Doctor of Philosophy (PhD)
Multiple antibiotic resistance regulator (MarR) family proteins include numerous ligand-responsive transcriptional regulators that modulate diverse biological functions in pathogenic bacteria. HucR is a MarR homolog that negatively regulates the expression of a gene encoding urate oxidase in Deinococcus radiodurans by binding a cognate site in the gene promoter. In the presence of the effector molecule urate, repression is attenuated. This aspect of HucR has been instrumental in designing urate-responsive synthetic devices that can trigger production or release of urate oxidase upon sensing pathological levels of urate. The structure of HucR shows that two histidine residues are stacked at the pivot point of the long intersecting helices of the dimer interface, raising the possibility it is a pH sensor. My studies reveal that protonation of stacked histidine residues leads to reversible formation of molten globule state and attendant DNA binding attenuation at physiological temperatures. Further, the binding of urate to symmetrical sites in each protein lobe is conveyed to the DNA-binding motif via the dimer interface, which results in loss of DNA binding. Taken together, my findings suggest that HucR not only responds to urate, but also to pH and that this system could be used to design pH-sensitive synthetic devices. The second aspect of this dissertation focuses on elucidating the gene regulatory mechanism of PecS from the plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes. In related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression and controls secretion of the antioxidant indigoidine. PecS downregulates the expression of both pecS and pecM genes; PecM is responsible for effluxing indigoidine. DNase I footprinting shows that PecS binds two palindromic sites in the pecS-pecM intergenic region and induces significant distortion upon binding at neutral pH, the pH at which PecS represses the pecS promoter. At alkaline pH, DNA distortions are attenuated and derepression of the pecS promoter occurs. Likewise, attenuation of DNA distortions was observed by binding of oxidized PecS to DNA containing the pecS-pecM intergenic region. These findings indicate that PecS-imposed DNA distortions adversely affect the ability of RNA polymerase to bind or initiate transcription. I propose that PecS modulates gene activity by altering core promoter DNA topology in response to pH and cellular redox state. The physiological implication is that the PecS regulon is induced when P. atrosepticum colonizes the plant apoplast and encounters an elevated pH and reactive oxygen species.
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Deochand, Dinesh Kumar, "Gene Regulation by MarR Family Proteins that Responds to pH or Oxidative Stress." (2016). LSU Doctoral Dissertations. 4356.