Transcription Factor Discovery in the Opportunistic Pathogen Pseudomonas aeruginosa

Project Summary Pseudomonas aeruginosa is a gram-negative, aerobic/facultatively anaerobic, rod-shaped bacterium that is commonly found in freshwater environments. However, P. aeruginosa can become an opportunistic pathogen, causing increased morbidity and mortality in immunocompromised hosts, burn victims, and cystic fibrosis patients. For example, more than

two million people are infected by P. aeruginosa annually, resulting in almost 90,000 deaths. Notably, the environmental versatility of P. aeruginosa, its multiple mechanisms for providing antibiotic resistance, and its wide range of dynamic defenses (e.g., biofilm formation), render P. aeruginosa a most challenging organism to effectively treat, even in today’s hospital settings.

The genes responsible for P. aeruginosa virulence are regulated by a host of sequence-specific transcription factors. Unfortunately, our knowledge regarding their transcriptional regulatory networks and how they can be modulated is woefully incomplete. Our laboratory has developed an iterative selection method, Restriction Endonuclease Protection Selection and Amplification

(REPSA), that has proven highly successful in identifying consensus DNA binding sequences and target gene promoters for a variety of transcription factors in bacteria such as Escherichia coli and Thermus thermophilus. We now propose to use REPSA to identify the binding sites and genes regulated by the MerR-family of transcription factors in P. aeruginosa, which are thought

to be involved in mediating responses to environmental stimuli, e.g., oxidative stress, heavy metals, and antibiotics, involved in pathogenesis. Ultimately, such studies will illuminate global regulatory networks in this important pathogen, thereby facilitating the development of novel anti-P. aeruginosa therapeutics.

Notably, per the goals of the SuRE award mechanism, this project will support the research activities of the PI, a former NIH grant recipient, and will enhance the training opportunities of undergraduate and master’s graduate students at a primarily undergraduate/majority-minority institution, a major focus for the PI. An award will allow the PI to continue a record of training

students who have gone on to further biomedical education in graduate or professional schools and/or pursuing careers in STEM industries. It will also allow the PI to apply the skills honed through continuous NSF-funded basic research to questions of direct relevance for human health.