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Abstract
Pseudomonas aeruginosa is an important public health pathogen that is well-known for its high ability to adapt to a diverse range of environmental conditions, including those encountered in human hosts. The remarkably high ecological success, versatility, and pathogenicity of P. aeruginosa has been attributed to its harboring of a large repertoire of regulatory proteins coupled with an intricate genetic network. Yet, many aspects of the P. aeruginosa regulatory network remain largely unexplored for antibiotic drug discovery. Therefore, this dissertation describes studies that contribute to addressing some of the current unmet needs in P. aeruginosa regulatory network research. First, the methods utilized to produce high yields of highly pure and active P. aeruginosa RNA polymerase (RNAP) will be described, and previously unknown activity and kinetic information for the enzyme will be reported. The successful production of the central enzyme mediating transcription in P. aeruginosa sets the foundation to structural characterization of transcriptional regulation in the bacterium, which could provide novel insights into species-specific therapeutic drug development. For further probing transcriptional regulation in P. aeruginosa, this dissertation also focuses on two LysR-type Transcriptional Regulators (LTTRs) in the pathogen: FinR and CysB. Both proteins were found to be central to the P. aeruginosa virulence network through their regulation of sulfur metabolism in the pathogen. We identified a previously unreported role for FinR and found CysB to be a likely global regulator that mediated several virulent pathways. A central component of the experiments that led to these findings was our utilization of the P. aeruginosa RNAP to probe transcription in the bacterium. Overall, the findings from this dissertation provide the foundation for new studies targeting the P. aeruginosa regulatory network for antibiotic drug discovery and also provide a gateway for the elucidation of LTTR-mediated transcriptional regulation in P. aeruginosa.