature-independent, production of GmaR specifically at low temperatures was shown to confer temperature specificity to flagellar gene transcription. Transcription of gmaR was dependent on the DegU response regulator, as DegU activated the transcription of a flagellar operon encoding GmaR and basal body components. DegU-mediated activation of this operon appeared to constitute an assembly checkpoint regulating flagellar biogenesis and gene expression.

Interestingly, GmaR was also determined to possess β-O-linked N-acetyl glucosamine (O-GlcNAc) transferase (OGT) for flagellin. Since the glycosyltransferase activity of GmaR was dispensible for its anti-repression activity, these studies revealed GmaR as a bifunctional protein that regulates the production of its enzymatic substrate. Collectively, this thesis provides insights into how L. monocytogenes senses and integrates the temperature signal into the regulatory network governing flagellar motility and how the physical coupling of dual activities within a single protein can permit a glycosyltransferase to unexpectedly regulate gene expression.