Characterization of OmpR Gene Regulation

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DESCRIPTION (provided by applicant): Two component regulatory systems have emerged as a paradigm for adaptive responses. The simplest systems consist of a sensor and a response regulator. The two-component system in E. coli that regulates the porin genes responds to changes in osmolarity of the growth medium. EnvZ, the presumed osmosensor is phosphorylated by intracellular ATP and then phosphorylates OmpR. At low osmolarity, the major porin in the outer membrane is OmpF and at higher osmolarity, ompF transcription is repressed and ompC is activated. Two-component systems are intimately involved in the coordinate expression of virulence factors in many different pathogens and OmpR is an important global regulatory protein. In Salmonella, OmpR lies at the first step of a regulatory cascade that turns on a downstream two-component regulatory system and activates expression of a type three secretion system required for systemic infection. In the present application, the hypothesis to be tested is that EnvZ controls the concentration of OmpR approximately P by adjusting its phosphatase activity in response to the osmotic signal. Prior to the previous funding period, we discovered that DNA binding stimulates OmpR phosphorylation. This observation may have important mechanistic implications for signaling and it suggests that response regulators that function as transcription factors may be phosphorylated while bound to the DNA. In the first aim, we will attempt to isolate and characterize such an EnvZ/OmpR/DNA complex. An EnvZ-GFP fusion will be employed to examine signaling and transcription in intact cells and in spheroplasts to determine the role (if any) of the outer membrane. An OmpR approximately P dephosphorylation assay has been developed and will be used to test the role of EnvZ in OmpR-P turnover. In the previous funding period, we discovered that OmpR is capable of binding to DNA in a head-to-head orientation rather than the previously proposed head-to-tail mode. Our new model predicts that the recognition helix is actually helix 2 rather than helix 3. We will determine the role of helices 2 and 3 in DNA recognition and test the hypothesis that OmpR can bind to DNA in more than one orientation. In aim three, we propose to solve the full-length structure of OmpR, the structures of the isolated N- and C-terminal domains, the phosphorylated N-terminal domain and the C-terminal domain bound to DNA by NMR.