Pseudomonas aeruginosa is an opportunistic microbial pathogen that is the underlying cause of many serious and frequently fatal illnesses. It is the leading cause of acute pneumonia and urinary tract infections in hospital patients, has been linked to severe corneal infections, and is the source of acute and chronic infections in predisposed individuals such as burn victims and cancer patients. The type III secretion system (T3SS) of P.aeruginosa is instrumental in establishing and sustaining acute infections, and, therefore, is a potential target for antibiotic development. This application is focused on a feedback loop that ties transcription of all T3SS-related genes to the activation of the T3SS by controlling the activity of the central transcription factor ExsA. Through crystallographic and modeling studies we have made the fascinating discovery that ExsD might be a novel type of transcription factor belonging to the family of Gre-like proteins. Because Gre-like proteins do not usually bind to DNA, perhaps the most significant aspect of this discovery is the apparent physical and sequence-specific interaction of the ExsD protein with the DNA. We seek to answer the following specific questions: (1) Is ExsD a Gre-like but gene-specific transcription factor? (2) What is the molecular basis for the direct interactions between ExsD and ExsA? Both aims have the potential to make important contributions to the field. The discovery of a gene-specific Gre-like factor significantly expands the paradigm of how these factors might work. In addition, we propose the presence of a new promoter- element located downstream from the transcription start site. The studies involving ExsA may inspire investigations into similar mechanisms involving other members of this important transcription factor family. Lastly, we will determine the crystal structure of the regulatory domain of ExsA. This will be the first structure reported for this type of domain and may offer intriguing new insights into its functiona role. PUBLIC HEALTH RELEVANCE: The bacterium Pseudomonas aeruginosa is a major cause of hospital-associated infections and the leading cause of death for people with cystic fibrosis. Treatment is often complicated because the bacterium displays a high level of antibiotic resistance. The type III secretion system, which we are studying, is a key facilitator of P.aeruginosa infections. We aim to determine the central mechanism of how this system is regulated by the interactions of an intriguing transcription regulator, ExsD, with RNA polymerase and the transcription factor ExsA. This understanding will enable us to develop new therapeutic options for the treatment of P.aeruginosa-associated infections.