Grant: Discovery Grants Program - Individual

Post-translational modifications play a key regulatory role in bacterial physiology. A well known post-translational modification broadly conserved across the kingdom of life is protein phosphorylation. Protein phosphorylation of tyrosine residues has been well documented in eukaryotes, and plays a key role in regulatory cascades. Tyrosine phosphorylation in prokaryotes is poorly understood, and only a few classes of Bacterial tYrosine (BY)-kinases have been identified to date. However, recent proteomic studies have identified tyrosine phosphorylation as an abundant post-translational modification in prokaryotes, and studies in E. coli strongly suggest that unknown BY-kinases exist in the genome. During my postdoctoral studies I identified a new bacterial tyrosine phosphorylation system in E. coli that is conserved throughout laboratory, commensal, and pathogenic strains. Despite conservation of the BY-kinase and Protein Tyrosine Phosphatase (PTP), sequence alignments show genetic variation within the operon suggesting that  this system may be deferentially regulated between strains. Preliminary data supports this, providing the basis for this research program that seeks to determine the molecular mechanisms of tyrosine phosphorylation in bacteria using E. coli as a model system. I hypothesize that by characterizing bacterial tyrosine phosphorylation systems biochemically, I will uncover how post-translational modifications control key cellular pathways important for bacterial physiology. First, my lab will determine the mechanism of regulation of this new tyrosine phosphorylation system using luminescence- and fluorescence-based reporters. Secondly, my lab will biochemically characterize the newly identified BY-kinase and PTP through a combination of enzymology, structural biology, and biophysical characterization techniques. Third, my lab will investigate the structural basis for tyrosine phosphorylation-dependent regulation of major facilitator superfamily (MFS) transporters that are involved in the efflux of a variety of solutes including antibiotics, proline-rich peptides, bile salts, and other antimicrobial compounds. Together, these approaches will be invaluable for understanding the molecular basis for tyrosine phosphorylation in E. coli and uncover the functional differences between BY-kinases and PTPs compared to their eukaryotic counterparts. I anticipate that discoveries made by this proposal will significantly advance our understanding of cellular signaling and the role it plays in bacterial adaptation. This fundamental knowledge will be harnessed to identify first generation inhibitors of bacterial tyrosine phosphorylation and uncover novel targets for the design of antimicrobial control strategies that can greatly impact human and animal health, benefiting Canadians and our agriculture sector.